**2. Anti-neoplastic agent cardiotoxicity**

### **2.1 Anthracyclines**

Cardiovascular risk can be attributed to those things that directly damage the myocyte, those things that increase the likelihood of damage to the myocyte, or those things that impair the recovery following injury to the myocyte. Anthracyclines are effective anti-neoplastic agents, but with efficacy limited by cardiotoxic side effects. Clinically, there are specific guidelines based on serial cTnT levels to monitor of cardiotoxic effects, and experimentally, the cardiotoxicity is well-established enough that doxorubicin, a common anthracycline, is one of the most commonly used approaches to induce experimental heart failure independent of any neoplasm [40]. Anthracyclines cause a type I cardiotoxicity that is dose-limiting, often irreversible, and produces changes demonstrated on biopsy. While previous treatment for cancer is not considered a general cardiovascular risk factor per se, it is clear that at least some cancer therapeutics can produce persistent cardiac effects, and understanding those mechanism provides a great deal of insight regarding underlying mechanisms involved in increasing cardiovascular risk. This is particularly important when anthracyclines were used, but no overt toxicity was noted, which is to say only that ejection fraction, or CTnT NP remained within acceptable limits. It is worth reviewing the many ways that anthracyclines can induce injury so as to better appreciate how a sub-clinical event might have go undetected at the time, but could none-the-less create a vulnerable substrate for injury later.

Doxorubicin is toxic, in itself, and approximately half of the doxorubicin administered into the body is eliminated unchanged. However, the metabolized doxorubicin creates secondary effects/products that are at least as toxic as the parent compound.

#### *Predicting Risk of Emerging Cardiotoxicity DOI: http://dx.doi.org/10.5772/intechopen.105040*

Metabolism of doxorubicin occurs in one of three ways: two-electron reduction, one-electron reduction, and deglycosylation [5, 41, 42].

Two-electron reduction is the primary metabolic pathway for doxorubin and results in the formation of active secondary alcohol metabolites. The alcohol metabolites that are produced cause cardiotoxic side effects by disrupting intracellular and especially intramitochondrial calcium and iron homeostasis. Doxorubincinol, a secondary alcohol metabolite of doxorubicin, interacts with aconitase/iron regulatory protein 1 (IRP-1) and converts it to a "null protein". Normally, when iron levels are adequate, aconitase acts within the Krebs cycle to convert citrate to isocitrate. When iron levels are low, this enzyme does not contain a [4Fe-4S] cluster and will act as IRP-1, which has RNA-binding activity. IRP-1 helps to regulate the expression of genes involved in iron metabolism and homeostasis [5, 41, 42].

Doxorubicinol also is an inhibitor of a number of ATPases, including the mitochondria's proton pump and those which regulate intracellular calcium levels, which not only leads to inhibition of cellular oxidative phosphorylation and bioenergetic collapse, but also disrupts calcium homeostasis leading to intracellular calcium overload disruption of excitation-contraction coupling [5, 41, 42]. Anthracyclines, particularly doxorubicin, also increase activity of calpain and may suppress sarcomere protein synthesis via signaling pathways and transcription factors, such as GATA4 [5, 41, 42]. Increased Calpain activity likely is secondary to an increase in intracellular calcium. This increase in activity increases degradation of titin, a major myofilament of sarcomere, which further leads to an impaired diastolic relaxation. Calpain dysregulation is also known to activate caspase-12, which can cause apoptosis. Caspase-12 is localized in the endoplasmic reticulum and is often activated by ER stress [43].

Anthracycline alcohol metabolites also interfere with the activity of iron sequestering proteins which leads to an increase in intracellular free iron. Anthracyclines have a high affinity for ferric iron, forming a free radical that is capable of reducing oxygen [44]. This is one mechanism by which anthracyclines are capable of participating in "redox cycling". Doxorubicin also contributes to the generation of ROS by inactivating the cellular defense enzymes glutathione peroxidase (GPx) and superoxide dismutase (SOD) [5, 41, 42], essential enzymes critical to managing mitochondrial membrane potential.

