**2. The role of apoptosis in pathogenesis and treatment of cancer**

#### **2.1 Overview of apoptosis**

The term "apoptosis" is derived from Greek, meaning "dropping off" or "falling off" as leaves from a tree, was first used in 1972 to describe a morphologically distinct form of cell death. Apoptosis also known as programmed cell death is a highly regulated energy-dependent process that occurs normally during development and aging. It plays an important role as a homeostatic mechanism to maintain cell populations in the tissue of multicellular organisms. In addition to its importance in biological process, defects in apoptosis mechanism has been implicated in the pathophysiology of diseases including cancer [13, 14]. There are many factors, mostly proteins, involved in the activation and regulation of apoptotic mechanism. This highly complicated and regulated process involves an energy-dependent cascade of molecular events and includes the mitochondria-dependent (intrinsic) and death receptor-dependent (extrinsic) pathway. Caspases play a vital role in initiation and execution of both intrinsic and extrinsic pathways which is mediated through the cleavage of hundreds of proteins essential for normal cellular function [15].

#### *2.1.1 Caspases: key apoptotic proteins*

Caspases are a family of cysteine protease enzymes that are able to selectively cleave proteins at aspartic acid residues using the sulfur atom of cysteine in their catalytic site, hence, named as cysteine-aspartic proteases or caspases. They play an essential role in maintaining homeostasis through regulating cell death and inflammation. Caspases have been generally categorized by their known functions in apoptosis (caspase-2, -3, -6, -7, - 8, -9 and -10) and in inflammation (caspase-1, -4, -5 and -12) in human. Caspases involved in apoptotic cell death have been then subgrouped based on their position and mechanism of action in apoptotic signaling cascades and are either initiator caspases (caspase-8, -9 and -10) or executioner caspases (caspase-3, -6, and -7) in apoptotic pathway. Therefore, caspases as a conserved family of cysteine proteases, which are essential in initiation and execution of intrinsic and extrinsic pathways, are the main emphasis of apoptosis studies [16–18].

Caspases are initially synthesized as an inactive monomeric proenzyme, named zymogens or procaspases, containing a large subunit, small subunit, and prodomain that is only activated through proteolytic cleavage and dimerization following an appropriate stimulus. Therefore, this post-translational level of control provides rapid and tight regulation of the caspase enzyme activities [19, 20]. Initiator caspases have prodomains containing one of the two specific protein– protein interaction domain including caspase recruitment domain (CARD) and death effector domain (DED) that promote caspase dimerization through binding to adapter proteins. Two examples of activating multiprotein complexes include death-inducing signaling complex (DISC) and the apoptosome, which are formed during extrinsic and intrinsic pathway of apoptosis, respectively [19].

Once properly assembled into dimers, pro-caspases undergo cleavage by autocatalysis resulting in the removal of pro-domain and cleavage at the linker region between the large and small subunit resulting in the heterotetramer formation and provides the active-site loops to get a proper conformation for enzymatic activity [17, 19].

Although, initiator caspases are capable of autocatalytic cleavage and activation, effector caspases are cleaved by initiator caspases resulting in the formation of active heterotetramer. Each active caspase is a tetramer consists of two identical big subunits and two identical small subunits. Accordingly, activation of apoptotic caspases leads to the inactivation or activation of substrates, and therefore initiation of a protease cascade events in the apoptotic signaling pathway resulting in rapid cell death. Activated caspases trigger cytoplasmic endonuclease, cleave many vital cellular proteins and break up the nuclear scaffold and cytoskeleton as well as activate DNase, which further degrade nuclear DNA into 180 to 200 base pair. Collectively, caspase activity results in various morphological and biochemical changes in apoptotic cells [19, 21, 22].

