Recently, Hedhli and colleagues tested whether endothelial-derived Neuregulin1s are important for protecting the heart from ischemia-reperfusion injury (Hedhli et al., 2011). They generated mice with tamoxifen-inducible and endothelium-selective Neuregulin1 gene knockout by cross-breeding mice with VE-cadherin promoter driven Cre-ER (Monvoisin et al., 2006) and mice carrying homozygously floxed alleles of the Neuregulin1 gene (Yang et al., 2001). At the baseline, hearts from these knockout mice showed normal wall thickness, left ventricular chamber size, and systolic function. Cardiac morphology, including capillary density was not different from non-transgenic mice. However, after ischemia-reperfusion, the infarct area, the number of TUNEL positive cells, and the number of infiltrating leukocytes were significantly increased in knockout mice compared with controls. The activation of ErbB4 receptors was decreased in ischemia-reperfusion treated knockout mouse hearts. In addition, injection of a recombinant Neuregulin1 (EGF-like domain) reversed the adverse effects observed in the knockout mice. These results suggested that loss of endothelium-derived Neuregulin1 was the cause of worsening ischemia-reperfusion cardiac injury. This study has identified that Neuregulin1 is one of the protective factors derived from the cardiac endothelium.

#### **3.3.2 Transgenic mouse models with mutations of the ErbB receptors**

To understand the physiological roles of the ErbB receptors, transgenic mice with deletion of the ErbB2, ErbB3 or ErbB4 gene were generated (Gassmann et al., 1995; Lee et al., 1995; Erickson et al., 1997; Crone et al., 2002; Ozcelik et al., 2002). Deletion of the ErbB2 or the ErbB4 gene caused a similar cardiac phenotype as that observed in pan-Neuregulin1 knockout mice, which was the underdevelopment of cardiac ventricular trabeculae (Gassmann et al., 1995; Lee et al., 1995; Meyer and Birchmeier, 1995). ErbB2 or ErbB4 knockout mice died around E10.5 due to cardiac defects. Cardiac specific overexpression of the ErbB2 receptor in ErbB2 knockout mice restored normal ventricular trabeculation (Morris et al., 1999). These data suggest that the effects of Neuregulin1 proteins on cardiac development depend on both ErbB2 and ErbB4 receptors. They also suggest that ErbB2 and ErbB4 heterodimers are needed for propagating Neuregulin1 signals in the developing hearts. In mice with the ErbB3 gene knockout, however, the defect of ventricular trabeculation was not observed. Instead, cardiac cushions lacked mesenchyme and cardiac

Neuregulin1-ErbB Signaling in Doxorubicin-Induced Cardiotoxicity 75

Studies in animal models show that Neuregulin1 injections alleviate doxorubicin-induced cardiac dysfunction in mice. Further, studies show that the Neuregulin1-ErbB signaling protects the heart from doxorubicin cardiotoxicity via several mechanisms which include inhibition of doxorubicin-induced apoptosis, loss of mitochondrial integrity, loss of

**4.1 Neuregulin1 protects the heart from doxorubicin-induced cardiac dysfunction:**

that Neuregulin1 is necessary for protecting the heart from doxorubicin.

Studies from our laboratory show that doxorubicin induces a worsened cardiac dysfunction in Neuregulin1 knockout mice (Liu et al., 2005). We used heterozygous mice with Neuregulin1 EGF-like domain deletion (Meyer and Birchmeier, 1995). The RNA expression of Neuregulin1α and β isoforms was decreased in the hearts of knockout mice (unpublished data). These mice were fertile and no abnormalities were found in cardiac function and morphology at the baseline. When mice were injected with doxorubicin (20 mg/kg, i.p. once), however, survival was decreased in doxorubicin-treated knockout mice compared to wild type mice (two week survival: 13 vs. 33 %). Cardiac function as assessed by echocardiography and left ventricular catheterization showed worsened cardiac systolic function in doxorubicin-treated knockout mice. We further showed that the activation of ErbB2 and downstream signaling molecules Akt, mTOR and MAPK in the heart was more depressed in doxorubicin-treated knockout mice vs. wild type mice. These results suggest

Our laboratory and Liu et al. further demonstrated that injections of recombinant human Neuregulin1 proteins improve cardiac function in doxorubicin-treated animals (Liu et al., 2005; Liu et al., 2006; Bian et al., 2009). Comparing the two studies, we used recombinant human glial growth factor 2 (GGF2), and a subacute doxorubicin cardiotoxicity mouse model (doxorubicin, 20 mg/kg, i.p. once). GGF2 was injected subcutanously daily using the dosage of 0.75 mg/kg/day (Bian et al., 2009); while Liu et al. used a recombinant EGF-like domain (β2a isoform) of Neuregulin1 proteins, and a chronic doxorubicin rat model (3.3 mg/kg/week, i.v. for 4 weeks). Recombinant Neuregulin1 was injected i.v. daily for the first 7 days using the dosage of 20µg/kg/day (Liu et al., 2006). Despite the different isoforms of Neuregulin1, the dosage, the route of drug administration and the animal models used, both studies showed that injections of recombinant Neuregulin1 significantly improved survival and cardiac systolic function in doxorubicin-injured mice and rats. These studies demonstrate that Neureuglin1 injections can protect the heart from doxorubicin-

**4.2 Neuregulin1-ErbB signaling inhibits doxorubicin-induced cardiomyocyte**

The anti-apoptotic effects of the Neureuglin1-ErbB signaling pathway activation were assessed in doxorubicin-treated neonatal rat cardiomyocytes and adult rat cardiomyocytes in culture, in Neuregulin1 EGF-like domain knockout mice and in ErbB2 conditional

**4. Neuregulin1-ErbB signaling protects the heart from doxorubicin**

**cardiotoxicity**

**Studies in animal models**

induced heart failure.

**apoptosis**

knockout mice.

myofibrils, and impaired calcium homeostasis.

valves were underdeveloped (Erickson et al., 1997). These findings are consistent with the expression pattern of the ErbB3 receptor in the heart.

The studies in mice with ErbB2 cardiac-specific conditional knockout provided further information on the ErbB2 receptor in the adult heart (Crone et al., 2002; Ozcelik et al., 2002). Two independent groups generated these mice by crossbreeding animals carrying the Crecoding sequence (driven by myosin light chain 2v MLC2v or muscle creatine kinase promoter) and those with the loxP-flanked ErbB2 gene. Loss of the ErbB2 expression was observed perinatally in 50-60% of cardiomyocytes. Cardiac function was initially normal but progressively worsened. At the age of 3 months, these mice developed dilated cardiomyopathy with enlarged ventricular chambers, increase in the heart to body weight ratio and increase in atrial natriuretic factor and skeletal α-actin expression. Electron microscopy showed increased numbers of mitochondria and vacuoles in cardiomyocytes. Apoptosis as measured by TUNEL staining was increased in ErbB2 conditional knockout mice. Aortic stenosis caused more severe cardiac dysfunction in mutant mice. In addition, cardiomyocytes isolated from these mice were more susceptible to doxorubicin. Collectively, these data demonstrate that the ErbB2 receptor is essential for maintaining normal cardiac physiological function and morphology.

Table 1. Cardiac phenotypes in transgenic mouse models with disruption of the Neuregulin1 gene or the ErbB receptor genes

valves were underdeveloped (Erickson et al., 1997). These findings are consistent with the

The studies in mice with ErbB2 cardiac-specific conditional knockout provided further information on the ErbB2 receptor in the adult heart (Crone et al., 2002; Ozcelik et al., 2002). Two independent groups generated these mice by crossbreeding animals carrying the Crecoding sequence (driven by myosin light chain 2v MLC2v or muscle creatine kinase promoter) and those with the loxP-flanked ErbB2 gene. Loss of the ErbB2 expression was observed perinatally in 50-60% of cardiomyocytes. Cardiac function was initially normal but progressively worsened. At the age of 3 months, these mice developed dilated cardiomyopathy with enlarged ventricular chambers, increase in the heart to body weight ratio and increase in atrial natriuretic factor and skeletal α-actin expression. Electron microscopy showed increased numbers of mitochondria and vacuoles in cardiomyocytes. Apoptosis as measured by TUNEL staining was increased in ErbB2 conditional knockout mice. Aortic stenosis caused more severe cardiac dysfunction in mutant mice. In addition, cardiomyocytes isolated from these mice were more susceptible to doxorubicin. Collectively, these data demonstrate that the ErbB2 receptor is essential for maintaining normal cardiac

Mouse models Cardiac phenotype References

Homozygous die around E10.5 Absence of ventricular trabeculae; defect of endocardial cushion.   Heart failure

Normal cardiac function at baseline. Increased infarct area and apoptosis after ischemia-reperfusion

E10.5. Defect of ventricular

Survived to adulthood. Mice progressively developed dilated cardiomyopathy at the age of 3-6 months.

E10.5. Defect of ventricular

E13.5. Defect of cardiac cushions and valves.

trabeculae

trabeculae

(Meyer and Birchmeier, 1995)

(Kramer et al., 1996b)

(Liu et al., 1998a)

(Hedhli et al., 2011)

(Lee et al., 1995)

(Crone et al., 2002; Ozcelik et al., 2002)

(Gassmann et al.,

(Erickson et al.,

1995)

1997)

failure

expression pattern of the ErbB3 receptor in the heart.

physiological function and morphology.

 Cardiac phenotypes in transgenic mouse models with disruption of the

Cardiomyocyte-specific conditional

Neuregulin1 knockout (NRG1

KO)

#### **4. Neuregulin1-ErbB signaling protects the heart from doxorubicin cardiotoxicity**

Studies in animal models show that Neuregulin1 injections alleviate doxorubicin-induced cardiac dysfunction in mice. Further, studies show that the Neuregulin1-ErbB signaling protects the heart from doxorubicin cardiotoxicity via several mechanisms which include inhibition of doxorubicin-induced apoptosis, loss of mitochondrial integrity, loss of myofibrils, and impaired calcium homeostasis.

#### **4.1 Neuregulin1 protects the heart from doxorubicin-induced cardiac dysfunction: Studies in animal models**

Studies from our laboratory show that doxorubicin induces a worsened cardiac dysfunction in Neuregulin1 knockout mice (Liu et al., 2005). We used heterozygous mice with Neuregulin1 EGF-like domain deletion (Meyer and Birchmeier, 1995). The RNA expression of Neuregulin1α and β isoforms was decreased in the hearts of knockout mice (unpublished data). These mice were fertile and no abnormalities were found in cardiac function and morphology at the baseline. When mice were injected with doxorubicin (20 mg/kg, i.p. once), however, survival was decreased in doxorubicin-treated knockout mice compared to wild type mice (two week survival: 13 vs. 33 %). Cardiac function as assessed by echocardiography and left ventricular catheterization showed worsened cardiac systolic function in doxorubicin-treated knockout mice. We further showed that the activation of ErbB2 and downstream signaling molecules Akt, mTOR and MAPK in the heart was more depressed in doxorubicin-treated knockout mice vs. wild type mice. These results suggest that Neuregulin1 is necessary for protecting the heart from doxorubicin.

