**5.4. Apoptotic cell death induced by doxorubicin**

potentially harmful effect. However, superoxide radicals can be transformed by superoxide

lular enzymes, e.g., xanthine oxidase and microsomal NADPH-cytochrome P450 reductase that expresses in almost all cells. Mitochondrial NADH dehydrogenase, which mediates to produce ROS when DOX is present, is not present in other tissues, except cardiac tissue. This

Given more detailed knowledge regarding its structure and radical formation, DOX can be reduced at the C13 position from doxorubicinol. Although DOX can be transformed to doxorubicinone at its daunosamine sugar by acid-catalyzed hydrolysis, doxorubicinol can also undergo the same acid-catalyzed hydrolysis to form doxorubicinolone. Both can then experience protonation at C7, resulting in the formation of 7-deoxydoxorubicinone and 7-deoxydoxorubicinolone, respectively, by deletion of the sugar. After double reducing DOX, a tautomer of C7 deoxyaglycone, that is, C-7-quinone-methide, is produced. C7-quinone-

The drug can form ROS via two pathways: the first is iron dependent, and the second is redox cycling, which is catalyzed by NADPH oxidoreductases [30]. DOX has one of the paths, which produces ROS, and is mediated by iron (Fe). According to the Haber–Weiss reaction,

Oxidative stress produced by DOX relies on nitric oxide synthase (NOS) and nicotinamide adenine dinucleotide phosphate-oxidase (NOX). NOX and/or NOS can transform DOX to its semiquinone form, causing oxidative stress. When nitric oxide is produced by NOS, peroxynitrite, reactively oxidizing DNA, proteins, and lipids are produced as by-products. Moreover, two isoforms of NOS, namely endothelial NOS and inducible NOS (iNOS), have been reported to play a role in DOX's toxicity to produce RNS. Besides NOS, DOX can synthesize radicals by complexing with iron to produce hydroxyl radicals, which are also very dangerous for cells

is why DOX is highly toxic to heart tissue because it causes ROS to elevate [10].

O2

; this is called a Fenton or Haber–Weiss reaction, and the highly

. DOX can be reduced in some intracel-

O2

in existing iron. Another way is for DOX to directly inter-

, and then a hydroxyl

dismutase converting to H2

336 Mitochondrial Diseases

radical can be produced by H2

O2

**Acute DOX treatment Chronic DOX treatment** 1. Transient arrhythmias 1. More serious arrhythmias

3. Alternation of ECG 3. More severe ECG alternation 4. ROS production 4. High ROS production

6. Induction of apoptosis 6. Induction of apoptosis 7. Alteration of mitochondrial dynamics 7. Mitochondrial dysfunction

**Table 1.** Comparison of some parameters of acute and chronic doxorubicin toxicities.

5. Transient histopathological alternation 5. Permanent histopathological alternation

2. Transient hypotension 2. Hypertension

methide can connect to DNA and form free radicals [22].

the superoxide radical formed by DOX could be transformed into H2

play, resulting in a ferro (Fe2+) to ferric (Fe3+) form of abundant ROS [28].

and can have a detrimental effect on DNA, proteins, and especially lipids [20].

O2

toxic hydrogen radical can be produced from H2

Apoptosis plays a role in developmental and homeostatic mechanisms. So, uncontrolled apoptosis relies on an illness, e.g., cancer [64]. This is why apoptosis, known as programmed cell death [65], has a role in the development of cancer and cancer treatment [2]. Apoptotic pathways start as intrinsic or mitochondrial and extrinsic stimulus stimulated by the cell death receptor [64]. There are many ways to initiate the intrinsic apoptotic path, particularly nutrient deficiency, genotoxic damage induced by cytotoxic chemotherapies, and radiation [64].

Extensive research has been conducted on DOX's apoptotic pathways. The evidence supports a significant role of oxidative stress induced by DOX. The difficulties in determining DOX's apoptotic pathways are related to the drug's dosage, route [26], and duration of treatment [9]. It is almost impossible to explain a single, unique apoptotic pathway induced by DOX [26]. However, literature data have suggested that DOX has been reported to trigger apoptosis in both pathways, intrinsic or mitochondrial and extrinsic [57, 66].

