**4. Extrinsic and intrinsic apoptotic pathways**

70 Apoptosis and Medicine

rather, it refers to changes subsequent to any type of death. They prefer to use the term oncosis to describe a nonprogrammed or accidental type of cell death characterized by swelling [10]. Moreover, a new modality of cell death has recently emerged, termed

Apoptosis can be divided into three stages: initiation, integration/decision and execution/degradation [12]. The initiation phase mainly depends upon cell type and apoptotic stimulus. The integration/decision phase consists of the activation of proteases, nucleases and other effector molecules. The execution/degradation phase involves morphological and biochemical changes that are common to all apoptotic mechanisms, regardless of the stimulus that initiated the process. The classical apoptosis is defined as a type of programmed cell death characterized by the activation of zymogens known as caspases, which are **c**ystein-dependent **asp**artate-directed proteases. Both caspasedependent and caspase-independent cell death mechanisms share multiple characteristics, such as mitochondrial membrane permeabilization (MMP), DNA fragmentation, etc. Hence, (MMP) can commit cells to die even in the absence of caspase activation, by releasing factors

Caspases are the effector molecules of apoptosis in mammals. They were first discovered mediating programmed cell death during development in the nematode *Caenorhabditis elegans* [13]. The caspase family is characterized by their specificity for cleaving substrates after aspartic acid residues and for containing cystein in their active centre [14]. There are about fourteen caspases that used to be classified into two families: one involved in inflammation processes and the other taking part in apoptosis. However, some of the nonapoptotic caspases have some apoptotic roles, and conversely, some non-apoptotic caspases can induce pyroptosis [15]. They are synthesized as inactive precursors or zymogens (procaspases) that need to be proteolytically processed to become active. Once a caspase is activated, it can cleave another caspase creating thus an expansive hierarchical activating cascade that serves to amplify the apoptotic signal. Caspase activation is a complex and tightly regulated process. Within the apoptotic group, there are two types of caspases: upstream initiator or apical caspases and downstream effector or executioner caspases. The initiator group consists of caspases -2, -8, -9 and -10 and the effector group consists of -3, -6 and -7 caspases. Apical but not executioner caspases have long prodomains. Caspase-9 is considered the initiator of the mitochondrial pathway and caspase-8 is regarded as the originator of the dead receptor-mediated apoptotic pathway. Effector caspases carry out apoptotic programmes through direct processing of a variety of cellular substrates. The proteolytic cleavage of such substrates brings about a whole plethora of effects within the cell: disassembling of cytoskeleton, cytoplasm scaffolding, nuclear fragmentation as a consequence of laminin degradation, activation of endonucleases that cleave chromatin, inactivation of DNA damage repair proteins (PARP1, Rad9, etc.), phagocyte signalling, loss

**necroptosis** to indicate a regulated form of necrosis [11].

such as apoptosis inducing-factor (AIF) or endonuclease G (endo G).

**3. Caspases: Executioners of the apoptotic process** 

of cell-to-cell contact, etc. [16-18]

There are at least two main well characterized apoptotic routes: the death receptor or extrinsic pathway and the mitochondrial or intrinsic pathway. The extrinsic pathway plays a major role in tissue homeostasis and responds to external cues, coming especially from the immune system, whereas the intrinsic pathway is triggered as a response to various internal insults such as DNA damage, cytosolic calcium overload, starvation, oxidative stress, radiation, cytotoxic agents, etc., and involves mitochondrial destabilization [23,24].

The death receptor pathway is initiated by extracellular stimuli that are recognized by a subgroup of the tumour necrosis factor receptor (TNF-R) family named dead receptors (Fas/CD95/APO-1, TNFR1, TRAIL R1/DR4 and TRAIL R2/DR5). Upon binding of their ligands (FAS, TNFα and TRAIL) these receptors become activated and interact via their death domain with the protein motif **F**as-**a**ssociated **d**eath **d**omain (FADD) in adapter proteins, forming the **D**eath **i**nducing **s**ignalling **c**omplex (DISC), which binds to the prodomain of the initiator caspase-8 [25]. Thus, caspase-8 is activated by dimerization,

which leads to autocatalyisis and consequently activation of executioner caspases -3, -6 and - 7 [26].