Our laboratory and Liu et al. further demonstrated that injections of recombinant human Neuregulin1 proteins improve cardiac function in doxorubicin-treated animals (Liu et al., 2005; Liu et al., 2006; Bian et al., 2009). Comparing the two studies, we used recombinant human glial growth factor 2 (GGF2), and a subacute doxorubicin cardiotoxicity mouse model (doxorubicin, 20 mg/kg, i.p. once). GGF2 was injected subcutanously daily using the dosage of 0.75 mg/kg/day (Bian et al., 2009); while Liu et al. used a recombinant EGF-like domain (β2a isoform) of Neuregulin1 proteins, and a chronic doxorubicin rat model (3.3 mg/kg/week, i.v. for 4 weeks). Recombinant Neuregulin1 was injected i.v. daily for the first 7 days using the dosage of 20µg/kg/day (Liu et al., 2006). Despite the different isoforms of Neuregulin1, the dosage, the route of drug administration and the animal models used, both studies showed that injections of recombinant Neuregulin1 significantly improved survival and cardiac systolic function in doxorubicin-injured mice and rats. These studies demonstrate that Neureuglin1 injections can protect the heart from doxorubicininduced heart failure.

#### **4.2 Neuregulin1-ErbB signaling inhibits doxorubicin-induced cardiomyocyte apoptosis**

The anti-apoptotic effects of the Neureuglin1-ErbB signaling pathway activation were assessed in doxorubicin-treated neonatal rat cardiomyocytes and adult rat cardiomyocytes in culture, in Neuregulin1 EGF-like domain knockout mice and in ErbB2 conditional knockout mice.

Neuregulin1-ErbB Signaling in Doxorubicin-Induced Cardiotoxicity 77

myofilament disarray (Sawyer et al., 2002; Pentassuglia et al., 2009). Simultaneous treatments with doxorubicin and an anti-ErbB2 antibody, or a dual inhibitor that targets both ErbB2 and ErbB1, caused an additive effect on myofibril damage. Conversely, recombinant Neuregulin1 reduced this effect of doxorubicin. Neuregulin1 activated both PI3K and MAPK pathways in cardiomyocytes. Inhibition of ErbB2 prevented activations of these pathways by Neuregulin1. In addition, inhibition of MAPK, but not PI3K, induced myofrilament damage in adult rat cardiomyocytes similar to that observed in ErbB2 antibody or inhibitor treated cardiomyocytes (Pentassuglia et al., 2009). These results suggest that Neuregulin1 may prevent myofibril damage by activating ErbB2 and MAPK. In addition to preventing doxorubicin-induced myofibrillar disarray, the Neuregulin1-ErbB signaling also reduces doxorubicin-induced loss of cardiac troponin proteins (Bian et al., 2009). In doxorubicin-treated mouse hearts, cardiac troponin proteins (cTnI, cTnT and cTnC) were down-regulated. These were associated with a moderate increase in serum cTnI, but not cTnT, suggesting mechanisms other than releasing troponin proteins to serum may exist. Concurrent Neuregulin1 injections significantly prevented doxorubicin-induced loss

These phenotypes were again observed in cultured neonatal rat cardiomyocytes. Neuregulin1 co-treatment prevented doxorubicin-induced down-regulation of cTnI and cTnT proteins in cultured cardiomyocytes. These effects of Neuregulin1 were abolished by ErbB2, but not ErbB4, inhibition. These effects were also prevented by PI3K, Akt and mTOR

Further studies showed that doxorubicin reduced RNA expression of cTnI and cTnT in cardiomyocytes, while Neuregulin1 treatment reversed these effects of doxorubicin. The effect of Neuregulin1 on maintaining cTnI and cTnT proteins in doxorubicin-treated cardiomyocytes was reduced by cycloheximide (Bian et al., 2009). These results suggest that Neuregulin1 may maintain cTnI and cTnT levels by increasing transcription and translation of these proteins.

Neuregulin1 may also maintain cardiac troponins by decreasing doxorubicin-induced degradation of these proteins. Doxorubicin-activated caspases directly degraded cTnT. Doxorubicin also increased ubiquitination of cTnI. Neuregulin1 inhibited these effects of

Results from Neuregulin1 treated normal cardiomyocytes provided clues on how it may protect cardiomyocytes from stresses, such as doxorubicin. In cultured adult rat cardiomyocytes, Neuregulin1 induced lamellipodia formation and elongation of cardiomyocytes, which restored the cell-to-cell contact of cultured cardiomyocytes. This was associated with Neuregulin1 activation of focal adhesion kinase (FAK) and formation of a complex which includes ErbB2, FAK, p130CAS and paxillin. FAK is pivotal for formation of the focal adhesion complex, cell spreading and motility. Neuregulin1-induced activation of

In cultured adult rat cardiomyocytes, Neuregulin1 also increased the phosphorylation of GATA4, a transcriptional factor that regulates genes encoding cardiac sarcomeric proteins and important for cardiac development. Inhibition of the ErbB2 receptor or the MAPK pathway prevented phosphorylation of GATA4 by Neuregulin1. Further studies are needed to demonstrate whether these mechanisms are involved in the cardioprotective effects of

of cardiac troponin proteins in the heart.

inhibitors (Bian et al., 2009).

doxorubicin (Bian et al., 2009).

FAK was inhibited by an anti-ErbB2 antibody.

Neuregulin1 in doxorubicin-injured cardiomyocytes or hearts.

In cultured neonatal rat cardiomyocytes, Fukazawa et al showed that daunorubicin (anthracycline drug) significantly increased apoptosis as assessed by three different methods which were TUNEL staining, flow cytometric quantification of subG1 cell fraction and caspase3 activation. Co-incubation of recombinant Neuregulin1 (GGF2) with daunorubicin significantly reduced apoptosis. This effect of Neuregulin1 was associated with the activation of Akt. Adenoviral infection of a dominant negative Akt abolished this effect of Neuregulin1, suggesting Akt is necessary for neuregulin1's anti-apoptotic effects. In addition, activation of Akt by neuregulin1 was abolished by the ErbB4, but not ErbB2, inhibitor. Together, these observations suggest that Neuregulin1 protects cardiomyocytes from daunorubicin-induced apoptosis via activations of ErbB4 and Akt (Fukazawa et al., 2003). Another study by Rohrbach et al. also showed that Neuregulin1 inhibited daunorubicin-induced cytochrome c release, and caspase3 activation. In addition, Neuregulin1 inhibited daunorubicin-induced increase in the ratio of pro-apoptotic protein Bcl-xS to anti-apoptotic protein Bcl-xL (Rohrbach et al., 2005a).

In doxorubicin-treated mice, we observed increases of caspase3, 6, 9 and caspase8 activations in the heart, suggesting both intrinsic and extrinsic apoptosis pathways were activated in these hearts. Concomitant Neureuglin1 injections significantly reduced the activations of these caspases (Bian et al., 2009). However, decreased activation of the Neuregulin-ErbB signaling does not further increase anthracycline drug doxorubicin or daunorubicin–induced apoptosis as measured by TUNEL staining. In mice with heterozygous knockout of the Neuregulin1 EGF-like domain, the number of TUNELpositive cardiomyocytes was increased in doxorubicin-treated mouse hearts; however, there were no significant differences between doxorubicin-treated wild type and knockout mice (Liu et al., 2005). Similarly, in neonatal and adult rat cardiomyocytes, neither the anti-ErbB2 antibody nor the ErbB2 inhibitor further increased doxorubicin or daunorubicin-induced TUNEL staining (Fukazawa et al., 2003; Pentassuglia et al., 2009). These results, however, may be caused by the TUNEL staining method, which is relatively insensitive. In ErbB2 conditional knockout mouse hearts, very low levels of apoptotic cells was detected by TUNEL assay, while a more sensitive ligation-mediated PCR DNA fragmentation assay was able to detect the increase of apoptotic cells in ErbB2 knockout hearts (Crone et al., 2002).

Studies have also demonstrated that ErbB2 inhibition itself can induce mitochondrial dysfunction and increase apoptotic signals in cardiomyocytes as well as in adult hearts (Crone et al., 2002; Grazette et al., 2004). In cultured neonatal and adult rat cardiomyocytes, anti-ErbB2 antibody increased the ratio of Bcl-xS/Bcl-xL, translocation of BAX to mitochondria, cytochrome c release and caspase activation. These changes caused a loss of mitochondrial membrane potential and a decrease of ATP levels. Restoration of Bcl-xL levels prevented mitochondrial dysfunction (Grazette et al., 2004). In ErbB2 conditional knockout mice, apoptosis was increased in the heart. *In vivo* expression of the anti-apoptotic gene BclxL partially reduced chamber dilation and restored contractility in the ErbB2 knockout mice. Cardiomyocytes isolated from the ErbB2 knockout mice were more sensitive to doxorubicininduced cell death (Crone et al., 2002).

#### **4.3 Neuregulin1-ErbB signaling prevents doxorubicin-induced cardiac myofibrillar disarray and loss**

One of the pathological features of doxorubicin-induced cardiotoxicity is myofibril loss and disarray in cardiomyocytes. In adult rat cardiomyocytes, doxorubicin treatment resulted in

In cultured neonatal rat cardiomyocytes, Fukazawa et al showed that daunorubicin (anthracycline drug) significantly increased apoptosis as assessed by three different methods which were TUNEL staining, flow cytometric quantification of subG1 cell fraction and caspase3 activation. Co-incubation of recombinant Neuregulin1 (GGF2) with daunorubicin significantly reduced apoptosis. This effect of Neuregulin1 was associated with the activation of Akt. Adenoviral infection of a dominant negative Akt abolished this effect of Neuregulin1, suggesting Akt is necessary for neuregulin1's anti-apoptotic effects. In addition, activation of Akt by neuregulin1 was abolished by the ErbB4, but not ErbB2, inhibitor. Together, these observations suggest that Neuregulin1 protects cardiomyocytes from daunorubicin-induced apoptosis via activations of ErbB4 and Akt (Fukazawa et al., 2003). Another study by Rohrbach et al. also showed that Neuregulin1 inhibited daunorubicin-induced cytochrome c release, and caspase3 activation. In addition, Neuregulin1 inhibited daunorubicin-induced increase in the ratio of pro-apoptotic protein

In doxorubicin-treated mice, we observed increases of caspase3, 6, 9 and caspase8 activations in the heart, suggesting both intrinsic and extrinsic apoptosis pathways were activated in these hearts. Concomitant Neureuglin1 injections significantly reduced the activations of these caspases (Bian et al., 2009). However, decreased activation of the Neuregulin-ErbB signaling does not further increase anthracycline drug doxorubicin or daunorubicin–induced apoptosis as measured by TUNEL staining. In mice with heterozygous knockout of the Neuregulin1 EGF-like domain, the number of TUNELpositive cardiomyocytes was increased in doxorubicin-treated mouse hearts; however, there were no significant differences between doxorubicin-treated wild type and knockout mice (Liu et al., 2005). Similarly, in neonatal and adult rat cardiomyocytes, neither the anti-ErbB2 antibody nor the ErbB2 inhibitor further increased doxorubicin or daunorubicin-induced TUNEL staining (Fukazawa et al., 2003; Pentassuglia et al., 2009). These results, however, may be caused by the TUNEL staining method, which is relatively insensitive. In ErbB2 conditional knockout mouse hearts, very low levels of apoptotic cells was detected by TUNEL assay, while a more sensitive ligation-mediated PCR DNA fragmentation assay was able to detect the increase of apoptotic cells in ErbB2 knockout hearts (Crone et al., 2002).

