Author’s Accepted Manuscript

Molecular Mechanism of Doxorubicin-Induced

Cardiomyopathy – An Update

Kaviyarasi Renu, V.G. Abilash, P.B. Tirupathi

Pichiah, Sankarganesh Arunachalam

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PII: S0014-2999(17)30692-1
DOI: https://doi.org/10.1016/j.ejphar.2017.10.043
Reference: EJP71481
To appear in: European Journal of Pharmacology
Received date: 24 July 2017
Revised date: 11 October 2017
Accepted date: 20 October 2017
Cite this article as: Kaviyarasi Renu, V.G. Abilash, P.B. Tirupathi Pichiah and
Sankarganesh Arunachalam, Molecular Mechanism of Doxorubicin-Induced
Cardiomyopathy – An Update, European Journal of Pharmacology,
https://doi.org/10.1016/j.ejphar.2017.10.043
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 Molecular Mechanism of Doxorubicin-Induced Cardiomyopathy – An Update

Kaviyarasi Renu1, Abilash V.G1*, Tirupathi Pichiah P.B2, Sankarganesh Arunachalam3*

1
Department of Biomedical Sciences, School of Biosciences and Technology, VIT University,

Vellore, Tamil Nadu- 632014, India

2
Department of Animal Science, School of Life Sciences, Bharathidasan University,

Tiruchirappalli, Tamil Nadu- 620024, India

3
Department of Biotechnology, Kalasalingam University, Krishnankoil-626126, Tamil Nadu,

India

* Equal contribution for the corresponding author.

Corresponding Author:

1. SankarganeshArunachalam, Ph.D.,

Department of Biotechnology,

Kalasalingam University,

Anand Nagar, Krishnankoil

PIN 626126 Tamilnadu,

INDIA.

Mobile number: 9500861593.

Email: sankarganesh@gmail.com
2. Abilash V.G, Ph.D.,

Assistant Professor (Senior),

School of Bio-Science and Technology,

VIT University,

Vellore, Tamilnadu PIN 632014

India.

Mobile number: 9840364805.

Email: abilash.vg@vit.ac.in
Abstract:

Doxorubicin is utilized for anti-neoplastic treatment for several decades. The utility of

this drug is limited due to its side effects. Generally, doxorubicin toxicity is originated from the

myocardium and then other organs are also ruined. The mechanism of doxorubicin is intercalated

with the DNA and inhibits topoisomerase 2. There are various signalling mechanisms involved

in doxorubicin cardiotoxicity. First and foremost, the doxorubicin-induced cardiotoxicity is due

to oxidative stress. Cardiac mitochondrial damage is supposed after few hours following the

revelation of doxorubicin. This has led important new uses for the mechanism of doxorubicin-

induced cardiotoxicity and novel avenues of investigation to determine better pharmacotherapies

and interventions for the impediment of cardiotoxicity. The idea of this review is to bring up to

date the recent findings of the mechanism of doxorubicin cardiomyopathies such as calcium

dysregulation, endoplasmic reticulum stress, impairment of progenitor cells, activation of

immune, ubiquitous system and some other parameters.

Keywords:

Doxorubicin, Adriamycin, Cardiotoxicity, Signaling Mechanism, Oxidative Stress

1. Introduction

2. Oxidative stress associated doxorubicin-induced cardiomyopathy – molecular

consequences

2.1. Mitochondrial dependent ROS

2.2. Role of Iron regulatory protein in the production of ROS

2.3. Role of NADPH in the production of ROS

2.4. Role of Nitric Oxide in the production of ROS

2.5. Role of nrf2 in oxidative stress

2.6. Role of Lipid peroxidation in oxidative stress

2.7. Elevated oxidative stress and decreased antioxidant

2.8. Role of RAAS and AGE in oxidative stress

3. Involvement of sub-cellular organelles associated oxidative stress doxorubicin-

induced cardiomyopathy – molecular consequences

3.1. Ultra structural changes

3.1.1. Structural changes in sub-cellular organelles of myocardium

3.1.2. Suppression of genes in specific sub cellular organelles

3.2. Mitochondrial bio-energetic disruption in myocardium

3.2.1. Elevated fatty acid uptake and decreased glucose uptake in heart

3.2.2. Role of adenine metabolites in doxorubicin heart

3.2.3. AMPK signaling

3.2.4. Mitochondrial dysfunction

3.3.Endoplasmic reticulum stress and Impairment of calcium homeostasis in sarcoplasmic

reticulum
4. Immune response, oxidative stress, and tissue injury

4.1. NFĸB activation

5. Oxidative stress and cell death

5.1. Apoptosis

5.2. Necrosis

5.3. Autophagy

5.4. Fibrosis

6. Other mechanisms

6.1. Role of progenitor cells during cardiac injury

6.2. Activation of ubiquitin protease system

6.3. Neuregulin signaling

6.4. Role of natriuretic peptides

6.5. Inhibition of nucleic acid and protein synthesis

6.6. Role of β adrenergic receptors and adenylate cyclase

6.6.1. Release of vasoactive amines

6.7. Role of miRNA

7. Conclusion
 1. Introduction

Doxorubicin or Adriamycin® or Doxil® is most commonly used an effective

chemotherapeutic drug for the treatment of a wide range of cancers including both the solid and

hematogenous cancers since 1969 (Damiani et al., 2016). This antibiotic was first discovered

from a mutated strain of Streptomyces peucetius. The clinical utility of this drug is limited for its

side effects especially, cardiotoxicity when it exceeds the cumulative dosage of 400 mg/m2 - 700

mg/m2 for adults and 300mg/m2 in the cases of children. Upon crossing the mentioned

thresholds, the chances of developing cardiotoxicity increases, with 400 mg/m2 there is a 5%

incidence of cardiotoxicity, with 550mg/m2 26% risk, and with 700 mg/m2, there is a risk as high

as 48% (Li and Hill, 2014). Doxorubicin is biphasic in nature depends on the concentration of it

(Ondrias et al., 1990). Determination of acute toxicity within two or 3 days of drug

administration and chronic cardiotoxicity found in several weeks or even several months after

drug administration. The cardiotoxicity could be an arrhythmia, cardiomyopathy, left ventricular

dysfunction and congestive heart failure (Mitry and Edwards, 2016). Doxorubicin acquaintances

robustly with cellular nuclei and intercalate with deoxyribonucleic acid (DNA) bases to mediate

doxorubicin-DNA complexes, ensuing in cell demise (Cheung et al., 2015; Trouet and Deprez-

De Campeneere, 1979). Doxorubicin - DNA intercalation is connected with double-strand DNA

breakage and mitotic catastrophe generation (Eom et al., 2005). Doxorubicin mediates dsDNA

breakage through reticence of Top2β (Topoisomerase 2-beta), and knockout of Top2β gene in

mice displayed a delay in the propensity to doxorubicin-induced cardiomyocyte death (Zhang et

al., 2012). Reactive oxygen species (ROS) apparently plays a key role in doxorubicin-induced

cardiotoxicity. However, the precise mechanism of doxorubicin-induced cardiotoxicity is still

elusive. The other mechanism contributes to doxorubicin cardiotoxicity are iron regulatory
protein, nitric oxide (NO) release, Mitochondrial dysfunction, impaired adenosine triphosphate

(ATP ) level, hampered cardiac progenitor cells, calcium dysregulation, inflammatory mediators,

endothelial dysfunction, activation of ubiquitin protease system, autophagy and cell death.