In one-electron reduction metabolic pathway, doxorubicin's adverse effects are due to redox cycling. The quinone moiety accepts an electron from NADH or NADPH via the use of endothelial nitric oxide synthase (eNOS), leading to the formation of a semiquinone free radical. This free radical reduces oxygen and forms a superoxide radical. This contributes to the increase in ROS observed in cardiomyocytes after doxorubicin exposure [5, 41, 42]. Further, doxorubicin's interaction with eNOS shuttles the enzyme from a NO-producing enzyme to a superoxide-producing enzyme. This could contribute to an impaired cardioprotective mechanism. This "uncoupling of eNOS" has already been shown to be a contributor in heart failure [6].

The deglycosylation pathway is thought to be a less important contributor to anthracycline metabolism, but it creates a lipophilic molecule that has greater accumulation in the mitochondrial membrane than unmetabolized doxorubicin [5, 41, 42], and by interfering with such mitochondrial membrane proteins as cardiolipin, can also disrupt mitochondrial function, which can trigger any number of secondary effects, including energy depletion, ROS generation and DNA damage, as well as apoptosis [7–11].

Topoisomerase IIB is found in all quiescent cells, including cardiomyocytes. Anthracyclines inhibit this enzyme, leading to unrepaired ROS induced double-stranded breaks, DNA damage and transcriptome changes in tissue with little regenerative capability [5, 41, 42]. It was demonstrated that mice with cardiomyocyte specific Top2b deletion were protected from the progressive heart failure effects of doxorubicin [45], clearly suggesting that the inhibition of topoisomerase IIB plays a major role in the cardiotoxicity induced by doxorubicin.

Stress to the heart generally shifts metabolic energy production away from the preferred free fatty acid substrate to one that is glucose based and more prone to lactate production. Typically, those shifts are driven by limitations in perfusion and access to sufficient oxygen to support electron transport chains. The same is true for anthracyclines, except that anthracyclines also directly disrupt multiple aspects of mitochondrial function. Doxorubicin causes p53 activation via its inhibition on topoisomerase IIB, leading to repression of PPARg co-activator alpha and beta, which normally promote mitochondrial biogenesis. Their signaling inhibition leads to aging and heart failure. Thus, doxorubicin's inhibition of topoisomerase IIB leads to a mitochondrial dysfunction that that is known to contribute to development of heart failure in doxorubicin-treated patients [46] and in other models of heart failure as well [7–11].

It has also been suggested that anthracycline-induced cardiotoxicity is partially due to the action of matrix metalloproteinases (MMP's). MMP-2 is highly abundant in cardiac myocytes and increased plasma levels have been observed following heart failure. It is possible that doxorubicin increases the concentration of MMP-2 in cardiac cells. Activation of MMP-2 by doxorubicin has been shown to play a role in the degradation of titin, further suggesting that this mechanism plays a role in sarcomere disruption and cardiac remodeling involved with doxorubicin-induced cardiomyopathy [47]. MMPs play an active role in regulating fibrosis in the heart and dysregulation can lead to disruption of the extracellular matrix, adverse remodeling and impaired relaxation.

Doxorubicin can have genomic effects as well. GATA4 is a zinc finger transcription factor that regulates multiple genes, including anti-apoptotic gene Bcl-x. GATA4 is not only involved in sarcomere integrity, but is also an essential survival factor in cardiomyocytes and has been shown to rapidly deplete after doxorubicin exposure. GATA4 depletion leads to cardiomyocyte apoptosis and is essential for adaptive stress response in the adult heart [48] and decreased GATA4 can occur secondary to p53 accumulation caused by doxorubicin exposure [49]. A GATA motif also was identified in an angiotensin II receptor (ATII-R). When stimulated, this receptor helps regulate the hypertrophic response to pressure overload. Mutations introduced into this binding site eliminated this response, demonstrating a role for GATA4 in the regulation of cardiac hypertrophic response [43].