Studies have also demonstrated that ErbB2 inhibition itself can induce mitochondrial dysfunction and increase apoptotic signals in cardiomyocytes as well as in adult hearts (Crone et al., 2002; Grazette et al., 2004). In cultured neonatal and adult rat cardiomyocytes, anti-ErbB2 antibody increased the ratio of Bcl-xS/Bcl-xL, translocation of BAX to mitochondria, cytochrome c release and caspase activation. These changes caused a loss of mitochondrial membrane potential and a decrease of ATP levels. Restoration of Bcl-xL levels prevented mitochondrial dysfunction (Grazette et al., 2004). In ErbB2 conditional knockout mice, apoptosis was increased in the heart. *In vivo* expression of the anti-apoptotic gene BclxL partially reduced chamber dilation and restored contractility in the ErbB2 knockout mice. Cardiomyocytes isolated from the ErbB2 knockout mice were more sensitive to doxorubicin-

**4.3 Neuregulin1-ErbB signaling prevents doxorubicin-induced cardiac myofibrillar**

One of the pathological features of doxorubicin-induced cardiotoxicity is myofibril loss and disarray in cardiomyocytes. In adult rat cardiomyocytes, doxorubicin treatment resulted in

Bcl-xS to anti-apoptotic protein Bcl-xL (Rohrbach et al., 2005a).

induced cell death (Crone et al., 2002).

**disarray and loss**

myofilament disarray (Sawyer et al., 2002; Pentassuglia et al., 2009). Simultaneous treatments with doxorubicin and an anti-ErbB2 antibody, or a dual inhibitor that targets both ErbB2 and ErbB1, caused an additive effect on myofibril damage. Conversely, recombinant Neuregulin1 reduced this effect of doxorubicin. Neuregulin1 activated both PI3K and MAPK pathways in cardiomyocytes. Inhibition of ErbB2 prevented activations of these pathways by Neuregulin1. In addition, inhibition of MAPK, but not PI3K, induced myofrilament damage in adult rat cardiomyocytes similar to that observed in ErbB2 antibody or inhibitor treated cardiomyocytes (Pentassuglia et al., 2009). These results suggest that Neuregulin1 may prevent myofibril damage by activating ErbB2 and MAPK.

In addition to preventing doxorubicin-induced myofibrillar disarray, the Neuregulin1-ErbB signaling also reduces doxorubicin-induced loss of cardiac troponin proteins (Bian et al., 2009). In doxorubicin-treated mouse hearts, cardiac troponin proteins (cTnI, cTnT and cTnC) were down-regulated. These were associated with a moderate increase in serum cTnI, but not cTnT, suggesting mechanisms other than releasing troponin proteins to serum may exist. Concurrent Neuregulin1 injections significantly prevented doxorubicin-induced loss of cardiac troponin proteins in the heart.

These phenotypes were again observed in cultured neonatal rat cardiomyocytes. Neuregulin1 co-treatment prevented doxorubicin-induced down-regulation of cTnI and cTnT proteins in cultured cardiomyocytes. These effects of Neuregulin1 were abolished by ErbB2, but not ErbB4, inhibition. These effects were also prevented by PI3K, Akt and mTOR inhibitors (Bian et al., 2009).

Further studies showed that doxorubicin reduced RNA expression of cTnI and cTnT in cardiomyocytes, while Neuregulin1 treatment reversed these effects of doxorubicin. The effect of Neuregulin1 on maintaining cTnI and cTnT proteins in doxorubicin-treated cardiomyocytes was reduced by cycloheximide (Bian et al., 2009). These results suggest that Neuregulin1 may maintain cTnI and cTnT levels by increasing transcription and translation of these proteins.

Neuregulin1 may also maintain cardiac troponins by decreasing doxorubicin-induced degradation of these proteins. Doxorubicin-activated caspases directly degraded cTnT. Doxorubicin also increased ubiquitination of cTnI. Neuregulin1 inhibited these effects of doxorubicin (Bian et al., 2009).

Results from Neuregulin1 treated normal cardiomyocytes provided clues on how it may protect cardiomyocytes from stresses, such as doxorubicin. In cultured adult rat cardiomyocytes, Neuregulin1 induced lamellipodia formation and elongation of cardiomyocytes, which restored the cell-to-cell contact of cultured cardiomyocytes. This was associated with Neuregulin1 activation of focal adhesion kinase (FAK) and formation of a complex which includes ErbB2, FAK, p130CAS and paxillin. FAK is pivotal for formation of the focal adhesion complex, cell spreading and motility. Neuregulin1-induced activation of FAK was inhibited by an anti-ErbB2 antibody.

In cultured adult rat cardiomyocytes, Neuregulin1 also increased the phosphorylation of GATA4, a transcriptional factor that regulates genes encoding cardiac sarcomeric proteins and important for cardiac development. Inhibition of the ErbB2 receptor or the MAPK pathway prevented phosphorylation of GATA4 by Neuregulin1. Further studies are needed to demonstrate whether these mechanisms are involved in the cardioprotective effects of Neuregulin1 in doxorubicin-injured cardiomyocytes or hearts.

Neuregulin1-ErbB Signaling in Doxorubicin-Induced Cardiotoxicity 79

In another study, Pentassuglia et al. showed that ErbB2 receptor inhibition aggravated doxorubicin-induced decrease in cardiomyocyte fractional shortening (Pentassuglia et al.,

Taken together, these studies demonstrated that Neuregulin1-ErbB signaling is pivotal for inhibiting doxorubicin-induced mitochondrial dysfunction, apoptosis, myofibril disarray and loss, as well as disturbance of calcium homeostasis in the heart. They have provided mechanisms of Neuregulin1's protective effects in doxorubicin-injured hearts, as well as

Doxorubicin-induced cardiotoxicity is a severe side effect of, and therefore, a major clinical obstacle to, cancer therapy by using this drug. The discovery of the cardiac protective role of the Neuregulin1-ErbB signaling pathway in doxorubicin-injured hearts has opened a new area for developing methods to prevent or cure doxorubicin-induced heart failure. The Neuregulin-ErbB signaling is an evolutionally conserved signaling pathway which plays critical roles in the development of multiple organ systems, including the heart. In addition to the cardiac protective effects in heart failure, this signaling pathway is one of the most mutated pathways in cancer. Understanding how this pathway protects the heart from doxorubicin injury is a first and necessary step towards using Neuregulin1 and potentially

During the past 10 years, the cardioprotective effects of the Neuregulin-ErbB signaling have been tested in other cardiovascular diseases. Studies have demonstrated that in addition to doxorubicin-induced heart failure, Neuregulin1 also improves cardiac function in ischemiareperfusion, viral infection, and pacing induced heart failure (Liu et al., 2006; Bersell et al., 2009; Hedhli et al., 2011). In addition, Neuregulin1 promotes cell cycle reentry of differentiated adult cardiomyocytes, improves angiogenesis in the heart (Bersell et al., 2009; Hedhli et al., 2011), and promotes embryonic stem cell differentiation into the cardiac lineage (Sun et al., 2011). Clinical trials are ongoing using Neureuglin1 for the treatment of heart failure in patients (Gao et al., 2010). It is conceivable that Neuregulin1 may become a

This work is supported by NASA (Goukassian, NNX11AD22G) and American Heart

Alimandi, M., Romano, A., Curia, M. C., Muraro, R., Fedi, P., Aaronson, S. A., Di Fiore, P. P.

transformation and human mammary carcinomas', *Oncogene* 10(9): 1813-21. Anderson, B. and Sawyer, D. B. (2008) 'Predicting and preventing the cardiotoxicity of

Appelbaum, F., Strauchen, J. A., Graw, R. G., Jr., Savage, D. D., Kent, K. M., Ferrans, V. J.

and Kraus, M. H. (1995) 'Cooperative signaling of ErbB3 and ErbB2 in neoplastic

and Herzig, G. P. (1976) 'Acute lethal carditis caused by high-dose combination chemotherapy. A unique clinical and pathological entity', *Lancet* 1(7950): 58-62.

new drug for heart failure, including doxorubicin-induced cardiotoxicity.

cancer therapy', *Expert Rev Cardiovasc Ther* 6(7): 1023-33.

2009).

**5. Conclusions**

Trastuzumab-induced cardiotoxicity (Figure 2).

other ErbB ligands in cancer survivors.

**6. Acknowledgements**

**7. References**

Association Grant-In-Aid (Yan, 10GRNT4710003).

#### **4.4 Neuregulin1-ErbB signaling maintains calcium homeostasis in doxorubicintreated cardiomyocytes**

Doxorubicin disturbs calcium homeostasis and contractile function of the heart. In the acute phase, doxorubicin increases cardiac systolic and diastolic functions; while in the later stage, it causes heart failure in patients (Brown et al., 1989a).

Timolati et al. established a model in cultured cardiomyocytes which mimics this change (Timolati et al., 2006). When adult rat cardiomyocytes were treated with 1µM doxorubicin for 18 hours, cardiomyocytes calcium amplitude and fractional shortening were significantly increased. Intracellular diastolic calcium and time to 50% relaxation were modestly increased, suggesting increased cardiomyocyte systolic function in these cardiomyocytes. On the other hand, treatment of cardiomyocytes with 10µM doxorubicin for 18 hours caused significant decreases in calcium amplitude and fractional shortening as well as increases in intracellular diastolic calcium and time to 50% relaxation, suggesting systolic and diastolic function were decreased in these cardiomyocytes. In addition, doxorubicin (10µM) induced a down-regulation of SERCA proteins and SR calcium content.

Neuregulin1 treatment 3 hours prior to doxorubicin attenuated doxorubicin-induced alterations of contractility in cardiomyocytes (Timolati et al., 2006). Neuregulin1 decreased calcium amplitude and fractional shortening in 1µM doxorubicin-treated cardiomyocytes while increased these indices in 10µM doxorubicin-treated cells. Neuregulin1 also normalized the diastolic calcium and time to 50% relaxation in both 1 and 10µM doxorubicin-treated cardiomyocytes. In addition, Neuregulin1 increased SERCA proteins in 10µM doxorubicin-treated cardiomyocytes.

Fig. 2. The potential mechanisms of Neuregulin1-ErbB signaling in protecting the heart from doxorubicin-induced cardiotoxicity. The Neuregulin1-ErbB signaling protects cardiomyocytes from doxorubicin-induced apoptosis which include mitochondrial dysfunction, caspase activation and increase in the Bcl-xS/Bcl-xL ratio, myofibril damage which includes myofilament disarray and loss of cardiac troponins, and disturbance of calcium homeostasis which includes the increase of intracellular diastolic calcium and the decrease of SERCA2a.

In another study, Pentassuglia et al. showed that ErbB2 receptor inhibition aggravated doxorubicin-induced decrease in cardiomyocyte fractional shortening (Pentassuglia et al., 2009).

Taken together, these studies demonstrated that Neuregulin1-ErbB signaling is pivotal for inhibiting doxorubicin-induced mitochondrial dysfunction, apoptosis, myofibril disarray and loss, as well as disturbance of calcium homeostasis in the heart. They have provided mechanisms of Neuregulin1's protective effects in doxorubicin-injured hearts, as well as Trastuzumab-induced cardiotoxicity (Figure 2).