Though a number of mechanisms have been shown to be involved in cardiotoxicity the exact

mechanism is indistinct. This review is aimed at comprehending the cellular and molecular

mechanisms by which doxorubicin induces the catastrophe in cardiomyocytes leading to heart

failure. Spotlight more on the cellular and molecular mechanism of doxorubicin on the

myocardium with the rationale of innovative copious delineating the essential molecular

mechanisms that encourage cardiotoxicity.

2. Oxidative stress associated doxorubicin-induced cardiomyopathy – molecular

consequences

Oxidative stress is one of the major effects of doxorubicin-induced cardiotoxicity. Oxidative

stress is the imbalance between the production of reactive oxygen species, reactive nitrogen

species (RNS) and intrinsic antioxidant mechanisms. The reactive oxygen species, reactive

nitrogen species produced within the cardiomyocytes are not effectively removed/neutralized by

the inherent antioxidant mechanisms of the cells (Tham et al., 2015). When the cumulative

dosage of doxorubicin exceeds 500mg/m2 body surface area it leads to the increased oxidative

stress (C Pereira et al., 2011). This oxidative stress is triggered through any of the following

mechanisms.

2.1. Mitochondrial dependent reactive oxygen species

Mitochondrial alterations in cardiomyocytes are one of the subcellular organelle

modifications during doxorubicin-induced cardiotoxicity. 90% of ATP utilized by the

cardiomyocytes is produced by the mitochondria. During doxorubicin treatment, there is an

elevated reactive oxygen species production through the reduction of the redox cycling in

complex I of electron transport chain (ETC) (Alexieva et al., 2014; Davies and Doroshow, 1986;

Davies et al., 1983; Doroshow and Davies, 1986). Any structural modification in the

ultrastructure of mitochondria results in disruption of ATP synthesis. Cardiomyocytes are

equipped with 35 - 40% higher mitochondrial number when compared to other tissues. (Goffart

et al., 2004).

Cardiolipin, an important component of inner mitochondrial membrane plays a crucial role in

the development of doxorubicin-mediated pathologic conditions. Since cardiolipin has anionic

charge and doxorubicin has a cationic charge they have a mutual affinity. Therefore,

doxorubicin and cardiolipin make an irreversible complex. (Parker et al., 2001) Subsequently,

doxorubicin is held within the mitochondria. When the intra-mitochondrial concentration of

doxorubicin exceeds 50 - 100 μM there is an increase in the concentration of reactive oxygen

species(Sarvazyan, 1996). The increased reactive oxygen species production contributes to

oxidation of protein, fat and signalling molecules (Kuznetsov et al., 2011). Cardiolipin is

important for the activation of enzymes present in electron transport chain such as cytochrome C

oxidase, NADH cytochrome C oxidoreductase etc. As cardiolipin is already engaged with

doxorubicin it is unavailable for activating above mentioned enzymes which are essential

components complex II and complex IV of electron transport chain. In addition, oxidative

phosphorylation is also affected which altogether result in cardiotoxicity. (Goormaghtigh et al.,

1980).

The upregulation of manganese superoxide dismutase (MnSOD) prevents the formation of

free radicals from mitochondria (Pani et al., 2000). MnSOD is situated in a mitochondrial matrix
which acts as a superoxide scavenger in the mitochondria (Fridovich, 1995). Knock out of

MnSOD would decrease the survival and mitochondrial function and its integrity which leads to

cell death further myocardial dysfunction (Li et al., 1995). Doxorubicin-induced apoptosis is

through elevated superoxide dismutase (SOD) and haem oxygenase and preserving

mitochondrial membrane potential would be protected by calceolarioside (Kim et al., 2006a).

Doxorubicin thereby suppresses the mitochondrial metabolism and production of the end product

which leads to the apoptosis. This process can be retrieved by allowing the mice to inhale carbon

monoxide which is responsible for the coding of the gene responsible for the mitochondrial

metabolism and also up-regulating gene which is responsible for the heme oxygenase (Piantadosi

et al., 2008; Suliman et al., 2007). The result of increased reactive oxygen speciesproduction is

pathological alterations in the lipid, protein, nucleic acids and signalling molecules (Berthiaume

and Wallace, 2007a).

2.2. Role of Iron regulatory protein in the production of reactive oxygen species

The non-enzymatic reaction such as Fe3+ (ferric iron) reacts with the ketone and hydroxy

group of doxorubicin forms doxorubicin-Fe2+ free radical complexes (Olson and Mushlin,

1990). These complexes interact with the negatively charged cell membranes and cause lipid

peroxidation. Lipid peroxidation occurs when there is a high production of free radicals near to

the cell membranes which contain more phospholipid (Chichuk et al., 1998; Markin et al., 1996).

Doxorubicin increases the intracellular iron pool and normally, iron represents less than 5% of

total cellular iron (Xu et al., 2005). This transition iron metal is involved in the generation of free

radicals. To control the process of free radical generation, the iron bound to storage protein

and/or transport protein (Dorr, 1996). The doxorubicin-induced apoptosis in cardiac is due to the

changes in the iron homeostasis and accumulates iron in mitochondria. Homeostatic changes are
regulated by transferrin receptor (TfR) and this homeostatic changes induced by the release of

iron from ferritin (Shi et al., 2011).

ABC protein-B48 (mABC1) is a protein involved in mitochondrial iron homeostasis by

exporting iron from mitochondria by maturing cytosolic Fe/S protein. Intraperitoneal

administration of doxorubicin at different dosage such as 30mg/kg in 3 injections or

administration of 24mg/kg in 5 injections for 5 days would down-regulates the ABCB8 protein

thereby affecting the export of iron from mitochondria. Administration of iron chelators such as

DFO-50mg/kg and DXZ – 60mg/kg before 2hrs of administration of DOX would protect from

iron accumulation in mitochondria and reverses cardiac damage. Mitoferrin-2 (Mfrn-2) is a

transport protein involved in the import of iron into the mitochondria. There is no change in

Mfrn-2 expression levels and therefore uptake of iron into the mitochondria remains unaltered.

