Biomedicine & Pharmacotherapy 139 (2021) 111708

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Biomedicine & Pharmacotherapy

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Review

Doxorubicin-induced cardiotoxicity: An update on the molecular

mechanism and novel therapeutic strategies for effective management
Pushkar Singh Rawat a, Aiswarya Jaiswal a, Amit Khurana b, c, Jasvinder Singh Bhatti d,
Umashanker Navik a, *
a
Department of Pharmacology, Central University of Punjab, Bathinda, Punjab, 151401, India
b
Department of Veterinary Pharmacology and Toxicology, College of Veterinary Science, PVNRTVU, Rajendranagar, Hyderabad 500030, Telangana, India
c
Centre for Biomedical Engineering (CBME), Indian Institute of Technology (IIT), Delhi 110016, India
d
Department of human genetics and molecular medicine, School of health sciences, Central University of Punjab, Bathinda 151401, Punjab, India

A R T I C L E I N F O A B S T R A C T

Keywords: Doxorubicin (Dox) is a secondary metabolite of the mutated strain of Streptomyces peucetius var. Caesius and
Cardiotoxicity belongs to the anthracyclines family. The anti-cancer activity of Dox is mainly exerted through the DNA inter­
Doxorubicin calation and inhibiting topoisomerase II enzyme in fast-proliferating tumors. However, Dox causes cumulative
Oxidative stress
and dose-dependent cardiotoxicity, which results in increased risks of mortality among cancer patients and thus
DNMT 1
Histone deacetylase
limiting its wide clinical applications. There are several mechanisms has been proposed for doxorubicin-induced
micro RNAs cardiotoxicity and oxidative stress, free radical generation and apoptosis are most widely reported. Apart from
this, other mechanisms are also involved in Dox-induced cardiotoxicity such as impaired mitochondrial function,
a perturbation in iron regulatory protein, disruption of Ca2+ homeostasis, autophagy, the release of nitric oxide
and inflammatory mediators and altered gene and protein expression that involved apoptosis. Dox also causes
downregulation of DNA methyltransferase 1 (DNMT1) enzyme activity which leads to a reduction in the DNA
methylation process. This hypomethylation causes dysregulation in the mitochondrial genes like peroxisome
proliferator-activated receptor-gamma coactivator (PGC)-1-alpha (PGC-1α), nuclear respiratory factor 1 (NRF-1)
and mitochondrial transcription factor A (TFAM) unit in the heart. Apart from DNA methylation, Dox treatment
also alters the micro RNAs levels and histone deacetylase (HDAC) activity. Therefore, in the current review, we
have provided a detailed update on the current understanding of the pathological mechanisms behind the well-
known Dox-induced cardiotoxicity. Further, we have provided some of the most plausible pharmacological
strategies which have been tested against Dox-induced cardiotoxicity.

1. Introduction ejection fraction in 9% of the treated patients [2]. Apoptosis-mediated

Doxorubicin (Dox) is a secondary metabolite of a mutated strain of
Streptomyces peucetius var. caesius and belongs to the anthracyclines
family. Dox is used as an effective antineoplastic agent for multiple
cancer types. However, multiple systemic adverse effects restrain its
wide use in cancer chemotherapy. It is one of the established and
commonly used antineoplastic agents in treating different types of
cancers, including pediatric cancer, leukemia, breast cancer, etc. Dox is
an inhibitor of the DNA topoisomerase II enzyme and causes damage to
the DNA [1]. Anthracyclines cause dose-dependent cardiac toxicity, if
used above the dose range from 400 mg/m2-700 mg/m2 [1,2].
Anthracycline chemotherapy for a year caused impaired left ventricular
loss of cardiomyocytes and oxidative stress is stated as the main cause
of Dox-induced cardiomyopathy. Depletion of Sarcoplasmic Reticulum
Ca2+ is one of the early phase events in Dox-induced cardiomyopathy.
Cardiac cell apoptosis and heart failure are well correlated with altered
levels of Ca2+ and calmodulin-dependent kinase II [3]. Moreover, in­
hibition of mitochondrial biogenesis is thought to be the mechanism
behind Dox-mediated cardiotoxicity, mainly by activating cell death
pathways by inhibiting topoisomerase 2β [4]. Also, reactive oxygen
species (ROS) play a pivotal role in Dox-induced cardiotoxicity and
several other mechanisms like mitochondrial dysfunction, a perturba­
tion in iron regulatory protein, the release of nitric oxide, inflammatory
mediators, calcium dysregulation, autophagy and cell death are

* Correspondence to: Department of Pharmacology, Central University of Punjab, Bathinda, Punjab 151401, India.
E-mail addresses: ak3.khurana@gmail.com (A. Khurana), jasvinder.bhatti@cup.edu.in (J.S. Bhatti), usnavik@gmail.com, uma.shanker@cup.edu.in (U. Navik).

https://doi.org/10.1016/j.biopha.2021.111708
Received 26 March 2021; Received in revised form 4 May 2021; Accepted 5 May 2021
Available online 13 May 2021
0753-3322/© 2021 The Author(s). Published by Elsevier Masson SAS. This is an open access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).
P.S. Rawat et al. Biomedicine & Pharmacotherapy 139 (2021) 111708

reported to play significant role [1]. Several drugs are used in
Dox-induced cardiotoxicities, like dexrazoxane concomitant with Dox,
which binds at topoisomerase 2β and reduces cardiotoxicity and heart
failure in sensitive cardiac patients. Various approaches, including
angiotensin inhibitors, β-blockers and phytochemicals, show protective
action over Dox-induced cardiotoxicity and prevent cardiovascular
dysfunctioning [4]. Therefore, this review aims to discuss the detailed
mechanisms responsible for Dox-induced cardiotoxicity. Further, some
of the important pharmacological and phytochemicals agents which
show beneficial effects against Dox-induced cardiotoxicity are
described.
2. Cardiotoxicity and symptoms
Defects to the cardiac muscle, including heart failure (HF), structural
damage, and hypertension, is a known adverse effect of many conser­
vative chemotherapeutic agents. Also, it is defined as the occurrence of
signs of HF with an ejection-fraction reduction ≥ 5% to < 55% or the
lack of symptoms with an ejection-fraction reduction ≥ 10% to < 55%
[5]. Further cardiac damage can be classified as reversible, irreversible,
acute and chronic. Irreversible cardiac damage is further categorized as
type 1 and reversible damage as type 2 [5]. Type 1 (direct damage) is
generally produced by an aggregate dose (dose-dependent), whereas
type 2 damage is not associated with aggregate dose (dose-independ­
ent). Further, cardiotoxicity’s clinical symptoms include shortness of
breath, chest pain, heart palpitations, fluid retention in the legs,
distention of the stomach, and dizziness [5].
2.1. Epidemiology
The occurrence of chemotherapy-induced cardiotoxicity differs
broadly and it depends on the duration of the specific anti-cancer
treatment and underlying patient comorbidities. Dose-dependent, cu­
mulative and progressive cardiotoxicity appears after anthracyclines
administration. Historically, Anthracycline is well correlated with left
ventricular dysfunction, reduced left ventricular ejection fraction and
symptomatic heart failure in up to 5% of patients [6]. Reduction in the
aggregate dose or cumulative dose of anthracyclines has successfully
lowered the heart failure-associated complications. The cumulative dose
400 mg/m2 of anthracyclines produces an average of 3.5% of heart
failure cases [7] and the complication with anthracyclines increases
with the dose. The moderate cumulative dose of 250–400 mg/m2 of
anthracyclines causes asymptomatic left ventricular dysfunction up to
25% and 35%. If the cumulative dose of anthracyclines is 550 mg/m2
then its cardiotoxicity percentage is 7–16% and at the dose of 700
mg/m2 its percentage is 18–48% [8]. Overall, cases of heart failure over
the world are 4.5–7%. Cancer and heart diseases are currently the
leading cause of death in developed and developing countries [7,9].
Table 1 enlists some of the drugs which are known to cause
cardiotoxicity.
3. Doxorubicin and its pharmacology
Dox is a cytotoxic agent that comes under the class of anthracycline
antibiotics [19]. Dox is isolated from the culture of Streptomyces peuce­
tius, a bacterial species [20]. Chemically, Dox is composed of naph­
thacenequinone nucleus joined via glycosidic linkage to an amine sugar
called daunosamine. Dox is used to treat several forms of cancer,
including Kaposi’s sarcoma, acute lymphocytic leukemia, breast cancer,
lymphoma, and bladder cancer [21,22]. Dox inhibits DNA and RNA
synthesis through the inhibition of topoisomerase II. It attaches over the
nucleic acids of the DNA, apparently by particular insertion of the
anthracycline nuclei with the DNA of human cells [22,23]. In the
anthracycline structure, a lipophilic and saturated hydroxyl rich moi­
eties is neighbor to the amine sugar which is hydrophilic and makes the
drug soluble in both (hydrophilic and lipophilic) types of solvents. The
lipophilic part in the Dox shows acidic nature and the amino sugar in the
hydrophilic part demonstrated basic nature. These specially bind over
the plasma protein and cell membrane.
Dox show a toxic effect on malignant cells. Its detrimental action on
different body organs is supposed to be connected to nucleotide base
insertion and cell membrane by lipid binding activities of Dox. Insertion
of Dox blocks the nucleotide replication and thus the activity of DNA and
RNA polymerases. The interaction of Dox with the topoisomerase II to
form DNA-rupturable complexes seems to be an effective mechanism of
Dox as a cell-killing action [24]. Treatment with Dox over cells has
shown a marked specific morphological alteration in apoptosis or pro­
grammed cell death. Doxorubicin treatment in the patients shows both
effects like therapeutic and toxic effects [24].
3.1. Pharmacokinetics
Polovich et al. has analyzed cancer patients either by giving a single
or multiple drug medication have shown that Dox has a multiphasic
disposition after giving this drug via intravenously (IV). The dose range
of 30–70 mg/m2 of Dox presented the dose-independent pharmacoki­
netics profile in patients [25].
3.1.1. Absorption and distribution
It is unstable at gastric pH; hence, it is administered via IV route.