#### **5. Conclusions**

78 Cardiotoxicity of Oncologic Treatments

Doxorubicin disturbs calcium homeostasis and contractile function of the heart. In the acute phase, doxorubicin increases cardiac systolic and diastolic functions; while in the later stage,

Timolati et al. established a model in cultured cardiomyocytes which mimics this change (Timolati et al., 2006). When adult rat cardiomyocytes were treated with 1µM doxorubicin for 18 hours, cardiomyocytes calcium amplitude and fractional shortening were significantly increased. Intracellular diastolic calcium and time to 50% relaxation were modestly increased, suggesting increased cardiomyocyte systolic function in these cardiomyocytes. On the other hand, treatment of cardiomyocytes with 10µM doxorubicin for 18 hours caused significant decreases in calcium amplitude and fractional shortening as well as increases in intracellular diastolic calcium and time to 50% relaxation, suggesting systolic and diastolic function were decreased in these cardiomyocytes. In addition, doxorubicin (10µM) induced

Neuregulin1 treatment 3 hours prior to doxorubicin attenuated doxorubicin-induced alterations of contractility in cardiomyocytes (Timolati et al., 2006). Neuregulin1 decreased calcium amplitude and fractional shortening in 1µM doxorubicin-treated cardiomyocytes while increased these indices in 10µM doxorubicin-treated cells. Neuregulin1 also normalized the diastolic calcium and time to 50% relaxation in both 1 and 10µM doxorubicin-treated cardiomyocytes. In addition, Neuregulin1 increased SERCA proteins in

Fig. 2. The potential mechanisms of Neuregulin1-ErbB signaling in protecting the heart from doxorubicin-induced cardiotoxicity. The Neuregulin1-ErbB signaling protects cardiomyocytes from doxorubicin-induced apoptosis which include mitochondrial dysfunction, caspase activation and increase in the Bcl-xS/Bcl-xL ratio, myofibril damage which includes myofilament disarray and loss of cardiac troponins, and disturbance of calcium homeostasis which includes the increase of intracellular diastolic calcium and the

**4.4 Neuregulin1-ErbB signaling maintains calcium homeostasis in doxorubicin-**

**treated cardiomyocytes**

it causes heart failure in patients (Brown et al., 1989a).

a down-regulation of SERCA proteins and SR calcium content.

10µM doxorubicin-treated cardiomyocytes.

decrease of SERCA2a.

Doxorubicin-induced cardiotoxicity is a severe side effect of, and therefore, a major clinical obstacle to, cancer therapy by using this drug. The discovery of the cardiac protective role of the Neuregulin1-ErbB signaling pathway in doxorubicin-injured hearts has opened a new area for developing methods to prevent or cure doxorubicin-induced heart failure. The Neuregulin-ErbB signaling is an evolutionally conserved signaling pathway which plays critical roles in the development of multiple organ systems, including the heart. In addition to the cardiac protective effects in heart failure, this signaling pathway is one of the most mutated pathways in cancer. Understanding how this pathway protects the heart from doxorubicin injury is a first and necessary step towards using Neuregulin1 and potentially other ErbB ligands in cancer survivors.

During the past 10 years, the cardioprotective effects of the Neuregulin-ErbB signaling have been tested in other cardiovascular diseases. Studies have demonstrated that in addition to doxorubicin-induced heart failure, Neuregulin1 also improves cardiac function in ischemiareperfusion, viral infection, and pacing induced heart failure (Liu et al., 2006; Bersell et al., 2009; Hedhli et al., 2011). In addition, Neuregulin1 promotes cell cycle reentry of differentiated adult cardiomyocytes, improves angiogenesis in the heart (Bersell et al., 2009; Hedhli et al., 2011), and promotes embryonic stem cell differentiation into the cardiac lineage (Sun et al., 2011). Clinical trials are ongoing using Neureuglin1 for the treatment of heart failure in patients (Gao et al., 2010). It is conceivable that Neuregulin1 may become a new drug for heart failure, including doxorubicin-induced cardiotoxicity.

#### **6. Acknowledgements**

This work is supported by NASA (Goukassian, NNX11AD22G) and American Heart Association Grant-In-Aid (Yan, 10GRNT4710003).

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**5**

*1Portugal 2Serbia*

**Doxorubicin-Induced Oxidative**

*1Laboratory of Proteomics, Department of Genetics, National Institute of Health Dr Ricardo Jorge, Lisbon,*

> *2Center for Clinical Pharmacology, Military Medical Academy, Belgrade,*

 **Injury of Cardiomyocytes – Do We**

**Have Right Strategies for Prevention?**

Anthracyclines are among the most utilised antitumour drugs ever developed. The discovery of one of the leading compounds, doxorubicin (DOX) in early 1960s was a major advance in the fight against cancer. According to the WHO, it belongs to the group of 17 essential drugs that are used to treat curable cancers or cancers for which the cost-benefit ratio clearly favours drug treatment (Sikora et al., 1999). It is used, often with other antineoplastic, in the treatment of Hodgkin's disease, non-Hodgkin's lymphomas, acute leukaemias, bone and soft-tissue sarcoma, neuroblastoma, Wilm's tumour, and malignant neoplasms of the bladder, breast, lung, ovary, and stomach. The mechanisms of cytotoxicity of DOX in cancer cells is complex including: inhibition of both DNA replication and RNA transcription; free radicals generation, leading to DNA damage or lipid peroxidation; DNA cross-linking; DNA alkylation; direct membrane damage due to lipid oxidation and inhibition of topoisomerase II (Gewirtz, 1999; Minotti et al., 2004). Today, topoisomarase II is generally recognized to be the cellular target of DOX, which act by stabilizing a reaction intermediate in which DNA strands are cut and covalently linked to this enzyme (Simunek et al., 2009). It blocks subsequent DNA resealing. Failure to relax the supercoiled DNA blocks DNA replication and transcription. Furthermore, DNA strand breaks may trigger apoptosis of cancer cells. However, as with all traditional antineoplastic drugs, DOX administration is accompanied by adverse drug reactions arising from the limited selectivity of their anticancer action (Aronson et al., 2006; McEvoy et al., 2010). Particularly common are bone marrow depression, which may be dose-limiting. White cell count reaches a nadir 10 to 15 days after a dose and usually recovers by about 21 days. Gastrointestinal disturbances include moderate or sometimes severe nausea and vomiting; stomatitis and oesophagitis may progress to ulceration. Alopecia occurs in the majority of patients. Occasional hypersensitivity reactions may also occur. However, a cumulative-dose

dependent cardiac toxicity has been a major limitation of DOX use.

**1. Introduction**

Vukosava Milic Torres1 and Viktorija Dragojevic Simic2

### **Doxorubicin-Induced Oxidative Injury of Cardiomyocytes – Do We Have Right Strategies for Prevention?**

Vukosava Milic Torres1 and Viktorija Dragojevic Simic2 *1Laboratory of Proteomics, Department of Genetics, National Institute of Health Dr Ricardo Jorge, Lisbon, 2Center for Clinical Pharmacology, Military Medical Academy, Belgrade, 1Portugal 2Serbia*

#### **1. Introduction**

88 Cardiotoxicity of Oncologic Treatments

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A. (1998) 'Neuregulins promote survival and growth of cardiac myocytes. Persistence of ErbB2 and ErbB4 expression in neonatal and adult ventricular Anthracyclines are among the most utilised antitumour drugs ever developed. The discovery of one of the leading compounds, doxorubicin (DOX) in early 1960s was a major advance in the fight against cancer. According to the WHO, it belongs to the group of 17 essential drugs that are used to treat curable cancers or cancers for which the cost-benefit ratio clearly favours drug treatment (Sikora et al., 1999). It is used, often with other antineoplastic, in the treatment of Hodgkin's disease, non-Hodgkin's lymphomas, acute leukaemias, bone and soft-tissue sarcoma, neuroblastoma, Wilm's tumour, and malignant neoplasms of the bladder, breast, lung, ovary, and stomach. The mechanisms of cytotoxicity of DOX in cancer cells is complex including: inhibition of both DNA replication and RNA transcription; free radicals generation, leading to DNA damage or lipid peroxidation; DNA cross-linking; DNA alkylation; direct membrane damage due to lipid oxidation and inhibition of topoisomerase II (Gewirtz, 1999; Minotti et al., 2004). Today, topoisomarase II is generally recognized to be the cellular target of DOX, which act by stabilizing a reaction intermediate in which DNA strands are cut and covalently linked to this enzyme (Simunek et al., 2009). It blocks subsequent DNA resealing. Failure to relax the supercoiled DNA blocks DNA replication and transcription. Furthermore, DNA strand breaks may trigger apoptosis of cancer cells. However, as with all traditional antineoplastic drugs, DOX administration is accompanied by adverse drug reactions arising from the limited selectivity of their anticancer action (Aronson et al., 2006; McEvoy et al., 2010). Particularly common are bone marrow depression, which may be dose-limiting. White cell count reaches a nadir 10 to 15 days after a dose and usually recovers by about 21 days. Gastrointestinal disturbances include moderate or sometimes severe nausea and vomiting; stomatitis and oesophagitis may progress to ulceration. Alopecia occurs in the majority of patients. Occasional hypersensitivity reactions may also occur. However, a cumulative-dose dependent cardiac toxicity has been a major limitation of DOX use.

Doxorubicin-Induced Oxidative Injury

**2.3 Prognosis and treatment**

of Cardiomyocytes – Do We Have Right Strategies for Prevention? 91

that myocyte damage and ventricular dysfunction progress after the initial myocardial insult and may lead to late-onset cardiac decompensation. Although the DOX dose of 550 mg/m2 was suggested to be relatively safe, CHF was found to begin at a lower cumulative dose in the presence of other risk factors, such as age above 70 years, combination therapy (cyclophosphamide, dacarbazine etc.), previous or concomitant mediastinal radiotherapy, history of pre-existing cardiac disease, or liver disease (Ludke et al., 2009; Singal & Iliskovic, 1998; Sweetman, 2011). Although sensitivity to DOX-induced cardiotoxicity exhibit large interindividual variation, the risk of developing impairment in myocardial function increases with increasing cumulative dose, occurring in 3-5, 5-8, and 6-20% in those receiving cumulative doses of 400, 450, or 500 mg/m2, respectively, in schedules of rapid IV doses given every 3 weeks. The strong association between cumulative DOX dose and cardiotoxicity obviously is more important with increasing time after treatment (Lipshultz & Adams, 2010). Some clinicians suggest that late-onset cardiotoxicity can be clinically manifest in response to stressful situations (e.g. surgery, pregnancy), exercise and viral infection. Late-onset doxorubicin toxicity can be expected to be observed more frequently with the growing number of long-time survivors (survivors of childhood cancers) who have received DOX. Long term follow up shows that overt cardiac failure occurs from 0 to 16% of patients receiving anthracycline therapy. It depends on the cumulative dose, well recognized risk factor for late cardiotoxicity, radiation therapy involving the heart, age at diagnosis, greater length of follow up, higher dose rate and previous exposure to anthracycline (Kremer et al., 2002; McEvoy et al., 2010; Wouters et al., 2005). Therefore, recommendations for screening and management of late effects of therapeutic exposure used during treatment for pediatric malignancies are very important in every day practice.