With no change in import, iron export is affected leading to a disturbed state of iron homeostasis

in mitochondria (Ichikawa et al., 2014). There are also additional mechanisms which result in

altered iron homeostasis. Doxorubicin treatment in rat embryonic H9c2 would inactivate both

iron regulatory protein 1(IRP1) and IRP2 in cardiomyocytes permanently. Doxorubicin effects

the post-translational modification of IRP1. 4Fe/S cluster of IRP1 is affected and therefore IRP1

loses its ability to bind to iron-responsive element (IRE) thereby resulting in distortion of iron

homeostasis in mitochondria. Doxorubicin impedes ribonucleic acid (RNA) binding activity of

IRPs via post-transcriptional modification and communication with the 4Fe/S cluster in IRP1

(Kwok and Richardson, 2002; Minotti et al., 2001). Doxorubicin increases the iron incorporate

into the cells and decreasing the release of iron from sub-cellular organelles by altering the

protein trafficking inside the cells. Doxorubicin also affects the import of iron into the cells by
suppressing IRPs. Top-2β is also acting as a mediator in the doxorubicin cardiotoxicity

(Ichikawa et al., 2014).

Individuals having a condition of hemochromatosis (HH), a condition caused by a mutation

in human hemochromatosis protein (HFE) iron regulatory gene show heightened susceptibility to

doxorubicin-induced cardiotoxicity. (Miranda et al., 2003). Increased reactive oxygen species

ruptures the lysosomes rich in iron and also degrades ferritin in lysosomes which further leads to

the cardiac toxicity in hemochromatosis with increased iron (Terman et al., 2008). Even though

reactive oxygen speciesproduction acts as a major mechanism for doxorubicin inducing

cardiotoxicity, number: acts as a primary mechanism for doxorubicin-induced toxicity. Use of

many iron chelators like dexrazoxane (DXZ), deferoxamine (DFO), deferiprone, deferasirox

failed to export the iron from mitochondria during doxorubicin cardiotoxicity. So, the novel iron

chelator is needed to protect iron accumulation in mitochondria during doxorubicin

cardiotoxicity (Ichikawa et al., 2014).

2.3. Role of NADPH in the production of reactive oxygen species

Doxorubicin-induced toxicity is associated with free radical production ultimately leading to

the activation of reactive oxygen species. These free radicals are produced by the doxorubicin

redox cycle which is catalyzed by enzymes such as nicotinamide adenosine dinucleotide

phosphate (NADPH), mitochondrial NADH dehydrogenase (Bahadır et al., 2014). Angiotensin

II (Ang II) activates the level of NADPH oxidase and mediates reactive oxygen species

production during doxorubicin treatment at the 1μM concentration (Gilleron et al., 2009). The

inhibition of angiotensin-converting enzyme (ACE) protects from doxorubicin cardiotoxicity

(Sacco et al., 2009). Cardiomyoblast treated with doxorubicin shows an increased level of
NADPH oxidase (NOX) and flavin-containing enzymes such as P450 reductase, nitric oxide

synthase leading to increased level of reactive oxygen species and subsequently oxidative stress

(Gilleron et al., 2009).

2.4. Role of Nitric Oxide in the production of reactive oxygen species

Nitric Oxide is a vasodilator and it mediates contraction in heart significantly. Nitric oxide is

found in higher quantities in the diseased heart. Increased level of NO is found during

doxorubicin cardiotoxicity. The increased level of NO is induced by isoforms of NOS such as

endothelial NO synthase (eNOS), inducible NO synthase (iNOS) and neuronal NO synthase

(nNOS). Preferably, cardiac NO synthesis is carried by eNOS and iNOS. There is an increased

level of iNOS along with the NO quantity during doxorubicin treatment in cardiac cells. During

doxorubicin treatment, generation of superoxide anions by the activated NOXs reacts with the

NO leads to the formation of peroxynitrite by forming lipid peroxidation. The formation of

peroxynitrite oxides leads to mitochondrial oxidative stress, apoptosis, and necrosis. The

inhibition of iNOS by its inhibitors and amino guanidine protects form doxorubicin-induced

cardiotoxicity (Bahadır et al., 2014). Doxorubicin treatment for 3h, 15mg/kg would increase the

level of NO which subsequently decreases the level of cyclooxygenase-2 (COX-2) (FOGLI et

al., 2004).

2.5. Role of nrf2 in oxidative stress

Nuclear factor erythroid 2-related factor 2 (Nrf2), a basic leucine zipper protein, is involved

in regulation of the expression of a number of antioxidant proteins. Nrf2 also plays a crucial role

in doxorubicin-induced cardiomyopathy. Deficiency of Nrf2 aggravates cardiotoxicity and

cardiac function. Agents which can increase the expression of Nrf2 shows a protective effect
against doxorubicin-induced cardiomyopathy. For example, sulforaphane and dioscin have

recently been reported to prevent the development of doxorubicin-induced cardiac failure by

overexpressing Nrf2. Doxorubicin impairs the activity of autophagy which leads to toxicity in

the heart. Nrf2 prevents oxidative stress by inducing autophagy which happens during

doxorubicin treatment also. Nrf2 mediates the balance between oxidative stress and autophagy,

yet the exact mechanism remains elusive (Li et al., 2014).

2.6. Role of Lipid peroxidation in oxidative stress

Lipid peroxidation is the formation of a free radical chain by causing damage to phospholipid

layer of the cell through oxygen derived-free radicals (Chichuk et al., 1998; Markin et al., 1996).

Lipid peroxidation alters the membrane permeability and cell function. The formation of lipid-

lipid and lipid-protein moieties (Bruch and Thayer, 1983), the ratio of cholesterol to

phospholipid ratio is increased ( arcıa et al., 1 ). Upon exposure to doxorubicin, there is an

increase in the formation of free radicals which further leads to lipid peroxidation. Lipid

peroxidation occurs by both intracellular- and extracellular-derived free radicals. During high

concentration of doxorubicin, vacuoles are formed. Vacuoles formed are the product of lipid

metabolism and lipid peroxidation (Hiroyuki et al., 1984; Terman and Brunk, 2005). There is an

increased level of malondialdehyde which is the product of lipid peroxidation (Myers et al.,

1983; Singal and Pierce, 1986).

2.7. Elevated oxidative stress and decreased antioxidant

Antioxidant enzymes such as superoxide dismutase, catalase, glutathione peroxidase (GPx),

cytochrome P450 and glutathione transferases are found less in heart compared to the other

organs like kidney, liver. Selenium and zinc normally reduce the free radical production, which
acts as a cofactor for antioxidant enzymes (Koenig et al., 1997). Doxorubicin lowers the level of

catalase and inactivated form of selenium-dependent GSH-peroxide-1 (Doroshow et al., 1980;

Siveski-Iliskovic et al., 1995). The inactivated selenium-dependent GSH-peroxide-1 reduces the

activity of Cu/Zn superoxide dismutase, which results in the decreased level of the antioxidants

(Doroshow et al., 1979; Li et al., 2002; Siveski-Iliskovic et al., 1995).