The pathways are controlled under pre- and proapoptotic factors. Apoptosis can be triggered by proapoptotic factors such as Bax or Bak activation by BH3-only protein, BIM, and also BID. When Bax and Bak become oligomerized, mitochondrial outer membrane permeabilization occurs and results in releasing cytochrome-*c* to the cytosol. Then the apoptosome can be formed by cytochrome-*c* with apoptotic protease-activating factor-1 (APAF-1), resulting in a triggering caspase cascade, including caspase-3 (**Figure 4**). In contrast to proapoptotic factors, e.g., Bcl-2, Bcl-XL can prevent apoptotic pathways maintaining monomeric Bax/Bak or BH3 only proteins [64]. Caspase-8 participates in extrinsic pathways, whereas caspase-3 and -9 have a role in the intrinsic route [2].

Bax activation releases cytochrome-*c* by the mitochondrial permeability transition pore (PMT) activation, resulting in APAF-1 activation [26]. After the apoptosome complex is formed by APAF-1, cytochrome-*c*, dATP, and caspase-9, procaspase-3 can be transformed into its activated form by the apoptosome [67]. Alternatively, DOX can facilitate apoptosis through mitochondrial p53 by depolarizing MMP. Recently published data are suggests that p53 elevation by DOX treatment influences Bcl-2 decline and Bax expression [26, 67].

apoptosis, calcium homeostasis has a crucial function for cell metabolism. Several mitochondrial dehydrogenases, e.g., pyruvate dehydrogenase, isocitrate dehydrogenase, oxoglutarate dehydrogenase, and glycerol-3-phosphate dehydrogenase, are controlled by intracellular calcium concentration. As a result, the influx of calcium to mitochondria plays a central role in the regulation of cell metabolism. Therefore, any reason for preventing calcium entering mitochondria might also cause a drop-off in bioenergy production [69]. Furthermore, DOX at a low dose gives rise to a decline in the calcium storage capacity of mitochondria. The decline in mitochondria calcium storage capacity by DOX exaggerates its dosage. Eventually, the effects of DOX lead to the dissipation of MMP [15]. The other way that DOX disrupts both intracellular calcium homeostasis and mitochondrial calcium loading is via connexin 43 (Con-43). Con-43 is one of the essential gap junction proteins, and plays a role in the regulation of mitochondrial function. So, when Con-43 is blocked by a gap junction blocker it releases cytochrome-*c* and induces apoptosis. According to a recent study, DOX treatment enhanced Con-43 from cytosol to mitochondria through heat shock protein 90 and translocase of the

Mitochondrial Dysfunction Associated with Doxorubicin http://dx.doi.org/10.5772/intechopen.80284 339

MPT pore, one of the redox-sensitive proteins, has a function to regulate mitochondrial tasks [15]. It is thought that the pore consists of many proteins; however, this has been not fully understood yet [13]. So far, MPT is believed to contain VDAC [17], ANT [13], and Cyp D (also called Cyp F). The most crucial elements of the pore have been claimed to be Cyp D [17]. MPT is controlled by creatine kinase (CK), hexokinase, the Bcl-2 family, and peripheral-type benzodiazepine receptors [17]. DOX reduces mitochondrial calcium loading capacity based on triggering of the MPT pore [17]. So, oxidative stress through complex I [15], dissipation of MMP, and loss of mitochondrial calcium capacity trigger MPT, resulting in enhanced inner mitochondrial membrane permeability, and eventually augmentation of small molecules less than 1.5 kDa due to the opening of nonselective protein pores [13, 17]. Besides loss of MMP, opening the pore triggers apoptosis by releasing cytochrome-*c* and another apoptotic factor from mitochondria to cytosol and subsequent activation of caspase pathways [13]. This is why the opening of MPT initiates apoptosis by releasing cytochrome-*c* or SMAC/DIABLO. DOX prompts the opening of MPT through oxidation of thiol residues in mitochondrial proteins. The other way of initiating apoptosis by DOX is to delete GATA-4, which is a transcriptional factor encoding Bcl-XL antiapoptotic genes preventing mitochondrial function and integrity. Anthracycline also blocks AKT phosphorylation, resulting in GSK3β activation, leading GATA-4 suppression in the nucleus. DOX causes bioenergetic stress by reducing mitochondrial ATP production and damaging CK isoenzymes and AMPK [17]. DOX is shown to change Bax and Bcl-2 protein levels as well [13]. Moreover, MPT opening gives rise to mitochondriarelated osmotic swelling and structural detriment. The MPT formation is needed to clarify, so

