**2.2 The mitochondrial pathway of apoptosis (the intrinsic pathway)**

The intrinsic pathway contains the let off of cytochrome c from the mitochondria to the cytoplasm in response to cellular stress [26]. The cellular stress by the variety of factors such as DNA damage, deprivation of growth factors, defective ER, and several developmental factors activate the intrinsic pathway. In the mitochondrial pathway, the cleavage and activation of caspases begin with caspase-9, which leads to apoptosome machinery activation [19, 20]. The apoptosis proteaseactivating factor1 (APAF1) is a key molecule in the intrinsic pathway of apoptosis, besides, an important target for its pro-survival member, and scaffolding the assembled apoptosome [38]. In the Intrinsic apoptosis pathway, firstly, cytochrome c is discharged from mitochondria and enters into the cytosol, by ending with its

#### **Figure 1.**

*The caspase family proteases and apoptosis. The initial signal on the cell membrane or from the mitochondria activates the initiator caspases (caspase-2, caspase-8, and caspase-9), which act on execution caspases. The initiator caspases are produced inactively and composed of a pro-domain (containing a CARD or a death effector domain [DED]) and, a large and small subunit. On contrary, executioner caspases (caspase-3, caspase-6, and caspase-7) are activated by cleavage of the initiator caspases. Thus, dependent on initiator signaling. The active caspases including two small and large subunits.*

interaction to APAF1 [39, 40]. The cytochrome c, together with APAF1 forms the apoptosome, which leads to inactive caspase-9 monomers into active auto-process [26]. The active caspase-9, in turn, leads to activation of downstream effector caspases. Further, the mitochondria let off pro-apoptotic proteins includes apoptosis-inducing factor (AIF) and endonuclease-G [41]. Cytochrome c is only present in the mitochondrial intermembrane in healthy cells, to interact with APAF1, the apoptotic stimuli trigger the mitochondrial outer membrane (MOM) permeabilization (MOMP) [27]. Hereby, the MOMP conduce to discharge of the entire soluble proteins from mitochondrial intermembrane space to the cytosol. Additionally, rather than cytochrome c, two important pro-apoptotic proteins which are Smac and Omi, were also released meanwhile the above-mentioned procedure. These two proteins are crucial for increasing the potential of apoptosome via antagonizing the caspase inhibitor called X-linked inhibitor of apoptosis (XIAP) [26, 42]. In the lack of Smac and/or Omi, XIAP binding to caspase-9, and inhibits its catalytic activity in which also affects the caspase-3 and caspase-7 [42]. The intrinsic apoptotic pathway is regulated by the Bcl-2 family of proteins [5, 43]. The regulation of MOMP is tightly controlled by the Bcl-2 family proteins, they share BH region homology with their sequence, function, and structure. The Bcl-2 family can be sub-grouped into three categories: the pro-apoptotic proteins, Bax and Bak, play a key role for MOMP regulation; the anti-apoptotic proteins, Bcl-2, Bcl-xL, and Mcl-1, these are the blockers of MOMP; and the BH3-only proteins, Bid, Bim, Bad and Noxa, which are activators of pro-apoptotic proteins and neutralizers for Bcl2 anti-apoptotic proteins. Anti-apoptotic Bcl2 proteins control many other cellular processes such as autophagy and mitochondrial fusion [24, 44]. Bax and Bak are in charge of the loss of mitochondrial outer membrane integrity. Over their activation, they form broad oligomers, and by disturbing MOM enduing their entry into MOM. The disturbing process still unknown, but this lets off the release of entire intermembrane space proteins [45, 46]. Bax and Bak are acting on MOMP involuntarily but importantly at least one of them is needed for re-permeabilization of mitochondria. These proteins are generally being at an inactive state, and gets activated upon upstream stimuli [47–49]. The BH3-only proteins, at least two of them, Bim and active Bid activate Bax and Bak via their transient interaction [25, 50–52]. The antagonization of MOMP accrued by the Bcl2 anti-apoptotic proteins in which they bind and inhibit both Bax and Bak, also BH3-only proteins via interacting with their BH3-only domains [53]. The Bcl2 proteins are regulated both transcriptionally and posttranscriptionally. Especially, via the ubiquitin-proteasome system, the degradation of Mcl-1 involve in apoptosis due to many cellular stresses. Over DNA damage, Mcl1 ubiquitin ligase E3 (MULE) binds to Mcl1 and initiates it catalyzes [54]. Mcl1 is phosphorylated via mitogen-activated protein kinase (MAPK) upon cellular stress conditions [55]. Also, Mcl1 is degraded with growth factors stimuli such as IL3 and the deficiency of phosphoinositide 3-kinase (PI3K)-AKT signaling. This important process eases glycogen synthase kinase 3 (GSK3) from AKT inhibition. Then, GSK3 leads to phosphorylation of Mcl1 and its subsequent degradation [55, 56]. Intrinsic pathway signaling with pro-and anti-apoptotic proteins maintain many signaling processes in the important point of life and death decision. BH3-only proteins are essential upstream sensors of the mitochondrial apoptotic pathway. The regulation of several signaling pathways converge on the BH3-only proteins, and therefore, control and regulate their expression levels and activity [57, 58]. Such as the activation of Bid over cleavage by caspase-8 follow up DR ligation [59, 60]. Hereby, the Bid mediates the cross-regulation of both extrinsic and intrinsic apoptotic pathways. Also, the response to genotoxic stress can be given with Puma and Noxa, which are the direct target of p53 [61–64]. Also, the primary apoptotic factor Bim similarly can be up-regulated via forkhead transcription factor FOXO3A over