**2.2 Monitoring and markers of doxorubicin-induced cardiotoxicity**

Clinical manifestations found on physical examination, and/or changes found on electrocardiographic (ECG) monitoring are not specific enough to diagnose DOX-induced cardiotoxicity. However, a persistent reduction in the voltage of the QRS wave is generally indicative of the need to perform further tests. Non-invasive cardiac monitoring, by means of serial echocardiography studies or radionuclide angiography, is useful in predicting the development of cardiomyopathy. However, since it may give normal results until damage is quite advanced, sensitivity may be improved with exercise stress tests (Singal & Iliskovic, 1998; Steinherz et al., 1992). Endomyocardial biopsy is the most sensitive indicator of cardiomyopathy, but it is limited by its invasiveness, need for histologic expertise, and costs. Therefore, there is need for simple methods, like serum and plasma markers to identify patients at risk. There is some preliminary evidence to suggest that concentrations of cardiac troponins and natriuretic peptides could be used as predictive markers of myocardial damage (Ludke et al., 2009; Schimmel et al., 2004). Anyway, according to the Childrens' Cancer Study Group (Steinherz et al., 1992), standard echocardiogram should be performed before the beginning of the treatment and at the third, sixth and 12th month after its completion. Subsequent exam should be repeated every 2 years for the rest of the life.

Historically, DOX-induced cardiomyopathies have a poor prognosis and mortality rate as high as 61% (Allen, 1992; Von Hoff et al., 1979; Wouters et al., 2005). It is refractory to

#### **2. Doxorubicin-induced cardiotoxicity**

Cardiac toxic effects were first documented during early clinical evaluations of DOX, and in the late 1970s, first retrospective clinical studies showed convincingly that the observed cardiac disturbances could be directly attributable to repeated DOX administration. At the same time, these studies established the cumulative DOX dose received as the main risk factor of cardiotoxicity (Green et al., 2001; Lefrak et al., 1973; Von Hoff et al., 1979). Considering that more than 50% of long term-survivors of childhood cancer alone were treated with DOX or another anthracycline, anthracycline-induced cardiotoxicity is a widely prevalent problem that cannot be ignored. Namely, long-term survivors of childhood cancer are 8 times more likely than the general population to die from cardiovascular-related disease and, compared to sibling controls, they are 15 times as likely to suffer from heart failure (HF)(Lipshultz & Adams, 2010; Lipshultz et al., 1991). Despite very large numbers of new anthracycline compounds synthesized and tested, only few ones have been approved for clinical use, and none of them has fulfilled expectations of substantially improved cardiac safety. Therefore, a growing need to develop effective and cardioprotective strategies remains, in order to find a balance between the risks of cardiotoxicity and the benefits of oncologic therapy with DOX.

#### **2.1 Clinical presentation and risk factors of doxorubicin-induced cardiotoxicity**

DOX-induced cardiotoxicity is most often divided into 3 categories: acute changes, earlyonset chronic progressive cardiotoxicity and late-onset chronic progressive cardiotoxicity (Aronson et al., 2006; Schimmel et al., 2004; Wouters et al., 2005) Acute DOX-induced cardiotoxicity occurs during DOX administration or immediately afterwards. It typically involves transient electrocardiographic abnormalities such as non-specific ST-T changes and QT prolongation, vasodilatation and hypotension. Pericarditis-myocarditis syndrome and ventricular dysfunction which manifest 1 to 3 days after the DOX treatment are extremely rare, and were more frequently seen in early trials using very high doses of DOX. Most often, all of the disturbances attenuate after discontinuation of the therapy. Early-onset chronic cardiotoxicity, such as cardiomyopathy which progresses to congestive heart failure (CHF) usually occurs within 1 year after discontinuance of DOX therapy. CHF may occur as total cumulative dosage of DOX approaches or exceeds 550 mg/m2. Chronic cardiotoxicity reflects a progressive injury and loss of cardiac myocytes, with increasing cumulative DOX dose resulting in thinning of ventricular wall and decreased systolic performance. It is characterised by dilated (less often restrictive, mostly in children) cardiomyopathy, with subsequent development of left ventricular contractile dysfunction and congestive heart failure. Histopathological changes are quite unique and consist of distension of the sarcoplasmic reticulum of myocytes, cytoplasmic vacuolization, swelling of mitochondria and myofibrillar disarray and loss (Ferrans et al., 1997). Symptoms of the CHF include tachycardia, tachypnea, dilatation of the heart, exercise intolerance, pulmonary oedema, peripheral oedema and needs for treatment with diuretics and cardiac glycosides. These manifestations may respond to cardiac supportive therapy and may be self limiting, or may be irreversible and unresponsive to therapy and fatal (Aronson et al., 2006; Singal & Iliskovic, 1998; Sweetman, 2011; Wouters et al., 2005). Finally, late-onset chronic progressive DOX-induced cardiotoxicity occurs at least 1 year after the completion of therapy, sometimes even after decades of a prolonged asymptomatic interval. It has been suggested

Cardiac toxic effects were first documented during early clinical evaluations of DOX, and in the late 1970s, first retrospective clinical studies showed convincingly that the observed cardiac disturbances could be directly attributable to repeated DOX administration. At the same time, these studies established the cumulative DOX dose received as the main risk factor of cardiotoxicity (Green et al., 2001; Lefrak et al., 1973; Von Hoff et al., 1979). Considering that more than 50% of long term-survivors of childhood cancer alone were treated with DOX or another anthracycline, anthracycline-induced cardiotoxicity is a widely prevalent problem that cannot be ignored. Namely, long-term survivors of childhood cancer are 8 times more likely than the general population to die from cardiovascular-related disease and, compared to sibling controls, they are 15 times as likely to suffer from heart failure (HF)(Lipshultz & Adams, 2010; Lipshultz et al., 1991). Despite very large numbers of new anthracycline compounds synthesized and tested, only few ones have been approved for clinical use, and none of them has fulfilled expectations of substantially improved cardiac safety. Therefore, a growing need to develop effective and cardioprotective strategies remains, in order to find a balance between the risks of cardiotoxicity and the

**2.1 Clinical presentation and risk factors of doxorubicin-induced cardiotoxicity**

DOX-induced cardiotoxicity is most often divided into 3 categories: acute changes, earlyonset chronic progressive cardiotoxicity and late-onset chronic progressive cardiotoxicity (Aronson et al., 2006; Schimmel et al., 2004; Wouters et al., 2005) Acute DOX-induced cardiotoxicity occurs during DOX administration or immediately afterwards. It typically involves transient electrocardiographic abnormalities such as non-specific ST-T changes and QT prolongation, vasodilatation and hypotension. Pericarditis-myocarditis syndrome and ventricular dysfunction which manifest 1 to 3 days after the DOX treatment are extremely rare, and were more frequently seen in early trials using very high doses of DOX. Most often, all of the disturbances attenuate after discontinuation of the therapy. Early-onset chronic cardiotoxicity, such as cardiomyopathy which progresses to congestive heart failure (CHF) usually occurs within 1 year after discontinuance of DOX therapy. CHF may occur as total cumulative dosage of DOX approaches or exceeds 550 mg/m2. Chronic cardiotoxicity reflects a progressive injury and loss of cardiac myocytes, with increasing cumulative DOX dose resulting in thinning of ventricular wall and decreased systolic performance. It is characterised by dilated (less often restrictive, mostly in children) cardiomyopathy, with subsequent development of left ventricular contractile dysfunction and congestive heart failure. Histopathological changes are quite unique and consist of distension of the sarcoplasmic reticulum of myocytes, cytoplasmic vacuolization, swelling of mitochondria and myofibrillar disarray and loss (Ferrans et al., 1997). Symptoms of the CHF include tachycardia, tachypnea, dilatation of the heart, exercise intolerance, pulmonary oedema, peripheral oedema and needs for treatment with diuretics and cardiac glycosides. These manifestations may respond to cardiac supportive therapy and may be self limiting, or may be irreversible and unresponsive to therapy and fatal (Aronson et al., 2006; Singal & Iliskovic, 1998; Sweetman, 2011; Wouters et al., 2005). Finally, late-onset chronic progressive DOX-induced cardiotoxicity occurs at least 1 year after the completion of therapy, sometimes even after decades of a prolonged asymptomatic interval. It has been suggested

**2. Doxorubicin-induced cardiotoxicity**

benefits of oncologic therapy with DOX.

that myocyte damage and ventricular dysfunction progress after the initial myocardial insult and may lead to late-onset cardiac decompensation. Although the DOX dose of 550 mg/m2 was suggested to be relatively safe, CHF was found to begin at a lower cumulative dose in the presence of other risk factors, such as age above 70 years, combination therapy (cyclophosphamide, dacarbazine etc.), previous or concomitant mediastinal radiotherapy, history of pre-existing cardiac disease, or liver disease (Ludke et al., 2009; Singal & Iliskovic, 1998; Sweetman, 2011). Although sensitivity to DOX-induced cardiotoxicity exhibit large interindividual variation, the risk of developing impairment in myocardial function increases with increasing cumulative dose, occurring in 3-5, 5-8, and 6-20% in those receiving cumulative doses of 400, 450, or 500 mg/m2, respectively, in schedules of rapid IV doses given every 3 weeks. The strong association between cumulative DOX dose and cardiotoxicity obviously is more important with increasing time after treatment (Lipshultz & Adams, 2010). Some clinicians suggest that late-onset cardiotoxicity can be clinically manifest in response to stressful situations (e.g. surgery, pregnancy), exercise and viral infection. Late-onset doxorubicin toxicity can be expected to be observed more frequently with the growing number of long-time survivors (survivors of childhood cancers) who have received DOX. Long term follow up shows that overt cardiac failure occurs from 0 to 16% of patients receiving anthracycline therapy. It depends on the cumulative dose, well recognized risk factor for late cardiotoxicity, radiation therapy involving the heart, age at diagnosis, greater length of follow up, higher dose rate and previous exposure to anthracycline (Kremer et al., 2002; McEvoy et al., 2010; Wouters et al., 2005). Therefore, recommendations for screening and management of late effects of therapeutic exposure used during treatment for pediatric malignancies are very important in every day practice.

#### **2.2 Monitoring and markers of doxorubicin-induced cardiotoxicity**

Clinical manifestations found on physical examination, and/or changes found on electrocardiographic (ECG) monitoring are not specific enough to diagnose DOX-induced cardiotoxicity. However, a persistent reduction in the voltage of the QRS wave is generally indicative of the need to perform further tests. Non-invasive cardiac monitoring, by means of serial echocardiography studies or radionuclide angiography, is useful in predicting the development of cardiomyopathy. However, since it may give normal results until damage is quite advanced, sensitivity may be improved with exercise stress tests (Singal & Iliskovic, 1998; Steinherz et al., 1992). Endomyocardial biopsy is the most sensitive indicator of cardiomyopathy, but it is limited by its invasiveness, need for histologic expertise, and costs. Therefore, there is need for simple methods, like serum and plasma markers to identify patients at risk. There is some preliminary evidence to suggest that concentrations of cardiac troponins and natriuretic peptides could be used as predictive markers of myocardial damage (Ludke et al., 2009; Schimmel et al., 2004). Anyway, according to the Childrens' Cancer Study Group (Steinherz et al., 1992), standard echocardiogram should be performed before the beginning of the treatment and at the third, sixth and 12th month after its completion. Subsequent exam should be repeated every 2 years for the rest of the life.