Table 1
Agents which cause cardiotoxicity.
Drug Cardiotoxic effects Mechanism of cardiac toxicity Prevalence of cardiotoxicity Reference

Trastuzumab • Heart failure • Damage to mitochondria 1–28% [10]

Sunitinib, Sorafenib • Heart failure • Inhibit tyrosine kinase pathway 3–8% [11]
• Acute coronary syndrome
• Hypertension
5-Fluorouracil • Dilated cardiomyopathy • Vasospasm 1.2–18% [12]
• Ventricular arrhythmia • Autoimmune phenomenon
• Sudden cardiac death • Myocarditis
Imatinib, desatinib, nilotinib • Heart failure • Inhibit ABL tyrosine kinase in cardiac muscle cells 1.7% [13]
Bevacizumab • Hypertension • Unknown 50% [14]
• Congestive heart failure
Cisplatin • Hypertension • Generation of oxidative stress Unknown [15]
• Heart failure
Taxanes (Paclitaxel, docetaxel) • Heart failure • Direct cellular toxicity 2.3–8% [16]
• Lipid peroxidation
Cyclophosphamide • Inflammation of cardiac muscle • Unknown 7–28% [17]
Anthracyclines (Aggregate dose) • Heart failure • Production of free radicals 2% [18]
Doxorubicin (> 500 mg/m2)
Liposomal doxorubicin (> 900 mg/m2)
Epirubicin (> 720 mg/m2)

