**4.2. The mechanism of doxorubicin's anticancer activity**

It is agreed that the mechanism of its anticancer activity and its toxic impact follow different molecular mechanisms [13, 28, 29]. The anticancer activity of DOX relies on the interaction of the cell nucleus, mitochondria, and membranes. There are a number of reasons that explain the antineoplastic efficiency of the drug [17]:


DOX has been administered by intravenous infusion [13]. Peak plasma concentration and halflife have been reported to be 5–15 μmol/L and 20–30 h, respectively [13]. Another study, however, stated that the peak plasma concentration of patients treated with DOX is between 2 and 6 μM after bolus injection, but typically 1–2 μM [24]. DOX is reported to be very low when bound to plasma proteins [25]. The plasma clearance of DOX is measured between 324 and 809 mL/min/m2

half-life of the drug is around 5 min, which means that reuptake velocity is very high for tissues. However, elimination velocity is slow within the range 20–48 h [26]. After injection, DOX is dis-

The chemical structure of DOX is {(7*S*, 9*S*)-7-[(2*R*, 4*S*, 5*S*, 6*S*)-4-amino-5-hydroxy-6-methyloxan-2-yl] oxy-6, 9, 11-trihydroxy-9-(2-hydroxyacetyl)-4-methoxy-8, 10-dihydro-7*H*-tetracene-5, 12-dione (**Figure 2**). Due to structural specifications with a tetracycline moiety containing a quinone and a conjugated amino sugar residue, DOX can undergo metabolic modification by enzymes dominantly in the liver and kidneys during the elimination process. Some oxidoreductase enzymes, especially nicotinamide adenine dinucleotide phosphate (NADPH) dependent cytochrome P450 reductases at the endoplasmic reticulum (ER), nicotinamide adenine dinucleotide (NADH) dehydrogenase (complex I) at the electron transport chain (ETC), and cytosolic xanthine oxidase, have been suggested to play an important role in DOX elimination. The oxidoreductase enzyme can convert DOX to its semiquinone form by using

DOX's structure includes a glycoside group with anthraquinone moiety. The structure is responsible for its antineoplastic activity and also its toxicity [14]. DOX contains a tetracyclic ring with two quinone-hydroquinones and daunosamine. Though the tetracyclic sugar is nonsoluble in water, daunosamine sugar is soluble in water. DOX has been produced in a derivative form, e.g., daunorubicin. The difference between DOX and daunorubicin is only in the hydroxyl groups. Even though there is a slight difference between the drugs, their

**Figure 2.** The chemical structure of doxorubicin antibiotics. From Imstepf et al. [27].

dominantly by biliary excretion; the maximum volume is around 809–1214 L/m<sup>2</sup>

seminated to the heart, liver, kidneys, and intestine [25].

**4.1. Doxorubicin's chemical structure**

molecular oxygen [13].

326 Mitochondrial Diseases

,

. Moreover, the

**6.** Prevention of topoisomerase II (TOPII) results in elevation of DNA damage [17].

The anticancer effect of DOX is associated with intercalation of the DNA strands, regulatory protein, covalent binding to DNA, and condensation of histone protein. However, its toxic impact on tissue does not rely on DNA impact [15].

The therapeutic effects of DOX have been associated with binding and intercalation of DNA strands, resulting in the destruction of replication and transcription of DNA by topoisomerase inhibition [4, 13, 30]. The reason why enzymes are so crucial is because TOPII has a role to play in modulating the DNA superhelical state [31], relaxing accumulated positive supercoils, and unlinking intertwined DNA strands. Thus, proteins are vital for complete DNA replication [22].

Also, DOX's cardiotoxicity has been related to disrupting TOPIIβ [30]. DOX selects toxic cardiac mitochondria through selective accumulation and redox cycling. However, free DOX enters the nuclei of cancer cells without entering mitochondria, which causes lack of mitochondrial pathway therapy [31]. Besides nuclear DNA, DOX intercalates with the mtDNA double helix, binds to a protein, and has a role in DNA replication and transcription as well [23].