ER and mitochondrial dysfunction have been reported to include and follow the same apoptotic pathways [67, 71]. DOX also triggers apoptosis by ER dysfunction through activation of an ER stress sensor and transcription factor 6 [21]. Moreover, a study also found that apoptosis-related ER stress by DOX is instigated to elevate Ca2+, calpain-1 protein level, and caspase-12, which is a marker of ER stress [67]. Cardiac damage mediated by DOX can be merged with lysosome dysfunction causing autophagic flux as well. DOX damages mitochon-

outer membrane 20 pathways [70].

further studies are needed to evaluate MPT structure [17].

dria by tending to accumulate in it, triggering apoptotic cell death [21].

**Figure 4.** Apoptotic cell death by doxorubicin (DOX). Cyt C: Cytochrome-*c*. Modified from Meredith et al. [22].

DOX's toxicity is mainly thought to relate to ROS enhancement and TOPII inhibition [68]. Therefore, the therapy improves ROS production as described in the previous section. Cell death is stimulated based on transforming DOX to a semiquinone radical via complex I [15]. However, the heart has cardioselective external NADH dehydrogenase, which is a kind of alternative complex I, resulting in a long DOX redox cycle [14]. When semiquinone reverses to produce DOX, oxygen converts to a superoxide anion free radical. This superoxide radical can be scavenged by GSH, creating its oxidation form, glutathione disulfide [15]. It must be remembered that cardiac tissue has less antioxidant capacity than other tissues [14]. A decline in GSH causes oxidation of thiol groups in proteins, including MPT, resulting in depolarization of MMP. Enhancing MMP gives rise to decreased ATP production, and release of proteins from mitochondria to the cytosol, such as cytochrome-*c*. So, releasing cytochrome-*c* can trigger apoptosis and/or necrosis. Eventually, this causes cell loss [15]. DOX, therefore, leads to decreased heart muscle thickness [64] due to apoptotic cell death.

DOX's toxicity on mitochondria is dose and time dependent. The mitochondrial toxicity of DOX has been observed from 2 to 13 weeks at a low dose. Moreover, mitochondria play an essential role in the regulation of calcium homeostasis. Under the standard physiological condition, there is little impact of mitochondria on calcium homeostasis. However, the mitochondrial function of calcium homeostasis is essential under pathological circumstances to decrease cytosolic calcium. Calcium efflux into mitochondria causes MMP to depolarize. DOX's effect on calcium homeostasis in mitochondria is based on inhibition of the inward calcium flux and exaggeration of the release of calcium from mitochondria by swelling of the mitochondria–calcium-dependent pathways through MPT, resulting in enhanced calcium concentration at the cytosol [15]. Cytosolic calcium concentration at 30 nM is tightly controlled by several pumps, channels, and exchangers [69]. Besides initiating its role in apoptosis, calcium homeostasis has a crucial function for cell metabolism. Several mitochondrial dehydrogenases, e.g., pyruvate dehydrogenase, isocitrate dehydrogenase, oxoglutarate dehydrogenase, and glycerol-3-phosphate dehydrogenase, are controlled by intracellular calcium concentration. As a result, the influx of calcium to mitochondria plays a central role in the regulation of cell metabolism. Therefore, any reason for preventing calcium entering mitochondria might also cause a drop-off in bioenergy production [69]. Furthermore, DOX at a low dose gives rise to a decline in the calcium storage capacity of mitochondria. The decline in mitochondria calcium storage capacity by DOX exaggerates its dosage. Eventually, the effects of DOX lead to the dissipation of MMP [15]. The other way that DOX disrupts both intracellular calcium homeostasis and mitochondrial calcium loading is via connexin 43 (Con-43). Con-43 is one of the essential gap junction proteins, and plays a role in the regulation of mitochondrial function. So, when Con-43 is blocked by a gap junction blocker it releases cytochrome-*c* and induces apoptosis. According to a recent study, DOX treatment enhanced Con-43 from cytosol to mitochondria through heat shock protein 90 and translocase of the outer membrane 20 pathways [70].