#### **2.3 Prognosis and treatment**

Historically, DOX-induced cardiomyopathies have a poor prognosis and mortality rate as high as 61% (Allen, 1992; Von Hoff et al., 1979; Wouters et al., 2005). It is refractory to

Doxorubicin-Induced Oxidative Injury

of Cardiomyocytes – Do We Have Right Strategies for Prevention? 93

Fig. 1. A summary of potential mechanisms involved in doxorubicin (Dox)-induced

expression (blue), and impaired cardiac repair by inhibiting bone marrow, cardiac

protein, H-FABP heart fatty acid binding protein, OCTN organic cation/carnitine transporter, ARC apoptosis repressor with caspase recruitment domain, NFAT nuclear factors of activated T-cell, TauT taurine transporter, NRG neuregulin, ErbB epidermal growth factor receptor B, BMPC bone marrow cardiac progenitor cell, CPC cardiac

increases or decreases in function or expression, horizontal arrows indicate positive

regulatory sequence, blocks represent inhibition (Shi et al., 2011).

cardiomyopathy as described in the text. The major mechanisms involve ROS and iron (red). Meanwhile, Dox-induced cell death (necrosis, apoptosis, and autophagy) and activation of innate immunity (brown), gene changes that reduce cardiac-specific gene expression or trigger apoptosis, induction of cardiac premature senescence in cardiomyocytes (yellow), activation of the ubiquitin-proteasome system (UPS) causing the balance of the protein system to shift toward pro-apoptosis, downregulation of prosurvival gene (NRG1 and ErB4)

progenitor cell, and/or endothelial cell function (green) all emerge as potential mechanisms contributing to Dox cardiotoxicity. ROS reactive oxygen species, IRP iron regulatory protein, TLR toll-like receptor, TIR toll-interleukin-1 receptor, CARP cardiac Adriamycin-responsive

progenitor cell, EC endothelial cell, arrows pointing up or down (inside text boxes) indicate

conventional therapy, therefore cardiac transplantation is the only solution associated with improved survival (Aronson et al., 2006; Sweetman, 2011). Just recent study reports that the response of DOX-induced cardiomyopathy to the current standard medical therapy is better, especially in patients treated with β-blockers. Nowadays, in the treatment of CHF, including DOX-induced cardiomyopathy, β-blockers and angiotensin-converting enzyme (ACE) inhibitors, are proven Class I medications according to the American College of Cardiology/American Heart Association and Canadian Cardiovascular Society guidelines (Hunt, 2005; Malcom et al., 2008).

#### **2.4 Mechanisms of doxorubicin induced cardiac Injury**

Two important characteristics of doxorubicin induced cardiomyopathy are that is dose dependent and it is refractory to the commonly used therapeutic procedures (Lefrak et al., 1973; Singal & Iliskovic, 1998). Pathogenesis of cardiac injury caused by doxorubicin is multifactorial and it is in direct relationship with metabolism and antitumor activity of the drug (Figure. 1). Free radical stress, calcium overloading and mitochondrial dysfunction are main triggers in doxorubicin-induced cardiotoxicity. Subsequent gene expression changes and activation of the ubiquitin-proteasome system, cell death, as well as innate immunity activation all contribute to the toxicity (Shi et al., 2011). The level of the doxorubicin-induced oxidative stress is up to 10 times greater in the heart than in the other tissues (liver, kidney, spleen) (Davies & Doroshow, 1986; Doroshow & Davies, 1986; Gaetani et al., 1989; Lenzhofer et al., 1983; Milei et al., 1986; Mukherjee et al., 2003; Siveski-Iliskovic et al., 1995), and therefore oxidative injury of the heart is widely accepted theory presumed as a primary mechanism of the DOX-induced cardiotoxicity.

#### **2.4.1 Role of iron and free radicals**

Although doxorubicin-induced cardiotoxicity is multifactorial, formation of reactive oxygen species (ROS) has a leading role in promoting the oxidative myocardial damage. The high level of oxidative stress generated by anthracyclines is accounted to the molecular characteristics which allows drug to easily undergo redox reaction and subsequently free radical cascade.

Doxorubicin is belonging to the group of anthracycline antibiotics consist of naphthacenequinone nucleus linked through a glycosidic bond to an amino sugar, daunosamine (Figure. 2a). One-electron addition to the quinone moiety in ring C of DOX result in formation of a semiquinone that quickly regenerates its parent quinone by reducing oxygen to reactive oxygen species, like superoxide anion radical (O2 •-) and hydrogen peroxide (H2O2) (Figure. 2b).

This futile cycle is supported by a number of NAD(P)H-oxidoreductases [cytochrome P450 or -b5 reductases, mitochondrial NADH dehydrogenase, xanthine dehydrogenase, endothelial nitric oxide synthase (reductase domain) (Halliwell & Gutteridge, 2007; Minotti et al., 1999; Vasquez-Vivar et al., 1997). The dismutation of the O2.- is catalyzed by superoxide dismutase enzyme or it may occur spontaneously in the acidic environment - (pH<7). Hydrogen peroxide is low toxic molecule, which is eliminated from the body by enzymatic antioxidative defence system (catalase -CAT and glutathione peroxidase - GPx). However, in the presence of the transition metals, especially iron, H2O2 and O2•- can

conventional therapy, therefore cardiac transplantation is the only solution associated with improved survival (Aronson et al., 2006; Sweetman, 2011). Just recent study reports that the response of DOX-induced cardiomyopathy to the current standard medical therapy is better, especially in patients treated with β-blockers. Nowadays, in the treatment of CHF, including DOX-induced cardiomyopathy, β-blockers and angiotensin-converting enzyme (ACE) inhibitors, are proven Class I medications according to the American College of Cardiology/American Heart Association and Canadian Cardiovascular Society guidelines

Two important characteristics of doxorubicin induced cardiomyopathy are that is dose dependent and it is refractory to the commonly used therapeutic procedures (Lefrak et al., 1973; Singal & Iliskovic, 1998). Pathogenesis of cardiac injury caused by doxorubicin is multifactorial and it is in direct relationship with metabolism and antitumor activity of the drug (Figure. 1). Free radical stress, calcium overloading and mitochondrial dysfunction are main triggers in doxorubicin-induced cardiotoxicity. Subsequent gene expression changes and activation of the ubiquitin-proteasome system, cell death, as well as innate immunity activation all contribute to the toxicity (Shi et al., 2011). The level of the doxorubicin-induced oxidative stress is up to 10 times greater in the heart than in the other tissues (liver, kidney, spleen) (Davies & Doroshow, 1986; Doroshow & Davies, 1986; Gaetani et al., 1989; Lenzhofer et al., 1983; Milei et al., 1986; Mukherjee et al., 2003; Siveski-Iliskovic et al., 1995), and therefore oxidative injury of the heart is widely accepted theory presumed as a primary

Although doxorubicin-induced cardiotoxicity is multifactorial, formation of reactive oxygen species (ROS) has a leading role in promoting the oxidative myocardial damage. The high level of oxidative stress generated by anthracyclines is accounted to the molecular characteristics which allows drug to easily undergo redox reaction and subsequently free

Doxorubicin is belonging to the group of anthracycline antibiotics consist of naphthacenequinone nucleus linked through a glycosidic bond to an amino sugar, daunosamine (Figure. 2a). One-electron addition to the quinone moiety in ring C of DOX result in formation of a semiquinone that quickly regenerates its parent quinone by reducing

This futile cycle is supported by a number of NAD(P)H-oxidoreductases [cytochrome P450 or -b5 reductases, mitochondrial NADH dehydrogenase, xanthine dehydrogenase, endothelial nitric oxide synthase (reductase domain) (Halliwell & Gutteridge, 2007; Minotti et al., 1999; Vasquez-Vivar et al., 1997). The dismutation of the O2.- is catalyzed by superoxide dismutase enzyme or it may occur spontaneously in the acidic environment - (pH<7). Hydrogen peroxide is low toxic molecule, which is eliminated from the body by enzymatic antioxidative defence system (catalase -CAT and glutathione peroxidase - GPx). However, in the presence of the transition metals, especially iron, H2O2 and O2•- can

•-) and hydrogen

oxygen to reactive oxygen species, like superoxide anion radical (O2

(Hunt, 2005; Malcom et al., 2008).

**2.4 Mechanisms of doxorubicin induced cardiac Injury**

mechanism of the DOX-induced cardiotoxicity.

**2.4.1 Role of iron and free radicals**

radical cascade.

peroxide (H2O2) (Figure. 2b).

Fig. 1. A summary of potential mechanisms involved in doxorubicin (Dox)-induced cardiomyopathy as described in the text. The major mechanisms involve ROS and iron (red). Meanwhile, Dox-induced cell death (necrosis, apoptosis, and autophagy) and activation of innate immunity (brown), gene changes that reduce cardiac-specific gene expression or trigger apoptosis, induction of cardiac premature senescence in cardiomyocytes (yellow), activation of the ubiquitin-proteasome system (UPS) causing the balance of the protein system to shift toward pro-apoptosis, downregulation of prosurvival gene (NRG1 and ErB4) expression (blue), and impaired cardiac repair by inhibiting bone marrow, cardiac progenitor cell, and/or endothelial cell function (green) all emerge as potential mechanisms contributing to Dox cardiotoxicity. ROS reactive oxygen species, IRP iron regulatory protein, TLR toll-like receptor, TIR toll-interleukin-1 receptor, CARP cardiac Adriamycin-responsive protein, H-FABP heart fatty acid binding protein, OCTN organic cation/carnitine transporter, ARC apoptosis repressor with caspase recruitment domain, NFAT nuclear factors of activated T-cell, TauT taurine transporter, NRG neuregulin, ErbB epidermal growth factor receptor B, BMPC bone marrow cardiac progenitor cell, CPC cardiac progenitor cell, EC endothelial cell, arrows pointing up or down (inside text boxes) indicate increases or decreases in function or expression, horizontal arrows indicate positive regulatory sequence, blocks represent inhibition (Shi et al., 2011).

Doxorubicin-Induced Oxidative Injury

(Minotti, 1993; Minotti et al., 1999).

come along with the formation of O2

is the potent alkylating agent.

of Cardiomyocytes – Do We Have Right Strategies for Prevention? 95

generate highly toxic hydroxyl radical (OH•). This process is occurring during the Haber-Weiss reaction (1) which is actually quite slow. Presence of iron strongly catalyzes formation of OH•, in two step process. In the first step ferric ion (Fe3+) is reduced in to ferrous ion (Fe2+) by O2•- (2). Ferrous ion than reacts with hydrogen peroxide (generated during DOX

Unlike hydrogen peroxide and superoxide anion radical, hydroxyl radical is extremely reactive and cannot be neutralized by antioxidative enzymes. Instead, OH• reacts with polyunsaturated fatty acids forming lipid peroxides, conjugated dienes and malonyl dialdeyde. As a consequence, the structure of lipid bilayer is modified resulting in cell membrane damage followed by cell dysfunction. In addition ROS can seriously affected proteins and nucleic acids, particularly ion channels and ion transporters (Halliwell & Gutteridge, 2007). Zhu et al. proved involvement of ROS in apoptosis in cardiac myocytes through p38 MAP kinase which is inhibited by CAT and SOD scavengers (Zhu et al., 1999). Cells have very little or no free iron available for catalyzing free radical reactions. However, studies have shown that redox cycling of doxorubicin directly increases the intracellular iron pool (Minotti et al., 1999; Xu et al., 2005; Xu et al., 2008). Formation of DOX semiquinone followed by O2•- generation is accompanied by iron releasing from ferritine. Ferritines are sphere shaped complex of 24 protein subunits with central cavity which can store up to 4500 atoms of iron. Eight channels through the sphere are lined by hydrophilic amino acid residues and six more are lined by hydrophobic residues. These transprotein channels provide the route for iron entry and exit from ferritin shell (Harrison et al., 1986; Theil, 1987). Anthracycline semiquinone is too large for entering the ferritin. However, superoxide anion is small enough to penetrate the shell and has reduction potential lower than that of polynuclear ferric oxohydroxide stored in the ferritin core. Combination of steric and thermodynamic factors thus enables O2•- to reach and reduce the iron pool of ferritin promoting the release of iron in its Fe(II) form through transprotein channels

Furthermore, intracellular iron enrichment is mediated by doxorubicin and its metabolites. Cellular iron homeostasis is regulated by transferring receptor (TfR) and ferritin. Both TfR and ferritin are mainly regulated at a post-transcriptional level involving interactions of iron regulatory protein (IRP-1) and iron responsive elements (IRE) in target genes (Xu et al., 2005; Xu et al., 2008). Namely, DOX can disrupt the Fe-S cluster of cytoplasmatic aconitase and inhibit IRP-1 whose role is to adapt levels of cellular iron according to metabolic needs.