To give more detailed knowledge on drug intercalation, DOX is one of the great anticancer drugs that kills cancerous cells by interaction with the cells' DNA; it also produces covalent adducts, resulting in inhibition of DNA synthesis by DNA polymerase blocking. DOX could also interfere with DNA and TOPIIα, finally forming a TOPIIα/DOX/DNA complex. The interruption of DNA and TOPIIα by DOX causes DNA breakage and cell death. A special situation has been reported whereby heart tissue does not contain TOPIIα, but expresses TOPIIβ [20]. So, the TOPIIβ/DOX/DNA complex could only occur in the heart tissue [20, 28]. It is strongly supported that DOX's cardiotoxicity associates with TOPIIβ based on a TOPIIβ knockout mice study. When DNA damage occurs for some reason, e.g., by using DOX, the ataxia/telangiectasia-mutated protein is activated to trigger tumor suppressor protein p53. Activation of p53 by DOX has been indicated to elevate ROS production, double-strand DNA damage, and apoptotic cell death as well. Furthermore, another effect of p53 addresses one of the cardioprotective transcription factors, known as GATA-4. AMP-activated protein kinase (AMPK) plays the role of energy sensor to maintain enough available energy levels. If energy status drops too low by enhancing ROS production, and intracellular Ca2+ accumulates, AMPK can be activated and/or its phosphorylation can be elevated [20].

DOX are at high risk of developing cardiac damage. It seems that in children DOX causes some stem cells to vanish, including pluripotent, undifferentiated, and cardiac stem cells. The decreased stem cell ability in the heart by DOX results in decompensating for the decline of cardiac mass induced by the drug's treatment. However, DOX has been shown to accumulate

Mitochondrial Dysfunction Associated with Doxorubicin http://dx.doi.org/10.5772/intechopen.80284 329

Another factor associated with DOX toxicity is gender [36]. Unusually, female cancer patients treated with DOX have higher mortality vs. male cancer patients, but females develop cardiovascular disease 10 years later than males. However, after the menopause, females become

DOX is widely used for cancer therapy [38]. However, it has been recognized to have a toxic effect on noncancerous tissue such as the heart [13, 39], liver, kidneys [22], as well as the brain [40], and its poisonous effect is related to its dose [38]. This is why the drug's use for cancer treatment is limited based on its undesired impact on healthy tissue. Unfortunately, the mechanism of its toxic effect on noncancerous tissue has been not understood so far [38, 39]. DOX side effect symptoms are nausea, vomiting, alopecia, myelosuppression, stomatitis, and gastrointestinal disturbances [14], which are typical of cytotoxic chemotherapic agents [28]. The soft side effects of drugs include nausea, fever, and vomiting. Nausea, fever, and vomiting appear after DOX therapy as soft side effects. However, hypotension, arrhythmias, tachycardia, and congestive heart failure are also described after treatment as severe undesired side effects [17].

DOX's cytotoxicity includes two molecular mechanisms: intercalation of nuclear DNA and elevation of ROS production [41]. A cancer cell's DNA replication is well known to be faster than normal cells [41]. If DOX generates normal levels of ROS, it might selectively destroy heart pump function [41]. The most accepted mechanism leading to DOX's toxicity is oxidative stress, which causes damage to membrane lipid peroxidation products and decreases antioxidants as well. The most severe ROS generation by DOX is in the heart vs. other organs or tissues, e.g., kidneys, liver, etc. [10]. Extensive research has been suggested as to why DOX's cardiotoxicity relies on oxidative stress, mitochondrial dysfunction, and mitochondrial energy-forming disruption [16]. DOX treatment after 3 h has been reported to cause oxidative stress, lipid peroxidation, as well as lipid aldehydes in cardiac tissue [42]. Selective toxicity of the heart by DOX will explain these reasons. There is strong evidence supporting a critical role of oxidative stress on DOX's toxicological effect, though the molecular mechanism of its toxicity is still a mystery. According to study results from animal and human tissue, DOX