MPT pore, one of the redox-sensitive proteins, has a function to regulate mitochondrial tasks [15]. It is thought that the pore consists of many proteins; however, this has been not fully understood yet [13]. So far, MPT is believed to contain VDAC [17], ANT [13], and Cyp D (also called Cyp F). The most crucial elements of the pore have been claimed to be Cyp D [17]. MPT is controlled by creatine kinase (CK), hexokinase, the Bcl-2 family, and peripheral-type benzodiazepine receptors [17]. DOX reduces mitochondrial calcium loading capacity based on triggering of the MPT pore [17]. So, oxidative stress through complex I [15], dissipation of MMP, and loss of mitochondrial calcium capacity trigger MPT, resulting in enhanced inner mitochondrial membrane permeability, and eventually augmentation of small molecules less than 1.5 kDa due to the opening of nonselective protein pores [13, 17]. Besides loss of MMP, opening the pore triggers apoptosis by releasing cytochrome-*c* and another apoptotic factor from mitochondria to cytosol and subsequent activation of caspase pathways [13]. This is why the opening of MPT initiates apoptosis by releasing cytochrome-*c* or SMAC/DIABLO. DOX prompts the opening of MPT through oxidation of thiol residues in mitochondrial proteins. The other way of initiating apoptosis by DOX is to delete GATA-4, which is a transcriptional factor encoding Bcl-XL antiapoptotic genes preventing mitochondrial function and integrity. Anthracycline also blocks AKT phosphorylation, resulting in GSK3β activation, leading GATA-4 suppression in the nucleus. DOX causes bioenergetic stress by reducing mitochondrial ATP production and damaging CK isoenzymes and AMPK [17]. DOX is shown to change Bax and Bcl-2 protein levels as well [13]. Moreover, MPT opening gives rise to mitochondriarelated osmotic swelling and structural detriment. The MPT formation is needed to clarify, so further studies are needed to evaluate MPT structure [17].

DOX's toxicity is mainly thought to relate to ROS enhancement and TOPII inhibition [68]. Therefore, the therapy improves ROS production as described in the previous section. Cell death is stimulated based on transforming DOX to a semiquinone radical via complex I [15]. However, the heart has cardioselective external NADH dehydrogenase, which is a kind of alternative complex I, resulting in a long DOX redox cycle [14]. When semiquinone reverses to produce DOX, oxygen converts to a superoxide anion free radical. This superoxide radical can be scavenged by GSH, creating its oxidation form, glutathione disulfide [15]. It must be remembered that cardiac tissue has less antioxidant capacity than other tissues [14]. A decline in GSH causes oxidation of thiol groups in proteins, including MPT, resulting in depolarization of MMP. Enhancing MMP gives rise to decreased ATP production, and release of proteins from mitochondria to the cytosol, such as cytochrome-*c*. So, releasing cytochrome-*c* can trigger apoptosis and/or necrosis. Eventually, this causes cell loss [15]. DOX, therefore, leads to

**Figure 4.** Apoptotic cell death by doxorubicin (DOX). Cyt C: Cytochrome-*c*. Modified from Meredith et al. [22].

DOX's toxicity on mitochondria is dose and time dependent. The mitochondrial toxicity of DOX has been observed from 2 to 13 weeks at a low dose. Moreover, mitochondria play an essential role in the regulation of calcium homeostasis. Under the standard physiological condition, there is little impact of mitochondria on calcium homeostasis. However, the mitochondrial function of calcium homeostasis is essential under pathological circumstances to decrease cytosolic calcium. Calcium efflux into mitochondria causes MMP to depolarize. DOX's effect on calcium homeostasis in mitochondria is based on inhibition of the inward calcium flux and exaggeration of the release of calcium from mitochondria by swelling of the mitochondria–calcium-dependent pathways through MPT, resulting in enhanced calcium concentration at the cytosol [15]. Cytosolic calcium concentration at 30 nM is tightly controlled by several pumps, channels, and exchangers [69]. Besides initiating its role in

decreased heart muscle thickness [64] due to apoptotic cell death.