Cell Death and Cancer, Novel Therapeutic Strategies 73

fact, TNF-R1 and Fas (CD95/APO-1) are involved in apoptotic processes [30]. Death receptors are stimulated by death ligands: TNF; Fas ligand (FasL) and **T**NF-**r**elated **a**poptosis-**i**nducing **l**igand (TRAIL). Death receptors contain an intracellular globular interaction domain called death domain (DD). Upon ligand binding, death receptors aggregate forming trimers. As a consequence of this aggregation, they recruit adaptors such as FADD, which interacts with caspase-8 by virtue of its death effector domain (DED), forming the multi-protein complex known as DISC. However, in some instances, death receptors can oligomerize in the abscence of ligand binding. For example, they can be activated by UV radiation [31]. Fas and TNF-R1 can also recruit **R**IP (receptor-interacting protein) -**a**ssociated **I**ch-1/CED homologous [ICE (interleukin-1β-converting enzyme)/CED-3 (cell-death determining 3) homologue 1] protein with **d**eath **d**omain (RAIDD) which activates caspase-2 through a **c**aspase **a**ctivation and **r**ecruitment **d**omain (CARD). In addition, death receptors can participate in caspase-independent mechanisms. For instance, TNF-R1 can activate Extracellular signal-regulated kinase 2 (Erk2) through the **m**itogenactivated kinase **a**ctivating **d**eath **d**omain (MADD) protein [32]. Fas can bind Death-domain associated protein (Daxx) and activate c-Jun amino-terminal kinase (JNK) [33]. **T**NF-**r**eceptor **a**ssociated **d**eath **d**omain (TRADD) and RIP can activate Nuclear factor kappa-light-chainenhancer of activated B cells (NFkB), triggering a form of regulated necrosis named

MMP, a crucial event of the intrinsic mitochondrial apoptotic pathway, is considered a "point of no return" in the sequence of events leading to apoptosis. This phenomenon is associated with mitochondrial membrane potential loss (∆Ψm) that occurs as a result of assymetrical distribution of protons on both sides of the inner mitochondrial membrane. This irreversible process can take place before, during or after MOMP. The pore formation caused by Bcl-2 proteins induces MOMP, which leads to ∆Ψm dissipation, inhibition of ATP synthesis and ∆Ψm-dependent transport activities. Consequently, the respiratory change ceases, causing reactive oxygen species (ROS) generation and release of proteins confined within the inner mitochondrial space [35]. The contribution of the inner mitochondrial permeabilization, however, is controversial. MOMP can also result from the phenomenon called mitochondrial permeability transition that implies the opening of a non-selective pore in the inner mitochondrial membrane known as the mitochondrial permeability transition

*Bax/Bak pore formation*. The first proposed mechanism of MOMP is mediated by the Bcl-2 family of proteins that directly act on the outer mitochondrial membrane. This family consists of about 17 members, some of which are proapoptotic and others antiapoptotic. The antiapoptotic members (Bcl-2, Bcl-xL, Bcl-w, Mcl-1, A1, Bcl-B, etc.) contain 3-4 BH domains (Bcl-2 homology regions: BH-1, BH-2, BH-3 and BH-4) and are named "Bcl-2 like" proteins.

**necroptosis** [34].

pore complex [12].

**6.1. Mechanisms of MOMP** 

**6. Role of mitochondria in cell death** 

The intrinsic mitochondrial pathway is characterized by the action of B-cell lymphoma (Bcl-2) proteins. This family consists of proapoptotic and antiapoptotic proteins. The proapoptotic members promote mitochondrial outer membrane permeabilization (MOMP) and the antiapoptotic members counteract this action, so that the balance between these two groups of proteins determines the final outcome [27,28]. If the balance is in favour of the proapoptotic members, the outer mitochondrial membrane is permeabilized through pore formation and cytchrome c and other proteins such as Smac/DIABLO and Omi/HtrA2 are released to the cytosol. Then, cytochrome c binds to the adaptor protein Apaf-1 and dATP, forming the apoptosome, a catalytic complex that activates caspase-9 which in turn activates the executioner caspases.

The extrinsic and intrinsic pathways are interconnected through Bid. In some cases, when DISC formation is low, caspase-8 activation can induce MOMP through Bid cleavage, which translocates to the mitochondria and induces cytchrome c release, apoptosome formation and engagement of the caspase cascade [29].

**Figure 3.** The extrinsic and intrinsic apoptotic pathways.