2
P.S. Rawat et al.
Initially, the half-life distribution of Dox is 5 min because of the fast
tissue uptake of the drug. Later, the drug’s terminal half-life distribution
is slow to about 20–48 h, reflecting the slow elimination from the tissue
[25,26]. Conventional Dox is much distributed in the plasma and the
tissues. Absorption occurs into the cell and attaches to the cellular
components that further bind to the nucleic acid in the cellular DNA. It
does not cross the blood-brain barrier (BBB) and not attains the drug
concentration in the cerebrospinal fluid (CSF) [27].
Metabolism: After the Dox administration, it converted into its active
metabolite doxorubicinol by utilizing an enzyme NADPH-dependent
aldoreductases. Doxorubicinol is the active moiety that provides the
antineoplastic activity. These reductases are located in erythrocytes,
kidney and liver cells [28]. Dox metabolite is present more than 20% in
plasma after 5 min of Dox administration; after 30 min, it reaches 70%,
75% after 4 h and 90% in 24 h of drug administration [25].
Elimination: Chiefly, the drug and its metabolite are excreted from
bile in unchanged form and approximately 4–5% is excreted from urine
after 5 days of Dox administration in the unchanged form [25].
3.2. Pharmacokinetic study for a special population
3.2.1. In obese patient
High body mass index (BMI ≥ 30) in obese women patients leads to
high body surface area owing to their body weight. In such patients, the
clearance of the drug and its metabolite decreases. Reduction in clear­
ance leads to toxicity due to enhanced AUC levels in obese patients.
Limited evidence of cardiotoxicity appears in men in comparison to fe­
male obese patients [29].
3.2.2. In hepatic impairment patients
In liver compromised patients, the drug and their metabolite level is
increased because of a reduction in the clearance rate [30]. In hepatic
impairment, individuals with serum billirubin level in the range of 2 and
3 mg/100 ml, there is reduction in the 50% of dose of Dox, patients with
more than 3 mg/100 ml of serum billirubin dose need to be reduced by
75% and patients with more than 5 mg/100 ml of serum billirubin level
need to restrict the Dox treatment [30].
3.2.3. In children
The increased clearance rate was seen in the children of age above 2
years in comparison to adult ones. A slower clearance rate was seen in
the children of age below 2 years in contrast to older children [31].
4. Molecular mechanism of doxorubicin-induced cardiotoxicity
Several mechanisms have been proposed for Dox-induced car­
diotoxicity or heart failure. The cardiac complication of Dox are directly
related to increased oxidative stress in cardiac tissue. Increased oxida­
tive stress leads to ROS generation, which causes damage to the heart
muscle and arises due to the reduced level of antioxidants and sulph­
hydryl groups. Further, Dox-induced cardiotoxicity also involves altered
levels of Ca2+ ions, which causes apoptosis. Apoptosis occurs in the
cardiomyocytes and in the endothelial cells with the activation of cas­
pases [32]. Herein, we have compiled detailed molecular mechanisms
which are involved in Dox-induced cardiotoxicity.
4.1. Oxidative stress
Production of free radicals is the primary concern that produces
cardiac muscle cell injuries after Dox administration [33]. Dox-induced
cardiotoxicity appears via increased ROS production and lipid peroxi­
dation in cardiac tissues [34,35]. Aglycones and their iron complexes of
anthracyclines encourage the production of ROS [35]. From the last 30
years, several molecular mechanisms have been postulated for the
generation of ROS and its damaging effects on heart tissue. These
mechanisms have shown the involvement of various enzymes like NOS
3
Biomedicine & Pharmacotherapy 139 (2021) 111708
(nitric oxide synthase), NADPH oxidase (NOX),
mitochondrial-dependent ROS production and many more, which acti­
vates oxidative stress [32]. Herein, we have discussed all these mecha­
nisms below.
4.1.1. NOS dependent increased ROS production
Doxorubicin binds to the eNOS (endothelial nitric oxide synthase)
reductase enzyme and induces Dox semiquinone radical formation; this
radical reduces the free oxygen into the superoxide (O–2) free radical.
Enzymatic one-electron reduction type of reaction is involved in this
reactive conversion which shows cardiotoxic effects. Dox completes the
one-electron reduction reaction in the presence of flavoenzymes like
NADPH-cytochrome P450 reductase and mitochondrial NADH dehy­
drogenase [32,36]. When the drug binds to the eNOS reductase enzyme,
there is an imbalance in superoxide free radicals and nitric oxide levels.
Nitric oxide level decreases and the levels of superoxide increase which
leads to cardiotoxicity [37]. A study showed that the Dox administration
in the bovine aortic endothelial cell leads to increased eNOS mRNA and
proteins level, which activates the redox stimulation, which causes
apoptosis [38]. Interestingly, antisense eNOS mRNA reduced the stim­
ulation of the caspase-3 activity, indicating the cardioprotective effect
over Dox-induced cardiotoxicity. Likewise, transgenic eNOS mice show
enhanced Dox-induced cardiac ROS production, but in knockout mice,
cardiac ROS levels are decreased [39].
There are three types of NOS (nitric oxide synthase) in our system:
eNOS, i (inducible) NOS, and n (neuronal) NOS. Apart from eNOS, Dox
administration increases the iNOS transcription and protein expression
and induces the formation of nitrotyrosine (NT) with increased mito­
chondrial superoxide level in cardiac tissue [40–42]. Superoxide and
NT’s formation is based on the peroxynitrite level because it generates
potent oxidants like nitrogen dioxide and carbonates radical [42]. It
causes apoptosis of the cardiac muscle cells, decreased cardiac
contractility, and decreased CAT and glutathione peroxidase activity
after Dox administration. Chen et al., showed that in iNOS knockout
mice, the formation of NT and superoxide radicals were reduced after
the administration of the Dox and the same effects appear after the
administration of iNOS inhibitor like S,S˙-[1,3-phenylene-bis(1,2-etha­
nediyl)]bis-isothiourea (1,3-PB-ITU) [43].
In contrast, the effect of nNOS in Dox-induced cardiotoxicity is not
prominent. This isoform is less important because of poor oxidative
stress induction via Dox. No changes appear in nNOS mRNA level and
protein expression after Dox administration [42,44]. Hence, the effect of
nNOS in cardiotoxicity is less prominent in comparison to eNOS.
Moreover, nNOS shows the one-electron reduction of Dox after binding
at flavin domain and this domain plays an essential role in the action of
Dox [32,40].
4.1.2. Mitochondrial dependent ROS production
Dox mainly targets the cardiac cell’s mitochondria, which leads to
injury in the cardiomyocytes [43,45]. Due to the cationic nature of Dox,
it can easily engage inside the mitochondrial membrane and forms an
irreversible complex with cardiolipin protein [46]. Dox has a strong
affinity towards cardiolipin and it plays a crucial role in the proper
functioning of the electron transport chain (ETC) and forms the energy
for the body system. After the Dox administration, it bound to car­
diolipin and altered the cardiolipin functioning at the interface, which
causes the formation of superoxide (O-2) radical [43,47]. In mitochon­
dria, other proteins are also affected by the Dox treatment, leading to
impaired mitochondrial functioning [43,48].
Further, Dox reduces the oxidation of long-chain-fatty acid in mito­
chondria of the heart and increases glucose metabolism, showing
anaerobic metabolism apart from aerobic metabolism. This shift in the
metabolism may lead to heart failure or abnormal contractility and
relaxation; this metabolism shift appears only because of oxidative stress
[43,49–51]. Moreover, Dox affects the expression of the GATA-4 gene in
the mitochondria, leading to the suppression of mitochondrial synthesis
P.S. Rawat et al.
and their metabolism, thereby induces apoptosis [52]. Moreover, this
condition is reversed with the CO (carbon monoxide) inhalation to the
mice at low concentration because CO activates the gene required to
synthesize mitochondria and upregulates the nuclear-encoded heme
oxygenase (HO)-1. HO-1 is an antioxidant enzyme that protects against
cardiomyocyte apoptosis and HO-1/CO stimulates the Akt, deactivates
the glycogen synthase kinase-3β which allows the nuclear translocation
of the nuclear factor erythroid 2-related factor 2 (Nrf2) in the nucleus to
activates the GATA-4 gene and prevents the apoptosis [52].
4.1.3. Fe-Dox complex
Dox is a positively charged drug and plasma membrane is negatively
charged; thus, it can easily interact with each other [53]. The positive
charge of Dox facilitates good affinity towards the iron and forms the
Dox-Fe complex, which alters the metabolism of iron. This complex
further interacts with the free oxygen and enhances ROS production,
responsible for the induction of cardiotoxicity with this complex [54]. In
normal body physiology, the free iron content levels are not enough to
bind with Dox and therefore, no cardiomyopathy occurs [55]. Dox - Fe
interaction does not arbitrate alteration in the iron metabolism, but it
acts on the transferrin protein, which helps transport and binds to
intracellular iron [56]. Dox metabolite doxorubicinol removes the iron
from the catalytic Fe-S cluster of the cytoplasmic aconitase (also known
as iron regulatory protein- 1) and converts this enzyme into the