338 Mitochondrial Diseases

ER and mitochondrial dysfunction have been reported to include and follow the same apoptotic pathways [67, 71]. DOX also triggers apoptosis by ER dysfunction through activation of an ER stress sensor and transcription factor 6 [21]. Moreover, a study also found that apoptosis-related ER stress by DOX is instigated to elevate Ca2+, calpain-1 protein level, and caspase-12, which is a marker of ER stress [67]. Cardiac damage mediated by DOX can be merged with lysosome dysfunction causing autophagic flux as well. DOX damages mitochondria by tending to accumulate in it, triggering apoptotic cell death [21].

Extrinsic pathways involve death receptors, their ligand interaction, e.g., Fas/FasL, and then caspase-8 activation [57]. DOX also uses the extrinsic pathways for instigating apoptosis by elevation of Fas protein levels, caspase-8, and BID [67] (**Figure 4**). Even so, DOX's leading approach to initiate apoptosis is through intrinsic, called mitochondrial, pathways. The outer membrane of mitochondria has a central role in the natural apoptotic route because it has pro- and preapoptotic factors. The elevation of ROS and depolarization of MMP by DOX release proapoptotic factors to the cytosol, e.g., cytochrome-*c*. p38, p53, Bax, and caspase-3 have also been suggested to participate in the induction of apoptosis. p53 enhances the permeability of the outer membrane to release proapoptotic factors, such as Bax [57]. DOX has been reported to increase p53 in the nucleus and mitochondria from the heart. So, p53 localization is thought to associate with mtDNA. However, there is limited knowledge available of nuclear and mitochondrial p53 localization by DOX in cardiac tissue. DOX has been suggested to elevate 8-hydroxydeoxyguanosine (8-OHdG) and p53 levels in mitochondria within 3 and 24 h. Cytochrome-*c* release is an assessment of cytosolic/mitochondrial cytochrome-*c*. DOX enhances the ratio of heart tissue by around 35%. It can trigger apoptosis through p53 stabilization by MAPK [72].

in stimulation of chromatin condensation and DNA breakage, eventually triggering apoptosis by caspase-independent pathways. The other function of AIF is to repair and mature mitochondrial complex I and peroxide scavenging activities. Elevation of cytosolic AIF leads to release of cytochrome-*c*, resulting from depolarization of MMP. Why the molecular mechanism of DOX toxicity is so crucial is based on its effective utilization of therapy against cancer. This is why finding an effective therapy to counteract its toxicity, especially of the heart, will give hope to cancer patients treated with DOX to overcome nondesired effects. The other mechanism of cell death mediated with DOX decreases GATA, controlling apoptosis through

Mitochondrial Dysfunction Associated with Doxorubicin http://dx.doi.org/10.5772/intechopen.80284 341

Mitochondria have a role in regulating cell death or survival under cell stress or damage. The organelle has its own genome encoding 37 genes, of which 13 are complex I, III, IV; complex II is encoded by nuclear DNA [22]. So, mitochondrial dysfunction is associated with disease

Besides its nuclear effect, DOX has been reported to cause mitochondrial dysfunction, energy stress via disruption of the ETC [4]. It is well recognized that mitochondrial bioenergetics mechanism disruption has been thought to play an essential role in the development of the drug's toxicity, especially its cardiotoxicity. Adequate ATP production is not just necessary to maintain contractile function, it is also crucial for protein synthesis, the protein quality control function of ER, cytoskeletal function, and clearing cellular waste from lysosomes as well [4]. Moreover, DOX's mitochondrial effect is shown to change ultrastructure, swelling, and oxidative capacity. Furthermore, DOX tends to accumulate in nuclei and mitochondria vs. plasma [43]. All this is needed to explain why or how DOX selectively targets mitochondria in noncancerous tissue rather than cancerous tissue. One reason is that cancer has been reported to alter a cell's metabolic activation. A healthy cell produces energy by oxidative phosphorylation in mitochondria. However, a cancer cell synthesizes its energy by the glycolytic pathway, known as the Warburg effect. Enhancing glycolytic activity could be multifactorial, relying on mtDNA damage, oxidative phosphorylation defect, mitochondrial dysfunction, etc. [78]. Another reason could be that DOX is more toxic to mitochondria in noncancerous cells than in cancerous cells. Moreover, DOX could alter mitochondrial function in noncancerous and

The acute toxic effect of DOX on mitochondria has been reported to rely on its dose, especially redox cycling and ETC blocking. A low concentration of DOX treatment has been reported to have minimal alternation to ATP production and MMP, resulting from enhancing hydroxyl

has been emphasized, redox cycling is the primary process to augment ROS production; ETC blocking is the primary source of ROS manufacture at densities higher than 160 μM. Until it reaches a threshold, which means 480 μM, mitochondrial toxicity is progressively enhanced.