2.8. Role of RAAS and AGE in oxidative stress

In doxorubicin cardiomyopathy, no clear mechanism for the renin-angiotensin-aldosterone

system (RAAS) activation is available, but a recent report suggests that the RAS antagonist

prevents doxorubicin-induced cardiotoxicity (Akolkar et al., 2015). There is not much of

literature evidence regarding the involvement of hyperglycemia in advanced glycation end

products (AGE) activation, and the subsequent end result of cardiomyopathy. Doxorubicin

causes hyperglycemia and mimics type 2 diabetic condition (Arunachalam et al., 2013). There is

an increase in the AGE metabolites (Moriyama et al., 2010), and activation of the reactive

oxygen species (Shi et al., 2011) and also it causes the cardiomyopathy (Carvalho et al., 2014;

Octavia et al., 2012). Therefore, cardiomyopathy may be the result of increased AGE metabolites

and reactive oxygen specieswhich are attributable to hyperglycemia. The activated reactive

oxygen species level would mediate the sympathetic nervous system (SNS) and RAAS activity.

These mechanisms would elevate sympathetic tone, discharge of angiotensin-II, endothelins

(ET), vasopressin (VP), aldosterone, and dysregulation of NO release. The commencement of

endothelial cell activation leads to left ventricular dysfunction further induces cardiomyopathy

(Jungsuwadee, 2016).
 3. Involvement of sub-cellular organelles associated oxidative stress doxorubicin-

induced cardiomyopathy – molecular consequences.

3.1. Ultrastructural changes

3.1.1. Structural changes in sub-cellular organelles of myocardium

Doxorubicin-induced cardiotoxicity involves changes in the ultrastructure of subcellular

organelles of cardiomyocytes. The changes in ultrastructure of sub-cellular organelles includes

swelling of mitochondria, cytoplasmic vacuolization, myofibrillar loss (Singal et al., 2000a),

increase in the number of lysosomes (Minotti et al., 2004), disruption of cristae in mitochondria

(Ascensão et al., 2005), myocyte disruption, fibrosis (Bristow et al., 1981)(Torti et al.,

1986)(Billingham et al., 1978). Doxorubicin treatment to H9C2 cell lines caused degradation of

both the lamins A and B (Sardão et al., 2009) leading to changes in the morphology of nucleus,

chromatin decondensation, shrinkage of nucleoli, disruption in cytoskeleton, mitochondrial

depolarization, fragmentation of mitochondrial depolarization, fragmentation of mitochondrial

filaments and distortion of mitochondrial network (Grimmond and Beerman, 1982; Iwasaki and

Suzuki, 1991; Jang et al., 2004; Jones et al., 1990; Ueno et al., 2006)(Arola et al., 2000). These

effects ultimately result in a reduction in contractile force and myocardial dysfunction.

3.1.2. Suppression of genes in specific subcellular organelles

Doxorubicin reduces the expression of contractile proteins such as cardiac troponin I,

troponin C, alpha-actin. The specific gene which is responsible for the cell integrity and

contraction of the heart include α-tropomyosin (α-TM) and myosin heavy and light chains are

also down-regulated during doxorubicin treatment. Desmin, an intermediate filament, is another

contractile protein, found near the Z line of the sarcomere (Chen et al., 2008; Gewirtz, 1999).
 Connexin (CX) 40, CX43, CX45 are the gap junction protein and hemichannels. CX43, a

protein found in the heart in relatively higher quantities, is involved in protecting the heart from

insults. CX43 acts as a cardioprotective agent in doxorubicin-induced cardiotoxicity in vitro.

(Pecoraro et al., 2015) During chronic administration of doxorubicin, there is a decreased

expression of CX40 along with endothelial dysfunction (Idris-Khodja et al., 2013). In addition,

doxorubicin down-regulates some other genes including Ca2+ ATPase, ryanodine receptor 2

(RyR). In parallel, down-regulation of other genes includes mitochondrial iron-sulphur proteins,

phospholamban and calsequestrin (Takemura and Fujiwara, 2007).

3.2. Mitochondrial bio-energetic disruption in myocardium

3.2.1. Elevated fatty acid uptake and decreased glucose uptake in the heart.

Heart due to its extreme energy demands is equipped with a high number of mitochondria.

During doxorubicin-induced cardiomyopathy, energy metabolism is severely affected there is a

decreased level of phosphofructokinase (PFK), the key regulatory enzyme in glycolysis

(Jeyaseelan et al., 1997). Doxorubicin treatment reduces glucose uptake was determined using

fluorine-18F-deoxyglucose (positron emission tomography) in cardiomyocytes and relies solely

on fatty acid for survival (Takemuraet al., 2007). This might be possible since Adriamycin

(Doxorubicin-HCl) treatment affects adipocyte physiology and releases fatty acid(Arunachalam

et al., 2012). Since there is no hormonal regulation for fatty acid uptake by the heart (Ajay and

Prabhakaran, 2010; Bayeva et al., 2013) the main source of fatty acid for cardiomyocytes is

circulating fatty acids, the hematogenous chain fatty acid, triacylglycerol (TAG), cholesterol and

transporter for lipids such as very low density lipoprotein (VLDL) and low-density lipoprotein

(LDL) are also increased both in heart disease condition and doxorubicin cardiomyopathy(Hong

et al., 2002; Iliskovic and Singal, 1997) .

 3.2.2. Role of adenine metabolites in doxorubicin heart

Doxorubicin impedes the expression of genes encoding enzymes conscientious for energy

production in cardiomyocytes. The expression levels of certain key enzymes concerned in

electron transport chains such as adenosine diphosphate (ADP)/ATP translocase and Rieske iron-

sulphur protein are found to be abridged. (Jeyaseelan et al., 1997). As a result, there is a

decreased level of ATP, adenosine monophosphate (AMP), ATP/AMP ratio, phosphocreatine

(PCr) and total adenine nucleotide in cardiomyocytes. The reduced level of ATP impairs the

attraction of cardio-protective protein heat shock protein (HSP90) towards erEb2. This cardio-

protective protein is elevated during doxorubicin cardiotoxicity. (Wu et al., 2016)

3.2.3. AMPK signaling

During doxorubicin treatment, there is a suppression of adenosine monophosphate-activated

protein kinase (AMPK)signalling due to bio-energetic failure, genotoxic stress, and oxidative

stress and finally, leads to the increased energetic stress and hypertrophy. The AMPK inhibition

is due to the regulatory cross-talk such as AKT signalling (Gratia et al., 2012). In contrast, there

is an upregulation of AMPK signalling and leads to the activation of caspase 3 and ATP loss

leading to bio-energetic failure (Pointon et al., 2010).