non-functional protein [56,57]. Therefore, the stability of transferrin
mRNA increases and prevents the translation of iron sequestration
protein. Consequently, there is a reduction in the IRP-1 (iron regulatory
protein) gene, an increase in the free iron level leads to the generation of
the ROS and ultimately led to cardiotoxicity. The increase in sensitivity
to Dox-induced cardiotoxicity in mice leads to a decrease in the iron
regulatory gene HFE (Human homeostatic iron regulator protein) [58].
This iron regulatory gene defect leads to increased iron accumulation in
the myocardium and causes cardiotoxicity [59]. The HFE gene plays an
essential role in the regulation of circulating iron uptake [60].
It is essential to determine the alteration in the iron storage in cancer
patients because they are taking the chemotherapy treatment. In cancer
patients, there are abnormal blood losses and deficiency in nutrients;
therefore, they may need a blood transfusion and iron supplementation
[61]. The adult and pediatric patients of leukemia show increased iron
overload during chemotherapy [60]. Therefore, careful monitoring is
required in patients with Dox.
4.1.4. NADPH dependent ROS
Zhao et al., reported that NOX are the enzymes that were used as a
source of cardiac ROS generation [62]. This enzyme shows a
multi-component enzyme complex that comprises membrane-bound
cytochrome b-558. This cytochrome is a heterodimer of gp91phox and
p22phox, cytosolic regulatory subunits p47phox and p67phox, and Rac1
(small GTP-binding protein) [63]. These enzymes play an important role
in forming Dox semiquinone radical from the transference of one elec­
tron from the NADPH to Dox, which is similar in the NOS pathway [32].
There are seven isoforms of NOX and NOX2 isoform plays a crucial role
in Dox-induced cardiotoxicity. Studies demonstrated that NOX2 defi­
cient species or gp91phox knockout mice are resistant to Dox-induced
cardiotoxicity with reduced production of superoxide-free radical in
the cardiac tissue. In contrast, cardiac dysfunctioning like atrophy,
cardiomyocytes programmed cell death and interstitial fibrosis was seen
in wild-type mice [62,64–66].
One of the oxidative stress regulating genes (Rac1) also shows a
significant role in NOX stimulation. In this background, a study showed
that the deletion of Rac1 gene in cardiomyocytes leads to the alteration
in Dox-induced NOX2 stimulation, DNA fragmentation, programmed
cell death and recovers heart functioning [63]. Further, Rac1 inhibitor
NSC23766 shows protection against Dox-induced cardiotoxicity [67].
Moreover, SNP (single nucleotide polymorphism) in the NOX complex in
patients with Hodgkin-lymphoma causes cardiotoxicity. These patients
4
Biomedicine & Pharmacotherapy 139 (2021) 111708
developed CHF and acute arrhythmias after the Dox administration with
the impairment in NCF4, p22phox and Rac2 are NOX subunits [68].
4.1.5. Intracellular calcium dysregulation
Dox causes dysregulation of calcium levels, leading to an increase in
intracellular calcium, the generation of ROS, and induces apoptosis in
cardiomyocytes, whereas Ca2+ chelators inhibit the ROS generation and
apoptosis in cardiac muscle cells [69]. During the metabolism of Dox,
there is the formation of the toxic metabolite DOXOL, which inhibits the
sodium-calcium exchanger channel [70,71]. The sodium-calcium
exchanger channel plays an important role in regulating heart contrac­
tility through the sarcoplasmic reticulum calcium pump and increases
the L-type calcium channel activity [72]. Further, Dox-induced cardio­
myopathy involves alteration in the calcium exchange regulating gene,
which affects ryanodine receptor (RyR), calcium storage gene, Sar­
co/endoplasmic reticulum ATPase (SERCA2a), and phospholamban,
thereby causing impairment in both systolic and diastolic functions of
the heart. In addition, study reported that the elimination of Dox
metabolite is difficult in comparison to Dox. This metabolite is retained
on the cardiac muscle cell and causes significant calcium dysregulation,
affecting cardiac muscle [73].
Apart from this, one more pathway is involved with the rise in
intracellular calcium, wherein calcium activates the calpains, which are
calcium-dependent proteases. An increased amount of calcium stores in
the sarcoplasmic reticulum’s cardiac muscles was observed during
oxidative stress and causes leakage of calcium ions that activate the
calpains. These calpains cleave the caspase-12, one of the apoptotic
factors and induce apoptosis of cardiac cells and a similar mechanism
was reported with the administration of Dox [74]. Dox-induced car­
diomyopathy is also related to the destruction of cardiac muscle pro­
teins. This destruction occurs because of the stimulation of calpains.
These calpains degrade the titin that is the largest protein and principal
component of heart sarcomere [71]. Some positive cardiac response is
also seen via the inhibition of calpains and reported that the depletion of
CARP (cardiac ankyrin repeat protein) results in sarcomeric structure
stability. This protein is a negative regulating cardiac gene and causes
instability of sarcomere arrangement [71,75].
Dysregulation in the calcium channels leads to an increase in the
calcium burden, which causes a decrease in the mitochondria’s calcium
storage capacity. This leads to the activation of the cyclosporin-sensitive
calcium channel, which exacerbates the calcium burden [76]. Thus, an
increase in calcium cytoplasmic concentration leads to mitochondrial
dysfunction and apoptosis [77].
Apart from this, there are many other pathways involved in calcium
dysregulation and apoptosis through Dox. Dox increases the free Ca2+
levels, which activates the calcium-dependent activated signaling and
leads to cell death [78]. Further, increased Ca2+ leads to the activation
of the CaMKII (calmodulin activated protein kinase II) and the PLN
(phospholamban). It enhances the apoptosis of cardiomyocytes through
the activation of caspase 3 & 9, H2O2, increased terminal deoxy­
nucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)
positive cells, and ROS generation.
4.2. Role of AMPK (AMP-activated protein kinase) signaling in Dox-
induced cardiotoxicity
AMPK is a macromolecule protein complex that contains three sub­
units, like one catalytic site α and two regulatory sites are β and γ. α
divided into two subunits, present in the heart such as α1 in the endo­
thelial cells and α2 in the cardiac muscle cells. AMPK pathway is blocked
after the Dox administration and reduced the ACC (Acetyl- coA
carboxylase) enzyme activity in the heart. Dox-induced AMPK signaling
inhibition leads to activation of other pathways such as Akt and MAP
kinase through genotoxic stress and oxidative stress, which induces DNA
damage. Also, AMPK suppression leads to the enhanced energetic stress
and hypertrophy in the cardiac tissue [1,79].
P.S. Rawat et al.
4.3. Role of autophagy
Autophagy is a conserved process in eukaryotic cells involved in the
maintenance of cellular homeostasis and survival under normal and
stressful circumstances. It involves removing damaged and long-lived
organelles under both physiological and pathological conditions, thus
regarded as a protective mechanism. Importantly, in response to stress,
there is dysregulation of the autophagy process in the heart and can
cause cardiac dysfunctions and heart failure [80,81]. Dox causes
abnormal upregulation as well as downregulation of genes involved in
autophagy thereby results in cardiotoxicity. Dox treatment induces
autophagy initiation probably through the p53-mediated suppression of
GATA-4, which results in the down-regulation of the Bcl-2 protein. In
addition, Dox initiates the autophagy process by increasing the phos­
phorylation of Bcl-2 that inhibits the interaction of Bcl-2/Beclin1 [82,
83]. Dox administration induces the accumulation of autophagosome
and inhibition of flux through the upregulation of several autophagy
related gene (Atg) expression and downregulating the master tran­
scription factor EB with altered function of lysosomal biogenesis in
cardiomyocytes led to cell death [84]. Further, Dox inhibits mTOR,
possibly by stimulating the initiation of autophagy and it is also linked
with the upregulation of p-53 ultimately led to cardiomyocyte injury.
Moreover, the accumulation of autophagosomes results in ROS genera­
tion [85–87]. Dox administration upregulates the genes like Atg5,
Atg12, Atg4 and Bad and downregulates the beclin1 protein. Further,
LC3B is an important marker of autophagy that is also upregulated after
Dox administration and causes cardiac damage [88]. In contrast, cur­
cumin protects from cardiac damage by blocking apoptosis and enhance
autophagy by regulating the c-Jun N-terminal kinases (JNK) mediated
pathway [1]. Altogether, the impaired autophagy can induce car­
diomyocyte death.
4.4. Role of creatine kinase
Creatine kinase is a protein present in the number of tissues in the
body and acts as an energy reservoir. But in cardiac tissues, only CK-MB
isoenzyme of creatine kinase is expressed. Dox-induced oxidative stress
damages the creatine isoenzyme in the presence of ferrous iron, which
leads to the generation of the peroxynitrite free radical. This enzyme
aids in the preservation of the energy with the conversion of creatine to
phosphocreatine, which use ATP as a substrate [71].
4.5. Apoptosis
Dox stimulates both intrinsic as well as extrinsic pathways of
apoptosis [45,89]. These pathways are responsible for the apoptosis of