, and oxygen consumption. Although up to 160 μM DOX concentration

antiapoptotic Bcl-X gene activation [77].

and aging as well.

radical (\*OH), H2

O2

**6. Mitochondrial dysfunction induced by doxorubicin**

cancerous cells, resulting in different apoptotic pathways [79].

**6.1. The acute mitochondrial toxic effect of doxorubicin**

The MAPK family has extracellular signal-regulated kinases (ERK), p38 MAPK, and JNK [73]. While ERK1/2 predominantly operates cell proliferation, JNK and p38 participate in cell death pathways. DOX has been shown to kill prostate cancer cells by phosphorylation of p38 and JNK [74]. One of the MAPKs is p38, which has a pivotal role in cell growth, apoptosis, and inflammation. The apoptotic role of p38 depends on cell type, stimuli, or isoform activation of p38, which has four isoforms: p38α, β, γ, and δ. One study showed that DOX triggers apoptosis at the MCF-7 breast cancer cell line by elevation of caspase-3 and caspase-9 during 24 h of treatment [65]. So, p38 is one of the intrinsic pathway activators dependent on cellular stress, mitochondrial dysfunction, and caspase activation [57]. ERK1/2 probably has a role in the activation of caspase-3, Bax, p53, and cytochrome-*c* release. Moreover, ERK1/2 could contribute external apoptotic pathways at the caspase-8 level [75]. ERK1/2 could also phosphorylate p53. So, DOX activates apoptosis by the p53-dependent activation of caspases-2, -3, -8, -9, and -12 [66]. The release of caspase-12 activates caspase-3 [66].

Through extrinsic (receptor-mediated) or intrinsic (mitochondrial) pathways. Both pathways have a role in the trigger of apoptosis as upstream (initiator) caspase, e.g., caspase-8 and -9, and downstream (effector) caspase, e.g., caspase-3, -6 and -7 [76]. When MMP is depolarized and opened, mitochondrial apoptotic factors are released such as cytochrome-*c* and AIF to the cytosol [73]. Cytochrome-*c* can contain an apoptosome formation with APAF-1, caspase-9. Caspase-3 can be activated from both pathways [73]. The human fibroblast cell was used in one of the previous studies and reported that DOX at 3 μM concentration causes apoptosis through caspase-3, -7, and -9 by ROS [76]. DOX has been said to stimulate apoptosis via caspase-3-dependent pathways. The bcl-2 protein family is shown to play a role in apoptosis in cardiomyocytes as expected. Also, Bcl-2 and Bax can affect the MPT pore [68] (**Figure 4**).

DOX also stimulates apoptosis by an AIF. There are three sides of AIF: a NAD binding, FAD binding, and C-terminal. AIF is located at the intermembrane space or weakly binded to inner mitochondrial membrane and exhibits NADH oxidase activity. AIF can be released to the cytosol via PMT and translocate to the nucleus by poly (ADP-ribose) polymerase-1, resulting in stimulation of chromatin condensation and DNA breakage, eventually triggering apoptosis by caspase-independent pathways. The other function of AIF is to repair and mature mitochondrial complex I and peroxide scavenging activities. Elevation of cytosolic AIF leads to release of cytochrome-*c*, resulting from depolarization of MMP. Why the molecular mechanism of DOX toxicity is so crucial is based on its effective utilization of therapy against cancer. This is why finding an effective therapy to counteract its toxicity, especially of the heart, will give hope to cancer patients treated with DOX to overcome nondesired effects. The other mechanism of cell death mediated with DOX decreases GATA, controlling apoptosis through antiapoptotic Bcl-X gene activation [77].