cytokine deficiency or due to ER stress to unbind the accumulated proteins [65–67]. To this end, the activity of Bad regulated via several kinases' phosphorylation including AKT, which leads to its sequestration through 14–3-3- proteins [68, 69]. Also, when Bad is released, it acts against Bcl2 anti-apoptotic proteins over growth factor deficiency and/or lacking the AKT signaling [68, 70]. It is noteworthy that MOMP is obliged the cell to death even though the caspase activation is hindered which might be the conclusion of the loss of mitochondrial function, which causes the failure of energy [26, 71]. Some cells can survive under these conditions though [26]. Nevertheless, the MOMP activation through its downstream is an important point in the regulation of caspases and might be settled. Finally, mitochondrial apoptosis can be harassed through the phosphorylation and inhibition of caspase-9 by acting downstream of MOMP [72].

#### **2.3 The extrinsic pathway of apoptosis**

The extrinsic pathway of apoptosis can be initiated through the binding of ligands to the death receptors such as tumor necrosis factor receptors (TNFRs) and Fas receptor (FasR) on the cell surface [7, 17, 73]. These receptors possess death domains at the cytosolic side, which helps in recruiting adaptor proteins such as FAS associated via death domain (FADD), and also, for some receptors, TNFRSF1A is associated via death domain (TRADD) [74, 75]. The recruitment of these adaptor proteins occurs through the interaction of death domains present in both receptors and adaptors. The death effector domains of these adaptor proteins have different upstream procaspases such as caspase-8 and caspase-10 to form a death-inducing signaling complex (DISC), which then promote activation of these caspases [17, 76]. The downstream executioner caspases such as caspase-3, caspase-6, and caspase-7 activated through their upstream caspases to execute cell death [77]. The main feature of apoptosis is the lack of systemic damage to neighboring cells during its initiation and clearance of the dead cells. This feature of apoptosis is provided by the attentive wrapping of the cell membrane around the fragmented cell by forming apoptotic bodies. The release of ATP (adenosine 5′-triphosphate) and UTP (uridine- 5′-triphosphate) from apoptotic bodies display a signal for macrophages, leads to the migration of macrophages to the apoptotic site [78]. Besides, apoptotic cells also expose the eat me signal most likely phosphatidylserine (PtdSer) on their surface, which initiates phagocytes to engulf the must-remove cells [79].