cardiac muscle cells due to an imbalance in oxidant and anti-oxidant
[90]. Dox administration leads to the increase in oxidative stress,
which activates the HSF-1 (heat shock factor 1) thereby induces HSP-25
(heat shock protein), which stabilizes the p53 protein responsible for the
generation of proapoptotic factors such as FasL, Fas, c-Myc, and p53
which cause cardiac muscle cell death [90–93]. Moreover, Dox admin­
istration also increased the circulating HSP-70 and its expression in
cardiac tissue of mice thus causes inflammation and fibrosis [94]. In
contrast, other HSP proteins such as HSP-27, HSP-10, HSP-20, HSP-22
and HSP-60 are also protective and protect from apoptosis and maintain
myocardial functioning [89,95]. Recently Lan et al. reported that the
overexpression of HSP-22 in the cardiac tissue could reduce the cardiac
injury in response to Dox administration by inhibiting TLR-4/NLRP3
activation [96]. Increased level of HSP-27 shows the maintaining ac­
tion over the Dox-induced oxidative stress and mitochondrial dysfunc­
tioning [89]. Increased expression of HSP-10 and HSP-60 are
responsible for the increased post-translational modification of Bcl-2,
which leads to activation of the anti-apoptotic pathway [95]. HSP-20
also has a significant role in cardiac protection via activation of the
Akt pathway. It is an important pathway for cell survival and it prevents
5
Biomedicine & Pharmacotherapy 139 (2021) 111708
cardiac muscle cell apoptosis by inhibiting the pro-apoptotic factors like
Bad [97]. Moreover, Dox downregulates the Akt pathway and induces
caspase-3, which causes apoptosis [32,71,98].
Further, Dox upregulates the appearance of death receptors (DR) like
TNFR1, Fas, DR4, and DR5 at both protein and mRNA levels [99].
Toll-like receptor-2 (TLR-2) functions as a novel death receptor,
increasing the apoptosis through apoptotic factor, including caspase 8
and FADD (Fas-associated death domain). Antibodies of DR and TLR-2
receptor inhibitors blocks the binding of the HSP 60 protein to the
TLR-2 and it activates the NFκB (proinflammatory nuclear-factor κB),
which leads to the production of cytokine, programmed cell death and
altered cardiac functioning after the treatment of Dox. Further, TLR-4 is
also associated with Dox-induced cardiomyopathy, and it only happens
when HSP-70 interacts with the TLR-4 receptor and causes cardiomy­
opathy through the NFκB activation [100–102] and overall mechanisms
of Dox-induced cardiotoxicity is discussed in Fig. 1.
4.6. Endothelin-1
Endothelin-1 is also known as preproendothelin-1 (PPET-1) and it is
a potent vasoconstrictor produced from the vascular endothelial cells.
PPET-1 is an immature form of endothelin and endothelial cells convert
PPET to PET (Proendothelin) and then into their mature form
endothelin-1. After the Dox treatment, the levels of endothelin-1 in­
crease in cardiac muscle cells and cause hypertrophic cardiomyopathy.
Bosentan is an endothelin A/B receptor antagonist which produces
cardiac protective action over the Dox administration [103,104]. Their
antagonist shows cardioprotection via reduced TNF-α and Bax levels,
decreased lipid peroxidation and increase in the GATA4. Moreover, dual
antagonists of the receptor (ETA/ETB) produce more significant pro­
tection [104].
4.7. Fibrosis
Treatment with Dox in cancer patients leads to blockade of mRNA
transfer and inhibits protein synthesis of MMP-1 (Matrix metal­
loprotease -1) in tumor cells. It is helpful in cancer because of this
mechanism; there is a reduction in the tumor cell’s mobility. However, it
activates other MMP’s like MMP-2 and MMP-9, which show toxicity in
the heart by increasing collagen formation in the cardiac tissues [105,
106]. An increase in collagen leads to myocardium fibrosis. These two
MMP’s also activate the TGF-β and phosphor-SMAD3 signaling and this
enhanced collagen deposition in the cardiac muscle cells [1].
4.8. Role of epigenetics in doxorubicin-induced cardiotoxicity
Epigenetics is a heritable phenotypic change in which there is no
involvement in altering DNA base-pair sequence (genotype) [107].
Epigenetic modifications cause alteration in the gene function. It is a
natural process in which alterations occur regularly with several factors
like age, environment, external, and disease state. Two main epigenetic
modifications involved in many diseases and natural growth include
DNA methylation and histone protein modification. These two are
epigenetic mechanisms, which induce phenotypic changes and control
the gene expression by blocking proteins that attach to the silencer re­
gion of the DNA [108].
Every cell of the human body contains the same DNA molecule,
which develops from the individual cell at impregnation. Highly ar­
ranged epigenetic mechanisms facilitate the patterning required to
ensure normal human development and support stable regulation of
appropriate patterns of a natural phenomenon of expression in diverse
cell types. Regulation of these mechanisms by a more number of proteins
that begin, recite and erase specific epigenetic alterations. Thus, it de­
fines where and when the transcriptional machine can approach the
chief DNA sequences to run regular growth and differentiation in the
growing embryo and fetus [109]. A number of epigenetic marks work in
P.S. Rawat et al.
Fig. 1. This picture shows the different mechanisms of action of Dox-induced cardiotoxicity inside the cell and outside the cell.
whole human cells but run a specific gene expression. DNA methylation
occurs at the CpG island, covalent alterations of histone proteins, reg­
ulatory ncRNAs (noncoding) and other complementary mechanisms
maintaining higher order of chromatin association inside the cell nu­
cleus [110]. Following there are the three most important epigenetic
modifications reported behind Dox-induced cardiotoxicity.
4.8.1. DNA methylation
DNA methylation sometimes activates and inhibits gene expression
based on physiological or pathological conditions. The human genome
contains more than 30 million base pairs of CG dinucleotides (CpGs). For
methylation, CpG sites are an important target, comprised of the cova­
lent binding of a methyl group to the 5’ position of the cytosine (C). CpG
methylation arrangement is associated with transcriptional silencing
arrangement observed in 60–90% of the human genes [111]. It is a
process in which there is the addition of methyl moieties to the DNA
molecule and this addition leads to a change in DNA activity without
disturbing the DNA base pair sequence.
4.8.2. Histone modifications
Histones are one of the most important proteins of the body, which
provide a structural role. Highly basic proteins found in eukaryotic cell
nuclei that package and order the DNA into structural units called nu­
cleosomes [112,113]. Histones are the major protein component of
chromatin, acting as spools around which DNA wind, and have a sig­
nificant role in gene regulation. In the absence of histone proteins, un­
wound DNA in chromosomes would be very long.
4.8.3. ncRNA (non coding RNA)
A ncRNA acts directly as well as indirectly to regulate the epigenetic
modification. Even though transcription occurs up to 75% of genomic
DNA in eukaryotic genomes transcribe, but that only around 2% of these
transcripts are translated into protein; the majority are ncRNAs, which
can be classified according to size and function [114,115].
4.8.4. Dox-induced alterations in DNA methylation
In the DNA methylation process, there is a need for enzymes that can
add the methyl group or remove the methyl group for the body’s normal
functioning. Enzymes like TET (Ten eleven translocation) and methyl­
transferases and these enzymes require S-adenosyl methionine (SAM) or
alpha-keto glutarate (α-KG) for the development of 5-methylcytosine or
5-hydroxymethylcytosine. These formations directly show two pro­
cesses like DNA methylation and DNA demethylation. SAM and α-KG
develops in the mitochondria by a specific metabolic pathway and
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Biomedicine & Pharmacotherapy 139 (2021) 111708
mitochondrial dysfunctioning is one of the important mechanisms of
Dox induced cardiotoxicity [116].
Dox downregulates the DNA methyltransferase 1 (DNMT1) enzyme
activity which leads to reduction in the DNA methylation process. This
hypomethylation leads to dysregulation in the mitochondrial genes like
peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1-
alpha (PGC-1α), nuclear respiratory factor 1 (NRF-1) and mitochondrial
transcription factor A (TFAM) unit in the rat heart after the adminis­
tration of Dox. These gene products are encoded with both nuclear and
mitochondrial genomes [117]. In DNA methylation, mitochondrial ge­
nomes are regulated by DNMT1, but there is downregulation of DNMT1
after Dox exposure because Dox increases the oxidative stress, down­
regulates the DNMT1 and leads to mitochondrial dysfunctioning that
affects the DNA methylation [118,119].
4.8.5. Alterations in histone modification
Apart from DNA methylation, histone modifications are also
responsible for Dox-induced cardiotoxicity. Suppression of histone
deacetylase (HDAC) produces cardiac toxicity through the initiation
Rac1, GTP-binding protein, a subunit of NOX and p53 upregulation. The
initiation of these units leads to apoptosis and hypertrophy. After
treatment with Dox, there is abnormal upregulation and downregulation
of HDAC, leading to cardiotoxicity [67]. Dox causes the upregulation of
several HDACs, including HDAC4, HDAC5, HDAC6, HDAC7, HDAC10,