3.2.4. Mitochondrial dysfunction

The dysregulated lipid metabolism could also be attributed to impairment in the

mitochondrial function (Praet and Ruysschaert, 1993). (Boudina et al., 2007; Duncan, 2011;

How et al., 2006; Mazumder et al., 2004). The decreased ATP production is connected with the

augmented mitochondrial uncoupling (Tokarska-Schlattner et al., 2006) and improved

consumption of oxygen. Doxorubicin treatment mediate mitochondrial swelling which further

leads to apoptosis (Papadopoulou et al., 1999; Praet and Ruysschaert, 1993). Doxorubicin

impedes the mitochondrial membrane potential and mitochondrial permeability transition pore

(Wallace, 2003). Doxorubicin alters the respiratory components and also decreases respiratory

state 3 (Muhammed et al., 1983). As discussed above that there is an increased uptake of fatty

acid by the myocardium. Doxorubicin also alters the ATP production (Neri et al., 1991; Pelikan

et al., 1986; Sayed - Ahmed et al., 2000) and also increases the oxidative stress in mitochondria

by enhancing the production of reactive oxygen species(Octavia et al., 2012). The lipid content

such as cardiolipin in mitochondrial is enhanced by the oxidative stress (Minotti et al., 2004;

Tokarska-Schlattner et al., 2005). Doxorubicin-induced perturbations in calcium handling by

mitochondria leads to changes in the Ca2+ homeostasis of the myocardium (Cardoso et al., 2008;

Outomuro et al., 2007; Singal et al., 2000b). It also affects the fatty acid oxidation (FAO) which

is occurring in mitochondria (Berthiaume and Wallace, 2007b). The abnormality of mitochondria

is induced by fatty acid metabolism, calcium signalling, oxidative stress, lipid content leads to

mitochondrial dysfunction and apoptosis during further leads to doxorubicin cardiomyopathy

(Represented in figure 2 and 3).

3.3. Endoplasmic reticulum stress and Impairment of calcium homeostasis in sarcoplasmic

reticulum

Under pathological conditions of the heart, there is myocardial endoplasmic reticulum stress

(ER) which triggers apoptosis of cardiomyocytes and thereby contributes to cardiomyopathy

(Lakshmanan et al., 2013; Li et al., 2007; Xu et al., 2009). Doxorubicin treatment induces ER

stress as marked by the elevation of 78-kDa glucose-regulated protein (GRP78) and C/EBP

homologous protein (CHOP) (Wang et al., 2012). Increased ER stress also triggers the

activation of apoptosis in the heart (Chua et al., 2006).

Doxorubicin cardiotoxicity is associated with the dysregulation of calcium level. The impaired

calcium homeostasis acts as a reason for reactive oxygen speciesgeneration. Doxorubicin

decreases the exchange of sodium-calcium exchanger in sarcolemma (Caroni et al., 1981).

During doxorubicin cardiomyopathy, there is an altered expression of the calcium exchange

regulating genes such as RyR,Sarco/endoplasmic reticulum ATPase (SERCA2a),

phospholamban, and calcium storage gene such as calsequestrin in rabbits (Arai et al., 1998;

Kim et al., 2006b). The change in the expression of calcium regulating genes impairs the both

systolic and diastolic changes. During pathological conditions there is a glucose-dependent

modification through post-translational mechanisms (modification or oxidation of O-linked N

acetyl glucosamine) such as calcium / calmodulin-dependent protein kinase 2 (CAMK II) which

leads to the changes in genes involved in calcium signaling (Erickson et al., 2013; Luo et al.,

2013; Pereira et al., 2013). Similarly, there is a change in CAMKII in doxorubicin

cardiomyopathy (Wallace, 2003). These changes cause an alteration in the calcium homeostasis

(Represented in figure 3).

4. Immune response, oxidative stress, and tissue injury

Doxorubicin treatment induces the immune system to release a variety of cytokines. The

Further it stops the natural killer (NK) cell activity, stimulates the responses of cytotoxic T

lymphocytes (CTL) and decreases the differentiation of macrophages. Collective changes in an

immune cell affect the cardiac function during doxorubicin treatment. (Ehrke et al., 1984;

Haskill, 1981; Maccubbin et al., 1992).

Toll-like receptors (TLR) plays an important role in pathologic changes induced by

doxorubicin. These TLRs produce an immediate response to pathogenic and non-pathogenic

ligand produced by damaged tissue. Toll-like receptors 2 and Toll-like receptors 4 are mainly
involved in cardiac pathogenesis. Cardiomyopathy induced by the doxorubicin has an increased

oxidative stress associated with increased Toll-like receptors 2. These toll-like receptors 2

induces the nuclear factor kappa B (NF-kappa B) which ultimately leads to apoptosis (Frantz et

al., 2001; Wang et al., 2002).

The toll-like receptors 4 during doxorubicin treatment causes apoptosis, oxidative stress, and

cardiac inflammation and increased endothelial – 1 causes left ventricular dysfunction. The

knockdown of toll-like receptors 4 decreases the formation of reactive oxygen species in the

myocardium and prevents transcription factor GATA-4 (GATA-4) down-regulation. With the

aforementioned mechanism, the toll-like receptors 4 is involved in extracellular degradation by

causing the viscous cycle. (Riad et al., 2008) The toll-like receptors 4 also increases the level of

tumour necrosis factor (TNF – α) (Tacar et al., 2013).

Toll-like receptors 3 receptor has the ability to stimulate the type1 interferon (IFN) based on

the efficacy of doxorubicin and also produces the IFN – stimulated genes. The dying tumour

cells release the self RNA during doxorubicin treatment. This self RNA further triggers the toll-

like receptors 3 receptor signalling (Sistigu et al., 2014). The recombinant human interleukin

receptor antagonist reduces the doxorubicin-induced cardiomyopathy by reducing the

microstructural changes (Zhu et al., 2010).

During doxorubicin cardiomyopathy, there is elevated level of inflammatory markers such as

IL-1β (Guo et al., 2013a; Sauter et al., 2011), IL-6 (Guo et al., 2013a; Sauter et al., 2011;

Zordoky et al., 2011), TNF-α (Guo et al., 2013a; Riad et al., 2009; Sauter et al., 2011; Zordoky et

al., 2011) and p38 mitogen-activated protein kinase (p38 MAPK)/NF-κB (Guo et al., 2013b).

The inhibitors such as SB203580, an inhibitor of p38 MAPK or siRNAs specific for p38 (sip38)
or pyrrolidine dithiocarbamate (PDTC) reduced the inflammation caused by doxorubicin (Guo et

al., 2013a). Doxorubicin also increases the level of macrophage (Haskill, 1981). Recombinant

IL-1 receptor antagonist protects form doxorubicin-induced cardiotoxicity (Zhu et al., 2010). The

treatment with sodium hydrosulfide (NaHS) ameliorates inflammation caused by the doxorubicin

(Guo et al., 2013b).

4.1. NFĸB activation

Doxorubicin activates NFĸB through oxidative stress, which eventually mediates Bmx

SMAT3 pathway and enhances inflammatory mediators such as IL-1, IL-6, IL-7, TNF receptor

2, vascular endothelial growth factor (VEGF) and matrix metalloproteinases (MMP2). Activation

of SMAT3 pathway induces stress-mediated cardiac remodelling and left ventricular dysfunction

through mTOR. The establishment of cardiac remodeling leads to cardiomyopathy (Chau et al.,

2002; Gottar-Guillier et al., 2011; Holopainen et al., 2015; Mitchell-Jordan et al., 2008; Suwei et

al., 2002; Tavora et al., 2014; Tsai et al., 2000; Zelarayan et al., 2009).