Doxorubicin has a high affinity for the phospholipid cardiolipin. Cardiolipin is a mitochondrial membrane protein involved in many cellular mechanisms, including mitochondrial cristae morphology, electron transport chain function, steroid synthesis, mitophagy, and apoptosis. Cardiolipin turns over at a slower rate than other phospholipids of the mitochondrial membrane and a change in the cardiolipin pool is observed in multiple cardiovascular diseases and in the aged heart [5, 50]. When complexed to doxorubicin, cardiolipin is unable to perform some of its regular functions such as anchoring cytochrome c [7]. This high affinity for cardiolipin also enables doxorubicin to accumulate in the mitochondria. Additionally, it is possible that anthracyclines possess an affinity to intercalate into mitochondrial DNA and may have a higher affinity for mitochondrial DNA than for nuclear DNA [44], leading to mitochondrial genomic injury as well.

#### *Predicting Risk of Emerging Cardiotoxicity DOI: http://dx.doi.org/10.5772/intechopen.105040*

The intimate involvement of mitochondrial dysfunction in progression of cardiovascular injury is becoming increasingly evident [7–11]. It should not be surprising that anthracyclines also interfere with cellular energy metabolism in numerous ways, some of which already have been described. Doxorubicin is known to directly interfere with complex I of the electron transport chain and causes a dose-dependent opening of the mitochondrial permeability transition pore, preventing the mitochondria from creating a proton gradient [7–11]. Furthermore, doxorubicin acutely inhibits AMPK and causes a net metabolic shift from fatty acid oxidation to glucose oxidation and lactic acid production, and the observed alterations in redox and metabolic pathways caused by doxorubicin have been shown to persist beyond the half-life of the drug [7], suggesting a durable effect that would increase both the severity of injury but also recovery from future cardiac events.

In addition to the intracellular mechanisms of apoptosis already discussed, it has been shown that doxorubicin upregulates the expression of several death receptors, including TNFR1, DR4, DR5, and FAS [51]. In one study, Adriamycin was shown to induce apoptosis in a p53-independent mechanism, via a Fas-mediated pathway [52]. The cardiotoxic side effects of doxorubicin are also thought to be partially due to an inflammatory response. Doxorubicin has been shown to increase several specific cytokines, including IL-6 and COX-2, and the inflammatory response induced by doxorubicin can be mediated in part by the activation of the p38 MAPK/NF-κB pathway [53]. The activation of p38 MAPK in cardiac cells has been associated with the accumulation of ROS and the onset of apoptosis in ischemia–reperfusion injured hearts [43] and strategies to limit those effects have been a major feature of cardioprotection in ischemia–reperfusion settings [29–32].

Studies have demonstrated that specific inflammatory biomarkers are predictive of cardiotoxicity in anthracycline-treated patients [44]. For example, one study showed myeloperoxidase levels had predictive value for ANT cardiotoxicity [54]. Another study showed that patients with high baseline levels of IgE were at a lower risk of developing cancer therapy-related cardiotoxicity from doxorubicin and trastuzumab treatment [55]. These results indicate that an inflammatory response likely plays a significant role in the development of anthracycline-induced cardiotoxicity.

#### **2.2 Additional cancer drug treatment related cardiovascular effects**

Anti-neoplastic drugs also capable of causing cardiac injury are not limited to the anthracyclines. For example, Trastuzumab causes a type II cardiotoxicity that is often reversible after cessation of therapy; however, some degree of persistent cardiac dysfunction has been documented in a portion of patients [56]. Trastuzumab is a human epidermal growth factor receptor 2 (HER2/ErbB2) inhibitor. In the adult heart, HER2 plays a cardioprotective role and inhibits apoptosis. When the heart is subjected to biomechanical stress, neuregulin-1 (NRG1) secreted from endothelial cells binds to HER2/HER4 heterodimers on cardiomyocytes and activates PI3K and MAPK pathways [57]. ErbB2 inhibition is associated with a significant increase in Bcl-2 family proteins [58]. It has been demonstrated that mice with a cardiac-specific deletion of ErbB2 displayed evidence of dilated cardiomyopathy, suggesting ErbB2 signaling is essential for prevention of dilated cardiomyopathy during remodeling [59]. Further, downregulation of both ErbB2 and ErbB4 has been observed in pathological remodeling of the failing myocardium in humans [60].