The second mechanism by which Fe promotes oxidative injury involves generation of doxorubicin-iron complexes (DOX-Fe). Anthracyclines chelate Fe(III) forming the 1:1, 2:1 or 3:1 iron complex with association constant of 1033 (Figure. 2b) (Beraldo et al., 1985; May et al., 1980). In the presence of reducing agents (as NADPH cytochrome P450 reductase, glutathione or cysteine) DOX-Fe (III) complex are converting into DOX-Fe (II). This reaction

semiquinone free radical. Products of this reaction are further involving in the iron catalyzed Haber-Weiss reaction resulting in production of highly aggressive hydroxyl radicals (eq. 1-3). The semiquinone radical can be transform into C7 deoxydaglicone which

•-and conversion of quinone form of DOX to

reduction) resulting with formation of hydroxyl radicals (Fenton reaction) (3).

b)

Fig. 2. Doxorubicin mediated redox cycling and free radical production

$$\bullet \bullet \bullet \, ^{\bullet} \bullet \bullet \, ^{\bullet} \bullet \bullet \, ^{\bullet} \bullet \bullet \, ^{\bullet} \bullet \, ^{\bullet}$$

$$\text{O}\_2^{\bullet} \star \text{Fe}^{\text{3+}} \longrightarrow \text{O}\_2 \star \text{Fe}^{2+} \tag{2}$$

a)

b)

Fig. 2. Doxorubicin mediated redox cycling and free radical production

generate highly toxic hydroxyl radical (OH•). This process is occurring during the Haber-Weiss reaction (1) which is actually quite slow. Presence of iron strongly catalyzes formation of OH•, in two step process. In the first step ferric ion (Fe3+) is reduced in to ferrous ion (Fe2+) by O2•- (2). Ferrous ion than reacts with hydrogen peroxide (generated during DOX reduction) resulting with formation of hydroxyl radicals (Fenton reaction) (3).

Unlike hydrogen peroxide and superoxide anion radical, hydroxyl radical is extremely reactive and cannot be neutralized by antioxidative enzymes. Instead, OH• reacts with polyunsaturated fatty acids forming lipid peroxides, conjugated dienes and malonyl dialdeyde. As a consequence, the structure of lipid bilayer is modified resulting in cell membrane damage followed by cell dysfunction. In addition ROS can seriously affected proteins and nucleic acids, particularly ion channels and ion transporters (Halliwell & Gutteridge, 2007). Zhu et al. proved involvement of ROS in apoptosis in cardiac myocytes through p38 MAP kinase which is inhibited by CAT and SOD scavengers (Zhu et al., 1999). Cells have very little or no free iron available for catalyzing free radical reactions. However, studies have shown that redox cycling of doxorubicin directly increases the intracellular iron pool (Minotti et al., 1999; Xu et al., 2005; Xu et al., 2008). Formation of DOX semiquinone followed by O2•- generation is accompanied by iron releasing from ferritine. Ferritines are sphere shaped complex of 24 protein subunits with central cavity which can store up to 4500 atoms of iron. Eight channels through the sphere are lined by hydrophilic amino acid residues and six more are lined by hydrophobic residues. These transprotein channels provide the route for iron entry and exit from ferritin shell (Harrison et al., 1986; Theil, 1987). Anthracycline semiquinone is too large for entering the ferritin. However, superoxide anion is small enough to penetrate the shell and has reduction potential lower than that of polynuclear ferric oxohydroxide stored in the ferritin core. Combination of steric and thermodynamic factors thus enables O2•- to reach and reduce the iron pool of ferritin promoting the release of iron in its Fe(II) form through transprotein channels (Minotti, 1993; Minotti et al., 1999).

Furthermore, intracellular iron enrichment is mediated by doxorubicin and its metabolites. Cellular iron homeostasis is regulated by transferring receptor (TfR) and ferritin. Both TfR and ferritin are mainly regulated at a post-transcriptional level involving interactions of iron regulatory protein (IRP-1) and iron responsive elements (IRE) in target genes (Xu et al., 2005; Xu et al., 2008). Namely, DOX can disrupt the Fe-S cluster of cytoplasmatic aconitase and inhibit IRP-1 whose role is to adapt levels of cellular iron according to metabolic needs.

The second mechanism by which Fe promotes oxidative injury involves generation of doxorubicin-iron complexes (DOX-Fe). Anthracyclines chelate Fe(III) forming the 1:1, 2:1 or 3:1 iron complex with association constant of 1033 (Figure. 2b) (Beraldo et al., 1985; May et al., 1980). In the presence of reducing agents (as NADPH cytochrome P450 reductase, glutathione or cysteine) DOX-Fe (III) complex are converting into DOX-Fe (II). This reaction come along with the formation of O2 •-and conversion of quinone form of DOX to semiquinone free radical. Products of this reaction are further involving in the iron catalyzed Haber-Weiss reaction resulting in production of highly aggressive hydroxyl radicals (eq. 1-3). The semiquinone radical can be transform into C7 deoxydaglicone which is the potent alkylating agent.

Doxorubicin-Induced Oxidative Injury

most significant pathological changes appear in the heart.

mechanism underlying cardiac impairment induced by DOX.

**2.5 Strategies for cardioprotection**

**2.5.1 Limiting cumulative dose of doxorubicin**

supplements are used.

of Cardiomyocytes – Do We Have Right Strategies for Prevention? 97

to the liver, the cardiac antioxidant defense system is moderate. There is 150 times less catalase and four times less superoxide dismutase in the heart (Doroshow & Davies, 1986; Doroshow et al., 1980). In addition, the unique structure of cardiomyocytes, in which 50% of the cell organelles are mitochondria, serving both as a source and target for ROS, may explain why anthracycline antibiotics are selectively toxic to the heart (Berthiaume & Wallace, 2007). Cardiac mitochondria possess a NADH dehydrogenase on the outer surface of the inner mitochondrial membrane. Reduction of an anthracycline to the corresponding semiquinone by this enzyme produces an extremely high level of oxidative stress because the anthracycline transfers an electron to molecular oxygen and forms superoxide radicals. In relation to oxidative reactions, it must be emphasized that doxorubicin possess high affinity for cardiolipin, cardiospecific phospholipid that is reach in polyunsaturated fatty acids also placed in inner mitochondrial membrane. This high affinity, besides accumulation of DOX in interior of cardiomyocytes, also enables formation of DOX-cardiolipin complexes which can act as the substrate for the initiation of lipid peroxidation (Goormaghtigh & Ruysschaert, 1984). Once initiated, peroxidation continues autocatalytically, and has a progressive course that results in structural and functional changes in the heart tissue. The ultimate damage to mitochondria is oxidative damage of the mitochondrial DNA, which interferes with the regenerative capacity of the organelle. Once this irreversible damage occurs, the cardiomyocytes are destined to undergo apoptosis or necrosis, an event that may not occur until months or years after chemotherapy is completed (Davies & Doroshow, 1986; Doroshow et al., 1980). Therefore, the

It is evident that there are still unknowns about exact mechanisms of the toxic effects of doxorubicin and its metabolites. In spite numerous investigations at the molecular level, iron mediated free radical injury and alteration in intracellular iron homeostasis have a key role in cardiotoxicity of anthracycline antibiotics and therefore considered classical

Today's oncologists must be fully aware of cardiovascular risks to avoid or prevent adverse cardiovascular effects. The primary goal in the prevention is to minimise cardiac toxicities and to maximise oncological efficacy (Dorr, 1991; Schimmel et al., 2004). Currently, several preventive measures, such as: limiting cumulative dose of DOX, altering its administration, using new anthracycline analogues; combination with protective drugs and nutritional

Before the association between higher cumulative dose of DOX and the greater risk of cardiotoxicity was established, cumulative DOX doses greater than 400 mg/m2 were administered to children with acute lymphoblastic leukaemia (ALL). Unfortunately, progressive cardiotoxic effects, clinically important, continued years after the DOX treatment completion (Lipshultz et al., 1991; Lipshultz et al., 2005). As a result, cumulative doses of DOX given to high-risk children and adolescent with ALL were reduced to a cumulative dose no more than 360 mg/m2.. Later on, after median follow up of 8.1 year, on the basis of obtained results, the cumulative DOX dose for high-risk ALL patients were

Fig. 3. Hypothesized mechanism of chronic cardiotoxicity mediated by DOXol and/or DOX-Fe complex

Different pathway of doxorubicin toxicity, other than oxidative injury, is involved in cardiac toxicity of the drug. Doxorubicinol (DOXol) is hydroxy metabolite of DOX formed upon two electron reduction on the C-13 carbonyl group of doxorubicin (Figure. 3). This reaction is catalyzed by NADPH dependent carbonyl reducatses (Forrest et al., 1991). DOXol is redox inactive and therefore not involved in free radical production. Notwithstanding, DOXol is involved in iron homeostasis (Licata et al., 2000; Minotti et al., 2001), myocardial energy metabolism, ionic gradients and calcium movements, impairing cardiac contraction and relaxation (Boucek et al., 1987; Olson, R. D. et al., 1988). Moreover, conversation of doxorubicin into DOXol significantly increases polarity of the parent drug molecule, thus favouring its retention within the cell. Considering the all aforesaid mechanisms of DOXol acting, it is logical conclusion that alcohol metabolite of doxorubicin is most likely responsible for the DOX induced chronic cardiomyopathy (Figure. 3), while iron mediated free radical injury promote acute cardiotoxicity This hypothesis is supported by findings of Miranda and co-workers which found that patients harbouring gene mutations in hereditary hemochromatosis show increased susceptibility to doxorubicin cardiotoxicity and exacerbated iron metabolism (Miranda et al., 2003). Numerous studies on transgenic mice with overexpressed antioxidants like catalase (Kang et al., 1996), superoxide dismutase (Abou El Hassan et al., 2003; Loch et al., 2009; Yen et al., 1996) and metallothionene (Kang, 1999; Kang et al., 1997; Merten et al., 2005; Sun & Kang, 2002; Sun et al., 2001) have shown beneficial against DOX induced cardiotoxicity, confirming strong involvement of free radicals in oxidative injury of the heart.