5. Oxidative stress and cell death.

5.1. Apoptosis

Under pathological conditions such as infracted and reperfused myocardium, diabetes, left

ventricular dysfunction and hypertrophy apoptosis occurs along with necrosis. (Freude et al.,

2000; Guerra et al., 1999; Sam et al., 2000; Tea et al., 1999; Zhou et al., 2000). Oxidative stress

results in the release of cytochrome c through voltage-dependent anion channels (VDAC) and

activation of caspase 3 in mitochondria leads to apoptosis in the myocardium. The result of

apoptosis is increased by b-cell lymphoma 2 –(Bcl 2): Bcl-2-like protein 4 (bax) ratio. The

efficiency of mitochondria is increased (P/O ratio) and an increase in the activity of SOD (Childs
et al., 2002). Reactive oxygen speciesgeneration increases apoptotic level by activating the

apoptosis signal-regulating kinase 1(ASK1) signalling (Gilleron et al., 2009). Doxorubicin

mediates apoptosis through the enhancement of p53, decrease the GATA-4 expression level and

p300 degradation (Aries et al., 2004; Kawamura et al., 2004; Kim et al., 2003; Liu et al., 2008;

Liu et al., 2004; Park et al., 2010; Poizat et al., 2005). In doxorubicin cardiotoxicity, there is an

activation of oxidative stress-dependent heat shock factor such as HSF-1 which further activates

HSP25 and subsequently, p53 is equalized leading to the increased production of proapoptotic

proteins (Vedam et al., 2010). During doxorubicin treated condition, there is an accumulation of

ceramide (ANDRIEU-ABADIE et al., 1999; D'Anglemont De Tassigny et al., 2004). Ceramide

accumulation changes the signalling pathway and elevates the apoptosis in cardiomyocytes by

either of the following two ways: mitochondrial L-carnitine and through the channel volume-

sensitive chloride ion. Doxorubicin activates MAPK, p38 and JNK (c-Jun-NH(2)-terminal

kinase) (Xie et al., 2009; Xu et al., 2005) which leads to apoptosis by impairing the Bcl2, Bax,

cleaved caspase-9 and cleaved caspase-3 (Chatterjee et al., 2010; Liu et al., 2004). Apoptosis is

due to the increased oxidative stress, calcium impairment, opening of the mitochondrial pore,

mitochondrial potential and mitochondrial swelling (Tokarska-Schlattner et al., 2006).

Doxorubicin elevates the expression of the death receptors (DR) such as tumour necrosis factor

receptor 1 (TNFR1), fas cell surface death receptor (Fas), DR4, and DR5 in cardiomyocyte. This

elevated expression contributes to activation of caspase cascade shepherd to apoptosis (Zhao and

Zhang, 2017).
 5.2. Necrosis

The fundamental process of necrosis is swelling of cells which lead to the leakage of cells

and also cause the inflammation in the adjutant area. This necrosis occurs during acute

doxorubicin-induced cardiotoxicity (Arola et al., 2000). Although complete mechanism of

necrosis is still unclear the mitochondrial dysfunction, mitochondrial DNA damage and

alteration in ATP level all these are associated with the necrosis (L'Ecuyer et al., 2006; Solem et

al., 1996; Wallace, 2003, 2007; Zhou et al., 2001).

5.3. Autophagy

Doxorubicin suppresses the AMPK and unc-51-like kinase 1 (Ulk1) pathway through which

there is diminished autophagy (Kawaguchi et al., 2012). In contrast, doxorubicin also causes a

down-regulation of GATA4 and Bcl-2. (Kobayashi et al., 2012). The depletion of GATA4 level

activates the S6 kinase beta-1 (S6K1) level and subsequently triggers autophagic genes leading

to induction of autophagy (Rubinstein and Kimchi, 2012). Some of the autophagy-related protein

(Atgs) genes such as Atg5, Atg12, Atg4, Atg4, and Bad are up-regulated during doxorubicin

treatment. (Smuder et al., 2013). The autophagy marker such as LC3B is up-regulated in

doxorubicin cardiomyopathy (Nordgren and Wallace, 2014). The integrin-linked kinase

administration degrades autophagy vacuole through down-regulation of beclin1 protein level

expression (Gu et al., 2012). Therefore we may conclude from comparing these studies in

doxorubicin cardiomyopathy, there is both the stimulation and depression of autophagy. Overall,

doxorubicin interrupts autophagic flux. Either inflation of autophagy or enervated autophagy is

likely to avert or remit the doxorubicin cardiomyopathy (Bartlett et al., 2017). Curcumin protects

from doxorubicin-induced cardiomyopathy by revoking apoptosis and evoking autophagy

through the regulation c-Jun N-terminal kinases (JNK) mediated pathway (Katamura et al.,
2014). UV radiation resistance-associated gene protein (UVRAG) insufficiency increases

doxorubicin-mediated dysregulated autophagic flux in the myocardium. In the termination,

unbalanced fasting reduces impaired autophagy and rescues the pathological changes in

doxorubicin cardiotoxicity (An et al., 2017).

5.4. Fibrosis

During doxorubicin cardiomyopathy, there is an interstitial fibrosis is observed (Carvalho et

al., 2014; Takemura and Fujiwara, 2007). Doxorubicin also increases the perivascular fibrosis.

There is an alteration in both the MMP-1 and MMP-2 in both the animal models and cell culture.

Doxorubicin inhibits the collagen transcription and translation in tumour cells (Goetzenich et al.,

2009; Octavia et al., 2012; Spallarossa et al., 2006; Tokarska-Schlattner et al., 2010).

Doxorubicin treatment significantly elevates expression of transforming growth factor-beta

(TGF-β) and phosphor-SMAD3 along with increased collagen deposition, an increment of

fibroblast to myofibroblast phenotypic transformation and pro-fibrotic signalling pathway. Even

though the increase in myofibroblast is evident the fate of endogenous stem cells remains elusive

(Represented in figure 2 and 4).