Trastuzumab has also been shown to trigger oxidative stress and induce the expression and activation of proapoptotic proteins, ultimately leading to mitochondrial dysfunction, the opening of mitochondrial permeability transition pores, and activation of the cell death pathways [57, 59]. Trastuzumab and anthracycline combination treatment can be powerful in its therapeutic effect, but also is known to significantly aggravate the cardiotoxic effects of anthracyclines. It is thought that anthracycline induced cardiac injury triggers the activation of the HER2 survival pathways. When anthracyclines are treated in combination with trastuzumab, these cardioprotective survival pathways are inhibited and less protection is provided to the heart [57], explaining the aggravated progression of cardiac injury.

Another fairly common therapeutic, cyclophosphamide (CP), is an alkylating agent used to treat diseases such as neuroblastoma, as well as systemic inflammatory conditions such as Systemic Lupus Erythmatosis (SLE), and rheumatoid arthritis [61]. At lower doses, CP has an immunosuppressive effect, while at higher doses, it causes cardiotoxic effects. Cyclophosphamide and its metabolites, 4-hydroxy cyclophosphamide, aldophosphamide, and acrolein are cardiotoxic, with acrolein being the most cardiotoxic [61]. Acrolein forms cytoplasmic and nuclear protein adducts, as well as adducts with lysine and cysteine. When adducted to lysine, Acrolein can react with glutathione to cause oxidative stress. When adducted with cysteine, it leads to activation of caspases and NF-κB. Activation of caspases causes apoptosis and activation of NF-κB causes the production of inflammatory cytokines [61]. Cyclophosphamide also alters the energy pool in cardiomyocytes. Many anticancer drugs, including CP, alter the expression of heart-type fatty acid binding protein (H-FABP) and carnitine palmitoyltransferase 1 (CPT-1), leading to inhibition of fatty acid oxidation and diminished ATP production. When deprived of enough ATP, cardiac tissue alters contraction and relaxation, accumulates calcium in the mitochondria, and undergoes ER stress [61].

Like many other cytotoxic agents, CP causes oxidative stress in cardiomyocytes by decreasing antioxidant levels and generating free radicals. Nrf2 is a leucine zipper protein involved in the regulation of antioxidants. CP decreases antioxidant levels is through its action on Nrf2 [61], and by suppression of intracellular GSH and SOD [62]. In CP-treated rats, additional treatment with cyclosporin-A had a cardioprotective role against CP-induced cytotoxicity [63]. Cyclosporin binds cyclophilin and forms a complex that then binds calcineurin and inactivates it, suggesting that CP induces calcineurin-mediated effects. It is thought that CP causes calcineurin-mediated dephosphorylation of NFAT, which is involved in the transcription of hypertrophic genes and apoptosis via FasL and death receptors. Additionally, calcineurin causes activation of Akt, which participates in phosphorylation of GSK-3B and leads to cardiac hypertrophy [61].

Cyclophosphamide also was shown to increase the expression of pro-apoptotic proteins and decrease the expression of antiapoptotic proteins [64]. p53 inhibition is associated with reduced apoptosis, but CP and anthracyclines both upregulate expression of p53, promoting apoptosis [61], and CP was shown to increase the expression of caspase-3 and decrease the expression of Bcl-2, also pro-apoptotic mechanisms [65, 66]. Ultrastructural changes were observed in rat cardiomyocytes when treated with CP, including lysis of myofibrils, dilation of vesicles in the sarcoplasmic reticulum, and destruction of mitochondria [67] suggesting substantial potential for persistent effects long after therapy has ended.