DOX's anticancer activity is associated with intercalation to DNA by decreasing TOPII activity after double-strand breakage, resulting in alleviation of DNA replication and protein synthesis. However, it is accepted that DOX's toxicity and anticancer efficiency are different from each other. The cytotoxic effect of DOX is produced by a mechanism such as ceramide synthesis by CREB3L1 activation, oxidative damage to DNA, losing mitochondrial membrane potential (MMP), caspase-3 activation, and p53 and c-Jun NH2-terminal kinase (JNK) activations. Nuclear factor-kappa B (NF-κB), a proapoptotic factor, could participate in DOX's cytotoxicity [33].

in cardiac tissue in elderly patients, resulting in reduced blood flow in the heart [17].

more vulnerable than males at the same age [37].

disrupts the myofibril, mitochondrial membrane [13].

**4.4. Cytotoxic effect of doxorubicin on noncancerous tissues**

#### **4.3. The risk factors of doxorubicin's toxicity**

DOX utilization is limited due to its toxic effect [27]. There are well-established factors that pointed to an increase in DOX-related heart damage. These factors are total cumulative dose, one course or a day's full dose, radiation, especially mediastinal, age, gender, other cardiotoxic drugs or chemicals, cardiovascular illness, and liver pathologies [17].

One of the riskiest factors for toxicity is the drug's cumulative dose [4]. Extensive reports are available in the literature. The mortality of congestive heart failure-induced DOX is around 50% at higher than a 500 mg/m2 cumulative dose [17]. This side effect is related to its dose, which is reported to be between 75 and 1095 mg/m2 , and the median dose of its toxicity is around 390 mg/m2 [10]. The risk of developing toxicity on noncancerous tissue has been reported to enhance the cumulative dose, e.g., at 400 mg/m2 with 3–5% [4, 10, 20], 550 mg/m2 with 7–26% [4, 10], 700 mg/m2 with 18–48% [4, 10, 20], or 950 mg/m2 with 50% [10]. Furthermore, the risk of toxicity has been reported to enhance at 550 mg/m2 [10]. When mice are given a total of 71 mg/m2 DOX, the risk of heart damage is almost 100% [20]. Also, cancer type determines the risk ratio. For example, around 20% of lung cancer patients treated with DOX have been reported to develop heart failure [32]. DOX, an anthracycline antibiotic, therapy has been indicated to develop side effects in almost 35% of patients [33]. Its clinical utilization is, therefore, limited because many tissues become toxic when patients are treated with a 550–600 mg/m2 cumulative dose [15]. DOXs toxicity can reach a 50% mortality rate at the highest cumulative dose [14].

The detrimental effect of DOX is related to its dosage and treatment duration. Its utilization is recognized to develop into cachexia and cardiotoxic impact over time [4]. DOX can even cause cardiomyopathy after years of treatment [4]. Cardiomyopathy has been claimed to occur following final DOX treatment of 0–231 days (median 23 days) and final daunorubicin treatment of 9–192 days (median 60 days). Cardiotoxicity due to DOX therapy has been seen even after 20 years [10]. In other words, its toxic effect is mostly dose and time dependent [13]. DOXinduced cardiotoxicity causes death in 50% of patients within 2 years [34].

DOX is used not only for childhood cancer patient treatment, but also for adults cancer patients [35]. So, age is an important factor in the development of cardiotoxicity of DOX [9]. For example, it is reported that patients over 65 years and under 4 years treated with DOX are more susceptible to cardiomyopathy. Children, adolescents, and the elderly treated with DOX are at high risk of developing cardiac damage. It seems that in children DOX causes some stem cells to vanish, including pluripotent, undifferentiated, and cardiac stem cells. The decreased stem cell ability in the heart by DOX results in decompensating for the decline of cardiac mass induced by the drug's treatment. However, DOX has been shown to accumulate in cardiac tissue in elderly patients, resulting in reduced blood flow in the heart [17].

Another factor associated with DOX toxicity is gender [36]. Unusually, female cancer patients treated with DOX have higher mortality vs. male cancer patients, but females develop cardiovascular disease 10 years later than males. However, after the menopause, females become more vulnerable than males at the same age [37].