6. Other mechanisms

6.1. Role of progenitor cells during cardiac injury

The heart is considered as a terminally differentiated organ, but still, it has some capacity to

repair it after some injury by progenitor cells such as bone marrow progenitor cell(BMPCs) and

cardiac progenitor cells (CPCs). Doxorubicin has been shown to impair the viability of

clonogenic c-kit positive CPCs in vitro. Doxorubicin induces apoptosis of CPCS and prevents

differentiating into myocytes. Doxorubicin also reduces the endothelial cardiac repair and also in
case of cardiac lineage cells it reduced the proliferation and differentiation (Huang et al., 2010;

Yasuda et al., 2010). Doxorubicin decreases the expression of an insulin-like growth factor-1

receptor (IGF-1R ) and tyrosine-protein kinase Met (c-Met) in human cardiac-derived

cardiomyocyte progenitor cells (hCPCs) and pessimistic property continues with time moment

(Piegari et al., 2013). Doxorubicin not only interrupts the ability to differentiate but also

mediates cell death, senescence and apoptosis. Doxorubicin impedes with the growth factor

which regulates the cardiac repair and increases the condition of heart stress. Healthy hCPCs

treated with doxorubicin fails to recuperate the structure and functionality of heart. (De Angelis

et al., 2015). The infantile revelation of doxorubicin with the low dosage also mediates the

senescence and enduringly obstructs the quantity of inhabitant CPC, telomerase activity. The cell

cycle inhibitor p16INK4a and SA- β –gal are elevated. Doxorubicin elevates the expression of the

protein which is involved in DNA damage response such as phosphor-ATMSer1981 kinase,

γH2AX, and phospho-P53 Ser15 further induce apoptosis (Piegari et al., 2013).

Doxorubicin treatment on endothelial progenitor cells (EPC) causes apoptosis at high dosage

and phenotype of early senescence at low dosage. Doxorubicin at sub-cytotoxic dosage elevates

the reactive oxygen specieslevel along with the changes in expression of the protein which

regulates cell cycle, cellular structural design and cytoskeletal adjustment (Maejima et al., 2008;

Spallarossa et al., 2010). The senescence and apoptotic regulators such as MAPKs38 and JNK

signalling pathway are elevated during sub-apoptotic dose exposure of doxorubicin (Wada et al.,

2008). The deregulation of TRF2 and activation of p16INK41 with impairment of telomere

function would supply senescence to EPC further it destroys the regenerative process of EPC (De

Angelis et al., 2016).

 Doxorubicin cardiotoxicity, the trigger of BMCs by granulocyte colony-stimulating factor

(G-CSF) are utilized for assessment of their movement competence in the neighbourhood of the

myocardium and their probable responsibility in hampering of the cardiac dysfunction (Tomita et

al., 2004). The toxic effect of doxorubicin on mesenchymal stem cells (MSC) impede with its

function and extra organ function (Oliveira et al., 2014; Yang et al., 2013).

6.2. Activation of ubiquitin protease system

The therapeutic dosage of doxorubicin activates ubiquitin protease system(UPS) mediated

proteasome both in-vitro and in-vivo in cardiomyocytes (Ranek and Wang, 2009). Briefly,

doxorubicin elevates E3 ligase which increases the structural protein i.e., myofibrillar, β-catenin

which is involved in UPS-mediated degradation and the release of survival factors like apoptosis

repressor with caspase recruitment domain (ARC ) and Bcl2 and p300 transcription factor. This

change leads to sarcomeric structure dysregulation (Shi et al., 2011). Administrating proteasome

inhibitors such as bortezomib which protects from toxicity so that we can administrate both

bortezomib and doxorubicin simultaneously to treat advanced malignant tumours Co-

administration of proteasome inhibitor such as bortezomib along with doxorubicin has been

shown to prevent from the toxicity (LoConte et al., 2008).

6.3. Neuregulin signaling

Neuregulin signalling (NRG) or ErbB signalling plays an important role in cardiac

development and function. The reactive oxygen species-mediated NF-κB activates the miR-146a

expression. The up-regulated miR-146a expression induces acute doxorubicin cardiotoxicity by

the decreasing the ErbB4 signalling in a dose-dependent manner. (Horie et al., 2010).

Doxorubicin increases the level of ErbB2 ligand and activates downstream Akt signalling. There
is an increased level of the ErbB2 stabilizer and chaperone called HSP90 during doxorubicin

treatment (Gabrielson et al., 2007). The recombinant NRG1 signalling protects from cardiac

dysfunction by suppressing the proteasome-mediated degradation like troponin and also reduces

the ErbB2-PI3 –Akt signalling (Bian et al., 2009; Engelman et al., 2007; Liu et al., 2005; Sawyer

et al., 2002).

6.4. Role of natriuretic peptides

Natriuretic peptides are hormones which are produced by the cardiomyocyte. Natriuretic

peptides in plasma are increased during cardiac hypertrophy and left ventricular dysfunction.

During anthracycline cardiotoxicity, there is an increased level of the atrial natriuretic peptide

(ANP) and brain natriuretic peptide (BNP) in plasma. During doxorubicin treatment, there is no

significant increase compared to the control in the level of ANP produced by the atria. But BNP

which is secreted from the ventricles is elevated in the plasma which acts as a cardiac biomarker

during doxorubicin treatment (Troponin and BNP act as predictors) (Chen et al., 1999).

6.5. Inhibition of nucleic acid and protein synthesis

Doxorubicin intercalates with the two base pair of DNA helices and inhibits the synthesis of

DNA replication and RNA replication by affecting the topoisomerase II (Alexieva et al., 2014).

The intracellular damage of DNA stimulates the mitochondrial death pathway which is mediated

by p53 (Campisi, 2005) Doxorubicin also damages mtDNA which results in the mitochondrial

bio-energetic failure by changing the redox cycle (Alexieva et al., 2014). In general, doxorubicin

targets nucleus, so mitochondrial targeting doxorubicin protects from damaging nucleus at

different concentrations and further protects from cardiotoxicity (Jean et al., 2015).
 6.6. Role of β adrenergic receptors and adenylate cyclase

The cell membrane is made up of a polar bi-lipid layer with the larger amount of

phospholipid, it has a hydrophilic face and hydrophobic head, integral protein and glycoproteins

are present on the external surface. The cell membrane is responsible for many reactions include

enzymatic reactions, transport, and signalling. The β adrenergic receptor is the G protein-coupled

receptor. The β adrenergic receptor has an important role in the heart. During Doxorubicin

cardiotoxicity, the β adrenergic receptor is highly affected. Upon doxorubicin treatment, there

are changes in the physical properties of the membrane which also affects the β adrenergic

receptor. The relationship between alterations in the physical properties of the membrane and β

adrenergic receptor remains elusive in doxorubicin-induced cardiotoxicity. The decreased

cardiac contractility with cirrhosis has a decreased membrane fluidity and β adrenergic receptor

signalling. In doxorubicin-mediated cardiotoxicity, there is an increase in the membrane fluidity

and there is an increase in the density of β adrenergic receptor without any changes in affinity of

isoproterenol to β adrenergic receptor it decreases the density of (Xu et al., 2001). The chronic

administration of doxorubicin affects the myocardial β adrenergic system. Doxorubicin

administration in rabbit affects the whole adrenergic pathway and leads to the decrease in

adrenergic cyclase (Calderone et al., 1991).

6.6.1. Release of vasoactive amines

Doxorubicin treatment increases the release of histamine in the peripheral tissues of dog.