HDAC11 and downregulation of HDAC2. Upregulation of HDAC6 leads
to the deacetylation of α-tubulin seen in the rat heart after the treatment
of Dox [120]. In addition, Dox also upregulates the histone lysine
demethylase (KDM3A) and downregulates lysine-specific demethylase 1
(LSD1) in the H9C2 cardiomyocyte cell line [121]. Moreover, it also
upregulates the histone lysine methyltransferases like lysine N-methyl­
transferase 7 (SET7) and SET- and MYND-domain-containing protein 1
gene (SMYD1) in H9C2 cell line [119].
4.8.6. Alteration in the ncRNA
A ncRNAs act directly as well as indirectly to regulate the epigenetic
modifications. [114,115]. Regulatory ncRNAs, including small inter­
fering RNAs (siRNAs), microRNAs (miRNAs), and long ncRNAs
(lncRNAs) which play significant roles in the regulation of gene
expression at several levels: transcription, mRNA degradation, splicing
and translation [122].
There is also abnormal upregulation and downregulation of the
ncRNA after the treatment with Dox. There are several ncRNA’s which
are upregulated after Dox administration, like miR-23a, miR-34a, miR-
140, miR-146a, miR-532, miR-15 and some are downregulated like
P.S. Rawat et al.
miR-29b and miR-30. The upregulation of miR-15 ncRNA in the H9C2
cell line is reported to induce apoptosis. This effect appears because of
the suppression of Bmpr1a, which is an important target of miR-15 and
BMP and the administration of Bmpr1a agonist protects the cardiac cells
through the downregulation of miR-15 [119].
microRNAs (miRNA/miR) play an important role in Dox-induced
cardiotoxicity because of their role in cardiac functioning like devel­
opment, electrical signal conduction and cardiac muscle constrictions
[123]. Several studies show that Dox causes an alteration in the
microRNAs and causes either upregulation and or downregulation of
microRNAs. There was upregulation of microRNAs in rodents heart
tissue such as miR-34a, miR-34c, miR-208b, miR-215, miR-216b, and
miR-367, miR-21, miR-34a, miR-208a, miR-208b, miR-221, miR-222,
and miR-320a after the Dox administration. In contrast, the down­
regulated microRNAs are Let-7 g, miR-30a, miR-30c, and miR-30e [71,
123,124].
Further studies show that miR21 level is increased in H9C2 cells. This
upregulation of microRNAs indicates the cardioprotective action via
reducing the Dox induces apoptosis. However, in miR21, deficient spe­
cies of rat shows cardiotoxic effects after Dox treatment. Car­
dioprotective action via miR21 because it alters the anti-proliferative
factor and B cell translocation gene 2 (BTG2) [125]. Dox administration
also upregulates the miR-208a, but this results in cardiotoxicity because
it downregulates the GATA4 gene and increased apoptosis in car­
diomyocytes. In deficient species of miR-208a mice, improvement in
GATA4 and Bcl-2 was seen, which leads to a reduction in apoptosis
[126]. Dox also upregulates the expression of miR-146a and down­
regulates the ErB2 receptor tyrosine kinase 4 (ErB4). This receptor is an
important component of neuregulin-1-ErB signaling. This signaling
pathway plays critical role in cell survival, but Dox treatment down­
regulates this pathway and promotes apoptosis in cardiomyocytes
[127].
GATA-6 gene is a transcription factor and highly expressed in the
heart and plays an essential role in heart development [128]. Moreover,
Dox also downregulates miR30 family microRNAs and alters the
beta-adrenergic and mitochondrial apoptosis pathway. GATA-6 gene
acts as a precursor for the downregulation of the miR30 and causes
apoptosis. Increased expression of this miR30 protects cardiomyocytes
through the modulation of GATA-6. Overall, miRNAs play an essential
role in Dox-induced cardiotoxicity [128].
5. Therapeutic strategies
5.1. Metformin activates AMPK signaling pathway to reduce Dox
cardiotoxicity
Metformin is a drug that belongs to biguanide class, with anti­
hyperglycemic property and widley used to treat type 2 diabetes mel­
litus [129]. Metformin shows cardioprotective effects in rat
cardiomyocytes in Dox-treated rats. Dox deactivates the AMPK signaling
pathway and decreases platelet-derived growth factor receptor (PDGFR)
expression. In contrast, metformin treatment activates the AMPK
signaling pathway by increasing the PDGFR expression [130]. Another
study showed that metformin reduces the levels of H2O2, which offers a
protective mechanism on mitochondrial damage through the AMPK
activation. It also maintains autophagy markers like LC3B-II and p62 in
Dox-treated rats, thus improving cardiac functioning [79].
5.2. Statins
Statins are the agent which inhibits the rate-limiting enzyme HMG
CoA reductase in cholesterol biosynthesis. Statins help in lowering of
lipid levels and are used as an antihyperlipidemic drug. Statins are
commonly used in cardiovascular disorders, including atherosclerosis
and coronary heart disease [131]. Reports suggest that statins also
activate the AMPK signaling in cardiac muscle and show
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Biomedicine & Pharmacotherapy 139 (2021) 111708
cardioprotective action. This signaling pathway is dependent on the RNS
(reactive nitrogen species) and Ras-related C3 botulinum toxin substrate
1 (Rac1) [132–134]. Pitavastatin blocks Rac1 was shown in a mouse
model of Dox cardiotoxicity and shows cardiac protective role via a
reduction in the cardiac muscle apoptosis and regulates the constriction
function. Other statins also show cardioprotection like lovastatin and
rosuvastatin [79].
5.3. β-blockers and angiotensin-converting enzyme (ACE) inhibitors
These drugs are mainly used in the treatment of particular heart and
kidney diseases. This drug combination are used to treat high blood
pressure [135]. The β adrenergic blockers and ACE inhibitors elicit an
important pharmacological role in heart failure management. These
agents preserve the diastolic heart function in the Dox-treated in­
dividuals but left ventricular contraction remains constant, and this
action is mainly achieved by carvedilol. Alone, β-blockers do not show
cardioprotective action because of lacking antioxidant properties but are
useful in combination with carvedilol. Their mechanism of action re­
mains unclear; how it improved cardiac muscle contraction [136].
5.4. Phosphodiesterases-5 (PDE-5) inhibitor
Drugs that block phosphodiesterase-5 (PDE-5) show a positive
response in cardiac protection by inhibiting the cardiac muscle
apoptosis, preserving the mitochondrial membrane potential, regulating
myofibrillar integrity and prevent ventricular dysfunction and protect
from ST-interval prolongation in Dox treated mouse model [137].
Likewise, Sildenafil, a PDE-5 inhibitor, demonstrated a protective effect
in Dox-induced cardiotoxicity by modulating the NO/cyclic GMP,
mitochondrial K+ATP channel and oxidative stress [138].
5.5. α1-Adrenergic agonists
This agonist comes under the sympathomimetic drug class that ac­
tivates explicitly the α adrenergic receptors. Cardiac α1-adrenergic re­
ceptors show the cardioprotective role against Dox-induced
cardiotoxicity. α1-adrenergic receptor agonists like phenylephrine are
useful because they reduce the apoptosis, interstitial fibrosis and cardiac
muscle impairment caused by Dox. This protective action is exerted
through the generation of the anti-apoptotic factors, Bcl2 and maintains
mitochondrial functioning [139,140].
5.6. Dexrazoxane
It is a cyclic derivative of EDTA that quickly penetrates through the
plasma membrane [141]. It is a free radical scavenger used as an
adjunctive agent and shows chelator action on iron-mediated oxygen-­
free radical activation as well as lipid peroxidation. With this chelate
reaction, there is a reduction in iron-free radicals levels; thus, it shows
the cardioprotective action [32,71,142].
5.7. Resveratrol
The polyphenolic stilbene 3,5,40-trihydroxy-trans-stilbene (resver­
atrol) is produced by many plants and is a constituent of red wine.
Resveratrol is a naturally occurring agent having antioxidant and po­
tential chemopreventive activities. Resveratrol shows some pharmaco­
logical effects, including anti-inflammatory effects and inhibits
cyclooxygenase and hydroperoxidase functions [143]. Moreover,
resveratrol has cardioprotective activity through the activation of AMPK
[79]. AMPK attenuates oxidative stress, apoptosis and ameliorating
fibrosis in Dox-induced cardiotoxicity. It is a potent antioxidant that
reduces ROS levels and enhances antioxidant levels like superoxide
dismutase (SOD) and catalase (CAT) activity and also shows the
anti-inflammatory activity [144]. In addition, Pterostilbene, a natural
P.S. Rawat et al.
analog of resveratrol, prevents Dox-induced cardiotoxicity through the
activation of PGC-1α, reduces oxidative stress via enhancing AMPK and
SIRT1 cascades [145].
5.8. Vitamin E
Four tocopherols and four tocotrienols are combined to make
vitamin E, which is fat-soluble [146,147]. Vitamin E having an antiox­
idant property that protects the cell membranes from ROS [147]. A
study shows the protective action of Vitamin E over Dox-induced car­
diotoxicity. Vitamin E is helpful in acute cardiotoxicity caused by Dox.
Other antioxidants show limited benefit over the cardiotoxicity like
vitamin C, selenoorganic compound PZ51 and reduced glutathione,
ambroxol, ursolic acid and oleanolic acid [71,148].
5.9. Probucol
Probucol is an antihyperlipidemic agent which lowers lipid levels
and also has an effective anti-oxidant action. It shows cardioprotective