The high oxidative metabolism and the poor antioxidative defence, compared with other organs, most likely explains why the doxorubicin is selectively toxic to the heart. Compared

Fig. 3. Hypothesized mechanism of chronic cardiotoxicity mediated by DOXol and/or DOX-

Different pathway of doxorubicin toxicity, other than oxidative injury, is involved in cardiac toxicity of the drug. Doxorubicinol (DOXol) is hydroxy metabolite of DOX formed upon two electron reduction on the C-13 carbonyl group of doxorubicin (Figure. 3). This reaction is catalyzed by NADPH dependent carbonyl reducatses (Forrest et al., 1991). DOXol is redox inactive and therefore not involved in free radical production. Notwithstanding, DOXol is involved in iron homeostasis (Licata et al., 2000; Minotti et al., 2001), myocardial energy metabolism, ionic gradients and calcium movements, impairing cardiac contraction and relaxation (Boucek et al., 1987; Olson, R. D. et al., 1988). Moreover, conversation of doxorubicin into DOXol significantly increases polarity of the parent drug molecule, thus favouring its retention within the cell. Considering the all aforesaid mechanisms of DOXol acting, it is logical conclusion that alcohol metabolite of doxorubicin is most likely responsible for the DOX induced chronic cardiomyopathy (Figure. 3), while iron mediated free radical injury promote acute cardiotoxicity This hypothesis is supported by findings of Miranda and co-workers which found that patients harbouring gene mutations in hereditary hemochromatosis show increased susceptibility to doxorubicin cardiotoxicity and exacerbated iron metabolism (Miranda et al., 2003). Numerous studies on transgenic mice with overexpressed antioxidants like catalase (Kang et al., 1996), superoxide dismutase (Abou El Hassan et al., 2003; Loch et al., 2009; Yen et al., 1996) and metallothionene (Kang, 1999; Kang et al., 1997; Merten et al., 2005; Sun & Kang, 2002; Sun et al., 2001) have shown beneficial against DOX induced cardiotoxicity, confirming strong involvement of free

The high oxidative metabolism and the poor antioxidative defence, compared with other organs, most likely explains why the doxorubicin is selectively toxic to the heart. Compared

Fe complex

radicals in oxidative injury of the heart.

to the liver, the cardiac antioxidant defense system is moderate. There is 150 times less catalase and four times less superoxide dismutase in the heart (Doroshow & Davies, 1986; Doroshow et al., 1980). In addition, the unique structure of cardiomyocytes, in which 50% of the cell organelles are mitochondria, serving both as a source and target for ROS, may explain why anthracycline antibiotics are selectively toxic to the heart (Berthiaume & Wallace, 2007). Cardiac mitochondria possess a NADH dehydrogenase on the outer surface of the inner mitochondrial membrane. Reduction of an anthracycline to the corresponding semiquinone by this enzyme produces an extremely high level of oxidative stress because the anthracycline transfers an electron to molecular oxygen and forms superoxide radicals. In relation to oxidative reactions, it must be emphasized that doxorubicin possess high affinity for cardiolipin, cardiospecific phospholipid that is reach in polyunsaturated fatty acids also placed in inner mitochondrial membrane. This high affinity, besides accumulation of DOX in interior of cardiomyocytes, also enables formation of DOX-cardiolipin complexes which can act as the substrate for the initiation of lipid peroxidation (Goormaghtigh & Ruysschaert, 1984). Once initiated, peroxidation continues autocatalytically, and has a progressive course that results in structural and functional changes in the heart tissue. The ultimate damage to mitochondria is oxidative damage of the mitochondrial DNA, which interferes with the regenerative capacity of the organelle. Once this irreversible damage occurs, the cardiomyocytes are destined to undergo apoptosis or necrosis, an event that may not occur until months or years after chemotherapy is completed (Davies & Doroshow, 1986; Doroshow et al., 1980). Therefore, the most significant pathological changes appear in the heart.

It is evident that there are still unknowns about exact mechanisms of the toxic effects of doxorubicin and its metabolites. In spite numerous investigations at the molecular level, iron mediated free radical injury and alteration in intracellular iron homeostasis have a key role in cardiotoxicity of anthracycline antibiotics and therefore considered classical mechanism underlying cardiac impairment induced by DOX.

#### **2.5 Strategies for cardioprotection**

Today's oncologists must be fully aware of cardiovascular risks to avoid or prevent adverse cardiovascular effects. The primary goal in the prevention is to minimise cardiac toxicities and to maximise oncological efficacy (Dorr, 1991; Schimmel et al., 2004). Currently, several preventive measures, such as: limiting cumulative dose of DOX, altering its administration, using new anthracycline analogues; combination with protective drugs and nutritional supplements are used.

#### **2.5.1 Limiting cumulative dose of doxorubicin**

Before the association between higher cumulative dose of DOX and the greater risk of cardiotoxicity was established, cumulative DOX doses greater than 400 mg/m2 were administered to children with acute lymphoblastic leukaemia (ALL). Unfortunately, progressive cardiotoxic effects, clinically important, continued years after the DOX treatment completion (Lipshultz et al., 1991; Lipshultz et al., 2005). As a result, cumulative doses of DOX given to high-risk children and adolescent with ALL were reduced to a cumulative dose no more than 360 mg/m2.. Later on, after median follow up of 8.1 year, on the basis of obtained results, the cumulative DOX dose for high-risk ALL patients were

Doxorubicin-Induced Oxidative Injury

the future (Kalaria et al., 2009).

**3. Dexrazoxane**

**2.5.5 Combination with protective drugs**

Links & Lewis, 1999; Simunek et al., 2009).

of Cardiomyocytes – Do We Have Right Strategies for Prevention? 99

lead to less drug exposure in sensitive organs. The ability to reduce the plasma levels of free DOX is probably the source of the reduced cardiotoxicity provided by its liposomal formulation (Fulbright et al., 2010). Also, the release of drug is slow, which may avoid high peak plasma concentrations. It exists both in pegylated lyposomal and, at the moment, these two formulations are approved for clinical use by the US Food and Drug Administration (Ludke et al., 2009; Sweetman, 2011). Several randomized clinical trial in adults have examined the activity and cardiotoxicity of liposomal DOX. Most of them indicated that the activity of liposomal DOX is similar to that of conventional, whether given alone or in combination with other drugs. At the same time, the risk of cardiotoxicity is markedly lower with liposomal DOX (Gianni et al., 2008; McEvoy et al., 2010; Safra, 2003; Smith et al., 2010). However, although clinically approved, conventional DOX continues to be the major drug in antineoplastic combinations used in the management of most solid tumors. Nanoparticles made of biodegradable polymers are particles with sizes in range from 20 to 200 nm that can encapsulate hydrophilic or hydrophobic drugs. Drugs delivered by nanotechnology delivery systems have a prolonged time in the circulation (Ferrari, 2005). Although carbon nanotubes and gold nanoparticles have demonstrated potential as a new drug delivery system, clinical use has been restricted due to concerns regarding their biodegradability (Liu et al., 2008). These particles should link with tumor-targeting ligands, such as peptides and small molecules. At the moment, DOX nanoparticles shows great promise in improving the oral bioavailability and reducing cardiotoxicity, but so many issues wait to be resolved in

Apart from cumulative dose limitations, several attempts have been made to develop chemoprotectants to prevent the cardiotoxicity of DOX, without attenuating its antitumor effect. Following the aforementioned free radical hypothesis, antioxidants used as free radical scavengers have been examined both in experiments, and in clinical trials. On the other hand, considering a very important role of iron and the DOX-iron complex, iron chelators have also been used for prevention of DOX-induced cardiotoxicity (Dorr, 1991;

One of the most examined protectors against DOX cardiotoxicity is dexrazoxane (DEX). Activity of DEX against DOX-induced cardiotoxicity may be attributable to the intracellular conversion of DEX to an open-ring derivative ADR-925 which chelates iron, since one means of generating oxygen-free radicals may involve intramolecular reduction of DOX-iron conjugate. The cardioprotective activity of DEX is thought to result primarily from chelation of free and/or DOX-bound ferric ions in the myocardium by ADR-925 and some other hydrolysis products of DEX (Dorr, 1996; Hasinoff, 1989). As a result, the pool of free iron in the myocardium is reduced and bound iron is displaced from its potentially damaging complexes with DOX, thus preventing the formation of superoxide radicals after redox recycling. DEX has been shown experimentally to reduce DOX-induced cardiotoxicity in mice (Alderton et al., 1990); in rats (Della Torre et al., 1999; Herman et al., 1994; Villani et al., 1990); in beagle dogs (Herman et al., 1988) and in miniature swine (Herman & Ferrans,

further reduced to 300 mg/m2 (Nysom et al., 1998). However, patients vary widely in sensitivity and these values represent relatively arbitrary choices on a continuum of risk: there is no single safe dose (Shan et al., 1996; Von Hoff et al., 1979). Studies of late-onset anthracycline cardiotoxicity in childhood cancer survivors indicate that doses of DOX as low as 100 mg/m2 increase the risk of reduced fractional shortening and higher afterload, whereas a cumulative dose of 270 mg/m2 increases the risk of such abnormalities 4.5-fold. Even at doses that produce no symptoms, subclinical myocardial damage may occur, and in children this may result in diminished cardiac reserve and heart disease in later life (Hale & Lewis, 1994; Lipshultz et al., 1991).

#### **2.5.2 Altering doxorubicin administration**

Alteration of the dosage schedule to weekly rather than three-weekly dosage, or the use of continuous infusion, has also been advocated as a way of reducing doxorubicin cardiotoxicity (Legha et al., 1982a; Lum et al., 1985; Torti et al., 1983). At the same time, it was believed that continuous infusion might provide some cardioprotective benefits, because long-term exposures to drugs at modest concentrations would be safer than a pulsed supply of the drug at higher concentrations. Therefore, it was incorporated in many pediatric protocols although the comparative efficacy of these dosage schedules in various cancers and long-term effects on the development of DOX-induced cardiotoxicity have not been established tz (Lipshultz et al., 2005; Lipshultz et al., 2010). However, recently published meta-analysis performed to clarify the risk of early and late cardiotoxicity of anthracycline agents in patients treated for breast or ovarian cancer, lymphoma, myeloma or sarcoma showed that DOX given as bolus significantly increased the risk of clinical and subclinical cardiotoxicity compared with continuous infusion.

#### **2.5.3 Use of doxorubicin-analogues**

Several anthracycline derivatives have been developed with the aim of reducing the inherent cardiotoxicity of this class of compounds, including esorubicin, quelamycin, rodorubicin, and detorubicin. However, although this strategy has had some success, it turned out that almost all these compounds exhibit some degree of cardiotoxicity (Aronson et al., 2006; Sweetman, 2011; Weiss, 1992; Wouters et al., 2005). Epirubicin, idarubicin and mitoxantrone are the most promising ones. In the recently performed meta-analysis epirubicin significantly decreased the risk of clinical cardiotoxicity OR 0.39 (0.16, 0.57; p0.008; I2 0.5%), as well as any cardiotoxic event compared with DOX (Smith et al., 2010).

#### **2.5.4 New drug-delivery systems and doxorubicin application**