Followed by the release of histamine there is an increased level of the catecholamine and

prostaglandins (Bristow et al., 1980). In contrast to that, chronic administration of doxorubicin

decreases the level of epinephrine and nor-epinephrine (Kawada et al., 2000). Chronic
Adriamycin treatment affects the β–adrenergic system. Adriamycin treatment affects entire β–

adrenergic pathway and a decrease in adrenergic cyclase in the heart (Calderone et al., 1991; Xu

et al., 2001).

6.7. Role of miRNA

The dysregulation of miRNAs found in a variety of pathological conditions of the heart. The

main enzyme for the formation of mature miRNAs is dicer. The altered expression of dicer in

cardiomyopathy leads to an abnormal profile of miRNAs (Asrih and Steffens, 2013). Under

doxorubicin treatment, the upregulation of miR-146a, miR-367, miR-215, miR-216b, miR-208b,

miR-34c cause apoptosis in cardiomyocytes further it leads to the formation of doxorubicin

cardiomyopathy (Roca-Alonso et al., 2012; Vacchi-Suzzi et al., 2012).

7. Conclusion

Doxorubicin brings about a cure efficiently for a variety of cancer types, but its rigorous side

effects can also be lethal. Mechanisms of doxorubicin-mediated cardiomyopathy are complicated

and multi-factorial. The pathophysiology of doxorubicin cardiomyopathy is predominantly

determined by oxidative stress. This oxidative stress is a disproportion between the oxidant and

antioxidant. There are different mechanisms involved in doxorubicin-induced cardiomyopathy

but the outcome is cell death such as apoptosis, necrosis, fibrosis, and autophagy. Different

signalling pathways (Represented in figure 1 and table 1) are involved in doxorubicin treatment,

the target of signalling pathways with inhibitors may protect from doxorubicin-mediated

cardiomyopathy. Though there are different inhibitors are determined for treatment of

doxorubicin-induced cardiotoxicity. Still, the defence activity of cardiotoxicity is limited.

Doxorubicin mediated cardiotoxicity is primarily due to nuclear and mitochondrial dysfunction.

Modulation of doxorubicin without affecting the nucleus and mitochondria might protect from

cardiomyopathy. Determination of several methods to profitably rearrange these toxicities

through anticipatory or substitute treatments would have an immense improvement on medical

oncology treatment.

Acknowledgments

This study was financially supported by DST- SERB, Government of India, and Project file

number SB/LS/YS-99/2013.

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Figure legend

Figure 1: Different mechanism involved in doxorubicin cardiomyopathy

Figure 2: Involvement of doxorubicin in oxidative stress and its molecular consequences

Figure 3: Pathways involved in mitochondrial dysfunction and endoplasmic reticulum stress

Figure 4: Signaling pathways involved in Doxorubicin induced cell death

Fig. 1

Fig. 2
Fig. 3

Fig. 4

Molecular Genes Concentration Doxorubicin Experimental Effects References

mechanism of doxorubicin exposure model system

time

1. Oxidative

stress

1. 1. 0-0.6mM 0-20min Fresh bovine Elevated (Davies K et

Mitochondrial heart (Gold reactive al., 1986)

dependent Star Meat) oxygen

reactive species

oxygen species production by

reducing

Complex I of

ETC

1.2 Role of ABCB8 30mg/kg 5 days Mice ABCB8 (Ichikawa et

Iron regulatory protein al., 2014)

protein in the protects from

production of mitochondrial

reactive iron

oxygen species accumulation

by proper

exporting

iron into the

mitochondria

Mfrn-2 30mg/kg 5 days Mice No change in (Ichikawa et

import of al., 2014)

iron into the

mitochondria

IRP1 and IRP2 5μM 16h Embryonic rat Impairs (Minotti et

heart H9c2 cell import of al., 2001)

sline iron into the

other cells

1.3. Role of NOXs 1μM - Embryonic rat Activates (Gilleron et

NADPH in the heart H9c2 cell reactive al., 2009)

production of line oxygen

reactive species
oxygen species production by

NOX

1.4. Role of COX-2 15mg/kg 3h Male Rat Inhibition of (FOGLI et al.,

Nitric Oxide in COX-2 2004)

the production would induce

of reactive intracellular

oxygen species NO and

involved in

reactive

oxygen

species

production

1.5. Role of Nrf2 25mg/kg - Male Mice Deficiency of (Li et al.,

nrf2 in Nrf2 would 2014b)

oxidative increase

stress oxidative

stress

2.

Involvement

of sub-cellular

organelles

associated

oxidative

stress

2.1. CX-40 5mg/kg 22 week Male Rat Decreases (Idris-Khodja

Suppression of endothelial et al., 2013)

genes in function

specific
subcellular

organelles

2.2.

Mitochondrial

bio-energetic

disruption in

myocardium

2.2.1. PFK 1μM 24h Neonatal rat Doxorubicin (Jeyaseelan et

Decreased cardiac selectively al., 1997)

glucose uptake myocytes depressed

in heart glycolysis

pathway by

decreasing

PFK

- 2mg/kg 10 weeks Male Rat Decreased Takemura et

glucose al., 2007)

uptake by

heart

2.2.2. - 12mg/kg 4 weeks Male Rat Decreases (Wu et al.,

Disturbed ATP, ADP 2016)

adenine and AMP

metabolites level in heart

2.2.3. AMPK AMPK 15mg/kg - Rat Decreases (Pointon et

signaling energy al., 2010)

metabolism,

ATP loss

2.2.4. - 2μM 2h Male Rat Increased (Tokarska-

Mitochondrial mitochondrial Schlattner et

dysfunction uncoupling al., 2006)

due to

decreased

ATP

2.3. GRP78 and 5μM 6,12,24h Embryonic rat Increased ER (Wang et al.,

Endoplasmic CHOP cardiac H9c2 stress 2012).

reticulum cell line

stress

2.3.1. RyR, SERCA2a, 24mg/kg 8 weeks Male Rabbits Changes in (Arai et al.,

Impaired phospholamban, calcium 1998)

calcium calsequestrin regulating

homeostasis genes leads to

systolic and

diastolic

changes

3. IL-1β, IL-6, 75 mg/m2 - Female Mice Activation of (Sauter et al.,

Inflammatory TNF-α immune 2011)

markers system

4. Cell death

4.1. Apoptosis TNFR1, Fas, 450μM 24h Human Augments the (Zhao et al.,

DR4, and DR5 pluripotent expression of 2017)

stem cell- dead cell

derived receptor

cardiomyocytes would lead to

the condition

called

apoptosis

4.2. UVRAG 24mg/kg 6weeks Mice Dysregulated (An et al.,

Autophagy autophagic 2017)

flux

5. Other

mechanisms

5. Neuregulin ErbB2 7.5mg/kg 3 weeks Female Mice Impairs (Gabrielson et

signaling cardiac al., 2007)

development

and its

function

Table 1: Effect of different concentration of a doxorubicin with exposure time and biological model system