**2.1 The extrinsic death receptor pathway**

This pathway begins when ligand binds to the death receptor, for example, with the attachment of extracellular ligands, like tumor necrosis factor (TNF), Fas ligand (Fas-L), TNF-related apoptosis-inducing ligand (TRAIL) to the extracellular domain of transmembrane receptors. There are six death receptors (TNFR1, Fas, DR3, DR4 [TRAILR1], DR5 [TRAILR2], and DR6) that have been identified in mammalian cells (**Table 1**). While signaling through TNFR1 and DR3 is proinflammatory in nature, signaling via other death receptors is predominantly pro-apoptotic. The death receptors bind via their intracellular death domain with adapter proteins such as Fas-associated death domain (FADD) and TNF receptorassociated death domain (TRADD). These adaptor proteins, called the Death effector Domain (DED) have another protein interaction domain [12]. These sequential steps leads to the formation of a death inducing signaling complex (DISC), that leads to auto-catalytic activation of procaspase-8 (**Figure 1**). Active caspase-8 activates effector/executioner caspases (*c*ysteine-dependent *as*partylspecific prote*ases*), which cause cell death by damaging the nucleus and other intracellular structures. Once caspase-8 is activated, the execution phase of apoptosis is triggered and activated. This type of apoptosis, which is death receptormediated apoptosis, can be inhibited by a protein called c-FLIP. c-FLIP bind to FADD and caspase-8, and turns them ineffective. A protein called Toso, has been shown to block Fas-induced apoptosis in T cells. This may be due to inhibition of caspase-8. The death receptors belong to the tumor necrosis factor (TNF) receptor gene superfamily. TNF family contains cysteine-rich extracellular domains and a cytoplasmic domain of about 80 amino acids called the "death domain". This death domain plays a critical role in transmitting the death signal from the cell surface to the intracellular signaling pathways [13–15].


#### **Table 1.**

*Death receptors and their cognate ligands.*

**Figure 1.** *Key components of the intrinsic and extrinsic pathways of apoptosis.*

#### **2.2 The intrinsic mitochondrial apoptosis pathway**

Intrinsic apoptosis is a form of regulated cell death that is activated in response to a variety of stimulus such as dearth of growth factors, DNA damage, endoplasmic reticulum (ER) stress, and overproduction of reactive oxygen species (ROS). The intrinsic pathway is affected by members of the Bcl-2 family proteins such as Bcl-2 and Bax. These proteins usually are bound to the outer mitochondrial membrane and act as antiapoptotic and pro-apoptotic proteins respectively [6]. In the intrinsic pathway the main consequence of proapoptotic signaling is mitochondrial membrane perturbation and release of cytochrome 'c' in the cytoplasm. Once released in the cytosol, cytochrome 'c' forms a complex with apoptotic protease activating factor 1 (APAF1) and inactive form of pro-caspase 9, commonly known as apoptosome (**Figure 1**). This complex hydrolyzes adenosine triphosphate (ATP) to cleave and activate caspase 9. The initiator caspase 9 then cleaves and activates the executioner caspase 3, caspase 6, and caspase 7, resulting in cell apoptosis. The antiapoptotic proteins Bcl-2 and Bcl-XL inhibit cytochrome 'c' release [8]. The Bcl-2 group of proteins share one to four Bcl2 homology (BH) domains (i.e., BH1, BH2, BH3, and BH4) [16]. Bax and Bak of the Bcl-2 family form pores across the outer mitochondrial membrane. Besides this, Bax also continuously cycles between the outer mitochondrial membrane (OMM) and the cytosol, and exhibits a quiescent inactive dimeric conformation [17]. Bak constitutively resides at the OMM, where it inserts within hydrophobic C-terminal of voltage dependent anion channel 2 (VDAC2). Upon apoptosis induction, mitochondrial pools of Bax and Bak undergo direct or indirect activation by pro-apoptotic proteins [16].