effect via a reduction in cardiac muscle injury and heart failure without
interrupting the antineoplastic effect of Dox [149,150].
5.10. Allicin
Allicin is an active constituent of garlic and it is an organosulfur
containing compound [151]. It has several pharmacological effects,
including antimicrobial, antioxidant, anticarcinogenic, and antifungal
[151]. It is a cytoprotective agent against Dox-induced cardiotoxicity. A
study confirmed that allicin efficiently reduces cardiac oxidative stress,
reduces inflammation, and reduces apoptosis in the presence of acute
Dox intoxification. Dox deactivates the antioxidant substances like
glutathione peroxidase, SOD and CAT and thus, causes oxidative dam­
age and cardiac apoptosis; however, allicin restores the antioxidant level
[152,153].
5.11. Adiponectin agonist ADP355 attenuates the Dox-induced
cardiotoxicity
Adiponectin is a protein hormone responsible for the regulation of
glucose and the breakdown of fatty acids. It is encoded as ADIPOQ gene
in humans. It is mainly located in the adipose tissue and also in muscles
and brain [154].
Adiponectin agonists prevent cardiotoxicity induced by Dox via
reducing the serum creatine kinase level, lactate dehydrogenase and
hydroxybutyrate dehydrogenase level. It also shows the antiapoptotic
action by lowering the TUNEL positive cells and cleaved caspase-3,
associated with the reduced Bcl2/Bax level in cardiac tissue. Another
protective mechanism of adiponectin agonist is to enhance the antioxi­
dant level, which is reduced with the induction of the Dox like Nrf2 and
SOD2. It also blocks ROS in the H9C2 cell of the cardiac muscle and
activates the sirtuin 2 signaling pathways, thereby protecting the car­
diac muscle from apoptosis and oxidative stress [155].
5.12. Erythropoietin
Erythropoietin is a hormone which is generated from healthy kidney
and a small amount by the liver. Erythropoietin is a glycoprotein cyto­
kine mainly secreted from the kidney at the time of cellular hypoxia
[156]. Some antianemic drugs also show cardioprotection against
Dox-induced cardiotoxicity. Erythropoietin is an antianemic agent
which is used in anemia caused by chemotherapeutic agents. Erythro­
poietin protects from Dox-induced cardiotoxicity via reducing apoptosis
and cardiomyopathy when administered prophylactically [157,158].
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Biomedicine & Pharmacotherapy 139 (2021) 111708
5.13. MiR-181c protects cardiomyocyte injury by preventing cell
apoptosis through PI3K/Akt signaling pathway
Apoptosis in the cardiac muscle is responsible for heart failure, which
results in an increased risk of mortality related to cardiovascular dis­
orders. Upon treatment with Dox, there is downregulation in the MiR-
181c, one of the microRNAs that helps in the muscle cell survival via
PI3K/Akt signaling pathway. MiR-181c, downregulation, leads to the
activation of the toxic markers like TNF-α, Fas and IL-6 and causes
cardiotoxicity, whereas treatment with MiR-181c inhibits apoptosis in
the Dox treated H9C2 cells [159].
5.14. Cardioprotective effects of 20(S)-ginsenoside Rh2 against Dox-
induced cardiotoxicity
From ancient times, ginseng has been commonly used as a recog­
nized traditional Chinese medicine. Its principal active constituent is
ginsenosides, which contain various pharmacological effects, including
activating immune function, enhancing cardiovascular health,
increasing resistance to stress, improving memory and learning, and
developing social functioning and mental health in healthy individuals
and chemotherapy. Ginsenoside Rh2 (Rh2) is one of the major bioactive
ginsenosides from Panax ginseng [160]. Pretreatment with Rh2 attenu­
ates Dox-induced cardiomyocyte injury’s cell-killing effect and enhances
the cardiac cell’s survival rate in a dose-dependent fashion. Further, Rh2
treatment restores the lactate dehydrogenase, creatine kinase and
aspartate transaminase in the serum, thus shows cardioprotective action
[161]. Treatment with Dox also decreases antioxidants like SOD, CAT
and glutathione and increased the malondialdehyde, whereas Rh2 re­
stores this imbalance [162].
5.15. Curcumin suppresses doxorubicin-induced cardiomyocyte
pyroptosis
Curcumin is a biphenolic compound and it is a major active con­
stituent of turmeric [163]. Curcumin is widely used as a coloring agent
and food additive and also curcumin has presented some therapeutic
effects [164]. Curcumin shows protective effects against Dox-induced
cardiotoxicity. Dox increases the levels of ROS, lipid peroxidation,
reduction in antioxidant levels and induction of pyroptosis. Dox also
downregulates the PI3K/Akt/mTOR pathway, which leads to car­
diotoxicity. Bcl2 is an antiapoptotic factor that is suppressed after Dox
treatment and promoted the expression of BAX, which causes mito­
chondrial cell death.
Curcumin is a natural compound derived from the plant Curcuma
longa and has an anti-inflammatory effect. After treatment with curcu­
min, it reduces Bax levels and prevents mitochondrial cell death [165].
Further, curcumin inhibits pyroptosis by modulating pro-pyroptosis
markers like NLRP3, caspase1 and IL-18 and downregulates the
inflammasome [166]. Curcumin also reduces the levels of CK, LDH and
AST in Dox-induced cardiac injury. Dox also increases autophagy in the
cardiomyocytes, which leads to cardiac dysfunction. In contrast, cur­
cumin reduced autophagosome levels (GFP-LC3 puncta and Beclin1),
which initiates the maintenance of cardiac functioning after the Dox
treatment [167].
Dox also downregulates the PI3K/Akt/mTOR pathway and this
pathway is necessary for cell survival and cell differentiation. The
downregulation of this pathway causes apoptosis in the cardiomyocytes.
Curcumin upregulates this pathway and shows cardioprotection over
the Dox-induced cardiac toxicity. Curcumin also reduced pyroptosis and
autophagy via activation of the Akt/mTOR pathway [167].
5.16. Visnagin-a new protectant against doxorubicin cardiotoxicity
Visnagin is a furochromone which is a derivative of chromone and
furan. Visnagin is a major constituent of Ammi visnaga. In traditional
P.S. Rawat et al.
medicine, visnagin has been used for kidney-associated problems [168].
A study shows the protective effect of visnagin in Dox-induced car­
diotoxicity through the inhibition of mitochondrial malate dehydroge­
nase (MDH2). Liu et al., identified the role of MDH2 as a novel target for
reducing Dox mediated cardiotoxicity [169]. MDH2 is an important
enzyme in the tricarboxylic acid (TCA) cycle. MDH is actively present in
glyoxysomes, mitochondria, peroxisomes, chloroplasts and the cytosol
[170] that bind with the malate-aspartate shuttle (MAS), which estab­
lishes the primary metabolic pathway for the transfer of reducing
equivalents from the cytosol into the mitochondria for oxidation. Dox
has an affinity towards the MDH2 enzyme and blocks this respective
enzyme activity, which possesses a cardioprotective activity in
Dox-induced cardiotoxicity [171].
5.17. Schisandrin B (Sch B) prevents doxorubicin-induced cardiotoxicity
Sch B the most abundant dibenzocyclooctadiene lignan present in
Schisandra chinensis (Turcz.) [172]. It has used in the treatment of ce­
rebral ischemia and Alzheimer’s disorder [173]. Sch B prevents
Dox-induced acute cardiotoxicity by increasing cardiomyocyte gluta­
thione redox cycling that eliminates excessive ROS formation [174]. Dox
administration causes weight loss in rats; in contrast, Sch B moderately
prevents weight loss. Furthermore, Dox blocks the mTOR signaling,
which causes a reduction in cardiac mass while the Sch B restores the
mTOR signaling [175]. Similarly, Xu et al. reported that Sch B partially
relieves mTOR inhibition in muscle or adipose tissue under Dox treat­
ment and improves the rat’s body weight. Therefore Sch B shows the
cardioprotective effect over the Dox treatment [176].
5.18. Treatment strategies focusing on mitochondrial modulation
Mitochondrial division inhibitor (mdivi-1) has been tried for several
disease conditions, including heart failure and ischemia and reperfusion
injury. Gharanei et al. demonstrated that mdivi-1 co-treatment with Dox
improves cardiac function and reduces the infarct size in both naïve
conditions and during ischemia/reperfusion injury. mdivi-1 also im­
proves the Dox-induced alterations in time taken to depolarisation and
hypercontracture of cardiac myocytes [177]. Moreover, Xia et al. re­
ported that LCZ696 a novel angiotensin receptor neprilysin inhibitor,
improves cardiac function in Dox-induced cardiotoxicty by restoring the
cardiac function, mitochondrial morphology, improves mitochondrial
respiration complex I activity with increased adenosine triphosphate
content. These protective effects were demonstrated by inhibiting
dynamin-related protein 1 (Drp1) mediated mitochondrial dysfunction
in mice with Dox-induced dilated cardiomyopathy [178]. Recently,
liensinine a mitophagy inhibitor attenuated Dox-induced cardiac
dysfunction and apoptosis through the inhibition of Drp1-mediated
maladaptive mitochondrial fission. In addition liensinine suppresses
mitochondrial fragmentation, mitophagy, oxidative stress, cytochrome
C leakage, cardiomyocyte apoptosis and improved mitochondrial func­
tion and cardiomyocyte contractile function in Dox-induced cardiac
injury [179].
Furthermore, Xu et al. demonstrated the protective effect of luteolin
in Dox-induced cardiotoxicity by augmenting autophagy in mitochon­
dria. Luteolin is a phytochemical derived from vegetables and fruits
