**6. Mitochondrial dysfunction induced by doxorubicin**

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

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

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

stabilization by MAPK [72].

340 Mitochondrial Diseases

release of caspase-12 activates caspase-3 [66].

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 and aging as well.

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