The BH3-only proteins are BCL2 binding component 3 or p53, Bcl-2-like 11 (BIM) [18]. Some internal stimuli cause an increase in mitochondrial membrane permeability and these stimulus send the signal to the mitochondria, that form mitochondrial outer membrane permeabilization (MOMP). MOMP is most commonly mediated via a variety of protein membrane and protein–protein interactions of the B-cell lymphoma 2 protein (Bcl-2) family [19–21].

Both extrinsic and intrinsic pathways terminate at the execution phase in the final stage of apoptosis. Activated execution caspases further activates cytoplasmic *Insights into the Role of Defective Apoptosis in Cancer Pathogenesis and Therapy DOI: http://dx.doi.org/10.5772/intechopen.97536*

endonucleases, proteases that degrade nuclear and cytoplasmic proteins respectively. The executioner caspases are caspase-3, caspase-6, and caspase-7 that cleave substrates like cytokeratins, PARP, and others that results in morphological and biochemical changes in apoptotic cells. The most important of the executioner caspases is caspase-3 and is activated by any of the initiator caspases (caspase-8, caspase-9, or caspase-10). Caspase-3 induces cytoskeleton reorganization and disintegration of the cell into apoptotic bodies [22].

#### **2.3 Perforin/granzyme pathway**

The granzymes belong to a family of serine proteases contained in cytotoxic granules of innate and adaptive immune killer cells. The key function of these enzymes is eliminating viruses and tumor cells. They also regulate immune cells and inflammation by controlling the survival of lymphocytes. Granzymes are expressed by three gene clusters. Granzyme A and granzyme B are the most abundant granzymes. Granzyme-mediated apoptosis is mainly used by lymphocytes to destroy virus infected cells. It is a kind of type IV hypersensitivity where sensitized CD8+ cells kill antigen-bearing cells. The FasL/FasR interaction is the predominant method of CTL-induced apoptosis. They also involve pathways that include secretion of the transmembrane pore-forming molecule perforin with a subsequent release of cytoplasmic granules that have serine proteases as well as granzyme B, through the pore and into the target cell [23]. Granzyme B cleaves proteins at aspartate residues and can activate pro-caspase-10. It can also cleave factors like ICAD (Inhibitor of Caspase Activated DNAse). Granzyme B can amplify the death signal by inducing cytochrome c release. It can also activate caspase-3 directly [24].

Therefore, in these types of pathways, signaling pathways that are upstream are bypassed and execution phase of apoptosis is directly influenced. It is suggested that both the mitochondrial pathway and direct activation of caspase-3 are critical for granzyme B induced killing activates both mitochondrial pathway and caspase-3 activation [24, 25]. The immune cells that express highly variable and regulated patterns of granzymes include natural killer (NK) cells, cytotoxic CD4 and CD8 T cells, and regulatory T cells (Tregs). The granules contain perforin, to deliver the granzymes into the target cell. When cytotoxic T lymphocytes (CTLs) and NK cells form an immune synapse with a target cell for its elimination, cytotoxic granules join immune synapse where its membrane fuses with the killer cell membrane. This action results in the release of the granule contents into the synaptic cleft. The granzymes are then initiate distinct pathways of programmed cell death [8, 26, 27].

#### **3. Bio markers of apoptosis**

A biomarker is measured and evaluated to indicate normal or diseased biological processes. It has the potential to enhance translational progress and accelerate drug development. They allow monitoring of drug efficacy and also help in preclinical drug evaluation. It also allows early detection of toxicity during drug evaluation. The fragmentated DNA on agarose gel is a usual marker to detect apoptosis. The poly (ADP- ribose) polymerase (PARP) cleaved form, observed in cells undergoing apoptosis. Other markers are cytokeratin-18 cleavage by caspase 3. It may be detected by using the antibody M30. Cleavage of various caspases can be detected using flow cytometry during apoptosis [28, 29]. The widely used biomarkers of apoptosis, their methods of analysis and the specimen needed for analysis are discussed in **Table 2**. However, the use of these biomarkers as a tool to predict the occurrence of apoptosis in the pathogenesis of different diseases states warrants further investigation.


#### **Table 2.**

*Biomarkers of apoptosis.*