having anti-oxidative, anti-tumorigenic, and anti-inflammatory prop­
erties. Luteolin improves the Dox-induced alterations in defective
mitochondrial autophagy, mitochondrial dysfunction and car­
diomyocyte injury and apoptosis via stimulating autophagosome for­
mation and improving lysosomal generation through a Drp1/mTOR/
TFEB-dependent mechanism [180]. In addition, melatonin also pro­
tects the cardiomyocytes injury after Dox administration by free radical
scavenger activity, modulates the autophagy, mitophagy, mitochondrial
bioenergetics, mitochondrial fission and fusion process and apoptosis
[181]. Melatonin is a natural hormone produced by the pineal gland.
Recently, Song et al. supporting this evidence, demonstrated that
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Biomedicine & Pharmacotherapy 139 (2021) 111708
melatonin also mitigates Dox-induced cardiotoxicity via reduction of
AMPKα2-dependent mitochondrial damage thus modulating the car­
diomyocytes apotosis [182]. In addition, Lu et al. reported that chrys­
ophanol protects the cardiomyocytes from Dox-induced injury through
the inhibition of cardiac apoptosis, mitochondrial damage and cellular
PARylation levels [183].
Further, Echinochrome A is a naphthoquinoid pigment derived from
sea urchins having antioxidant, antimicrobial, anti-inflammatory and
chelating abilities. Notably, co-treatment of Echinochrome A with Dox
improves the mitochondrial membrane potential, reduces ROS level,
preserve the function of mitochondrial oxidative phosphorylation and
adenosine triphosphate level [184]. In addition, Ghrelin also demon­
strated protective action against Dox-induced cardiotoxicity through the
modulation of TNF-alpha/NF-kappaB pathways and increased the
mitochondrial anti-apoptosis-related gene protein expression [185].
Overall this indicating that the mitochondria damage is an essential
pathological alteration in Dox-induced cardiotoxicity. Besides these
strategies, the administration of fresh mitochondria can be tried and
could be a novel treatment strategy for mitigating cardiac injury.
However, there are no studies were reported with Dox-induced car­
diotoxicity. Interestingly, a study by Shi et al. administered mitochon­
dria through the intravenous route to treat Parkinson’s disease in mice.
Notably, the administered exogenous mitochondria is well distributed in
several tissues such as brain, liver, kidney, muscle, and the heart, indi­
cating beneficial effects in multi-systemically mitochondrial diseases
[186].
5.19. Other recent strategies to prevent Dox-induced cardiotoxicty
In the present section, we have compiled the agents recently
explored to manage Dox-induced cardiotoxicity. Herein, Sadek et al.
demonstrated the protective effect of proanthocyanidin in Dox-induced
cardiotoxicity in rats by improving the anti-oxidant levels and inflam­
mation. Proanthocyanidin downregulates the expression of α-SMA, TGF-
β1 and upregulates the CDK4 and Rb protein expression with reduced
expression of the NF-kB signaling pathway [187]. Further, Nie et al.
reported that the hydrogen sulfide treatment decreases the endoplasmic
reticulum stress, autophagy and upregulates the PI3K/AKT/mTOR
protein expression resulting in reduced myocardial fibrosis in
Dox-treated rats [188]. Tranilast (anti-allergy drug) improves
Dox-induced alterations in myocardial hypertrophy and cardiac
dysfunction by suppressing chymase expression, reducing the angio­
tensin II levels, and modulating the antioxidant level, thus preventing
apoptosis and fibrosis of cardiomyocytes [189]. Moreover, Alzahrani
et al. demonstrated the protective effect of Kirenol (diterpenoid) a
phytochemical extracted from Herba Siegesbeckiae having
anti-inflammatory and anti-rheumatic properties. Kirenol treatment
decreases the cardiac oxidative stress, cardiac remodeling, modulates
the apoptosis process, activates the IGF-IR-dependent p-PI3K/p-AKT and
Nrf2 signaling, thereby preventing the Dox-induced cardiac damage
[190]. Notably, a medicinal plant Malva verticillata (Malvaceae) pos­
sesses anti-inflammatory and antioxidant activities. Its methanolic
extract from leaf exhibited beneficial effects in Dox-induced cardiac
injury by improving the levels of cardiac-specific biochemical parame­
ters [191]. Likewise, alk-A (N-p-coumaroyl-4-aminobutan-1-ol) and
alk-B (hippophamide) pretreatment also reduce oxidative stress,
decrease the expression of cleaved-caspase-3, activates Jun N-terminal
kinases and improves the mitochondrial function in Dox-induced car­
diotoxicity [192].
Moreover, Ahmed et al. presented the beneficial effects of methyl
gallate in Dox-induced alterations in oxidative stress, cardiac injury,
ECG changes and modulates the levels of cardiac-related biochemical
parameters in rats [193]. Further, a bioactive peptide Xinmailong
extracted from American cockroaches decreases Dox-induced oxidative
stress, increased antioxidant activity, upregulates HO-1 expression,
restored lysosomal function and improved autophagy flux block in
P.S. Rawat et al.
Dox-treated H9c2 cells [194]. Another peptide Szeto-Schiller (SS)31 is
known to have antioxidant activity and selectively target the inner
mitochondrial membrane to decrease the mitochondrial reactive oxygen
species generation. SS31 demonstrated the cardioprotective effect via
stabilization of mitochondrial membrane potential, reduces myocardial
apoptosis and fibrosis in Dox-treated groups [195]. Furthermore, oil
from seeds exerted cardioprotective effects against Dox-induced car­
diotoxicity in rats by increasing the glutathione, reducing the MDA, and
restoring the various cardia-related biochemical parameters (CK,
CK-MB, LDH, AST) and ECG changes with improvement in histopatho­
logical alteration [196]. Focusing oxidative stress, Hashish et al. re­
ported that pretreatment of quercetin decreases the oxidative stress,
MDA level and serum cardiac troponin I in Dox-treated rats. It also re­
stores the ECG changes, histopathological alteration with reduced
apoptotic index in the Dox-treated group [197].
Further, Dox treatment increases cardiac xanthine oxidase activity
leading to increased hydrogen peroxide production, upregulation of
apoptotic and ferroptosis pathways, ultimately leading to impaired left
ventricular function. Xanthine oxidases inhibition by drugs such as
febuxostat/topiroxostat restores all these alteration in Dox-treated mice
thus maintaining cardiac energy metabolism [198]. Wang et al. used
calycosin-triblock copolymer nanomicelles to treat Dox-induced car­
diotoxicity in H9C2 cells. Interestingly, treatment with nanomicelles
reduces the ROS and RNS generation, downregulates the expression of
Bax and p53 and upregulates the Bcl-2 and ERp57 in Dox treated car­
diomyocytes [199].
Chatterjee et al. explored the role of telomerase overexpression in
cardiotoxic effects of Dox. Telomerase canonically preserves telomeres
during cell division however is silenced in adult hearts. Cardiomyocytes
are non-dividing cells where telomerase convenes pro-survival traits and
probably exhibits in the ROS detoxification. In this study, the authors
used adeno-associated virus-mediated gene therapy for the long-term
expression of telomerase against dox-induced cardiotoxicity. The over­
expressed telomerase presented beneficial effects through the inhibition
of apoptosis, which improves cardiac function and mitochondrial
morphology [200]. Further, Compound DanShen Dripping Pill (CDDP)
also explored and demonstrated the protective effects in
Dox/Iso-induced (isoproterenol) induces cardiac damage. CDDP restores
the left ventricular ejection fraction, fractional shortening, increases
antioxidant enzymes and inhibits fibrosis, inflammation and cell
apoptosis [201]. Karabulut et al. demonstrated the protective effect of
thymoquinone in Dox-induced cardiotoxicity in rats. This beneficial
effect is exhibited through the maintenance of antioxidants level,
downregulation of HSP-70, HSP-90, GRP78 and caspase-3 levels that
was upregulated in cardiac after Dox treatment [202]. Altogether, it is
indicating that management of Dox-induced cardiotoxicity by phyto­
chemicals is an emerging approach.
6. Conclusion
The anthracycline anticancer drug doxorubicin (DOX) is widely
prescribed to treat tumors such as breast, ovary, leukemia, lymphoma
and other malignant tumors in both adults and children. However, the
Dox administration leads to cardiac toxicity, resulting in increased
mortality risks and thus limiting its wide clinical applications among
cancer patients. Multiple mechanisms are involved in Dox-induced
cardiotoxicity, including oxidative stress, impaired mitochondrial
function, alterations in iron regulatory protein, deregulation of Ca2+
homeostasis with impaired apoptosis. Therefore, targeting these alter­
ations using allicin, metformin, enalapril, adiponectin, visnagin and
schisandrin B and other novel phytochemicals has shown protective
effects against Dox-induced cardiotoxicity and could reduce the mor­
tality rate among cancer patients.
10
Biomedicine & Pharmacotherapy 139 (2021) 111708
Funding sources
Central University of Punjab, RSM grant-2020 (GP-25).
Conflict of interest statement
The authors declare that there are no conflicts of interest.
Acknowledgements
Authors would like to acknowledge the funding from Central Uni­
versity of Punjab, Bathinda, under the Research Seed Money (RSM)
grant-2020 (GP-25). All authors read and approved the final manuscript.
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