#### *2.1.2 Morphological changes in apoptosis*

Apoptotic cells are differentiated from healthy and necrotic cells based on certain cellular morphological changes. Characteristic features of apoptosis in the nucleus are chromatin condensation and nuclear fragmentation which are accompanied by cell shrinkage, membrane blebbing and formation of apoptotic bodies in the final stage of apoptosis which are rapidly engulfed by phagocytosis that avoids an inflammatory response in surrounding tissues [23–25]. The shrinkage of the cell is one of the most common morphological changes in apoptotic cell death resulted from the extreme alteration in intracellular water. Intracellular water plays a critical role in apoptotic and necrotic cell death. Although necrotic cells absorb the water resulting in enlarging the size and finally burst, apoptotic cells lose water leads to reduction in cellular volume. Therefore, the apoptotic cells become smaller in size, the cytoplasm is dense and the organelles are more tightly packed. Consequently, due to the occurrence of cell shrinkage, the cell will lose its contact

with neighboring cells, or the extracellular matrix and acquire more rounded morphology. Although the plasma membrane is intact during the entire process, at later stage of apoptosis, loss of membrane integrity and formation of the blebs at the cell surface due to the separation of the plasma membrane from cytoskeleton occur in apoptotic cells [26–28].

#### *2.1.3 Biochemical changes in apoptosis*

Apart from structural alterations, several biochemical changes also play key events in apoptosis. Apoptotic cells generally display major types of biochemical modifications such as caspase activation, protein and DNA cleavage, and plasma membrane alterations, which lead to phagocytic recognition [13]. Disruption of plasma membrane asymmetry is a common feature of apoptotic cells, independent of the form of apoptotic stimulus. The maintenance of lipid asymmetry of plasma membrane is regulated through transporters named flippases and floppases. In addition, the activated scramblase enzymes have an important role in the loss of lipid asymmetry and enhanced phosphatidylserine (PS) exposure to the outer leaflet of plasma membrane [13, 29].

Therefore, in a healthy cell, PS is limited to the inner layer of the plasma membrane. However, during apoptosis, effector caspases cleave and activate scramblase, as well as cleave and inactivate flippase, responsible for transmitting PS from the outer to the inner leaflet that lead to externalization of PS. Therefore, phosphatidylserine, which is normally localized in the inner membrane layer of cells is flipped out and externalized on the outer layer of the plasma membrane. This PS externalization not only is the indicator of loss of membrane asymmetry during apoptosis, but also allows early recognition by phagocytes and prevents the release of proinflammatory cellular components [29–31].

#### *2.1.4 Pathway of apoptosis*

As mentioned earlier, the mechanism of apoptosis involves an energydependent cascade of molecular events. Apoptotic cell death machinery includes the mitochondria-dependent (intrinsic) pathway and death receptor-dependent (extrinsic) pathway. The intrinsic pathway arises from intracellular signals like cellular stress and DNA damage and relies on the release of proteins from the intermembrane space of mitochondria. However, the extrinsic pathway is activated through the binding of extracellular ligands to death receptors at the cell surface that trigger the multiprotein complex formation known as death-inducing signaling complex (DISC). These two mitochondria- and death receptor-mediated pathways are interconnected and the molecules in one pathway can affect another pathway [32, 33].

#### *2.1.5 The intrinsic mitochondrial pathway*

As its name implies, the intrinsic pathway is activated in response to internal stimuli such as hypoxia, severe DNA damage and oxidative stress and mitochondria play a critical role throughout this apoptosis signaling pathway [34, 35]. The intrinsic pathway is mainly controlled by the members of Bcl-2 family proteins, which regulate the permeabilization of mitochondrial outer membrane (MOM) and are structurally and functionally classified into three groups. BH3-only proteins, like Bim and Bik, that sense cellular stress and directly or indirectly activate the executioner proteins, like Bax, Bak, Bid, that are able to oligomerize in

#### *The Role of Apoptosis as a Double-Edge Sword in Cancer DOI: http://dx.doi.org/10.5772/intechopen.97844*

and permeabilize the MOM. The oligomerization of these pro-apoptotic proteins leads to component release form the intermembrane space to the cytoplasm and activation of effector caspases of apoptosis. The first two groups are known as the pro-apoptotic proteins of Bcl-2 family. The third group is the anti-apoptotic proteins, like Bcl-2 and Bcl-xL that hinder the overall process by inhibiting proapoptotic proteins. However, not the absolute quantity but rather the relative levels and balance between the pro- and anti-apoptotic proteins regulates whether the apoptosis event would be initiated. Although the excess of pro-apoptotic proteins makes the cells sensitive to apoptosis, excess of anti-apoptotic proteins makes the cells resistant and prevents the occurrence of apoptosis [36–38]. However, in the presence of apoptotic stimuli, the death signal is sensed initially by the BH3-only protein, which then interacts with the downstream mediators of apoptosis such as Bax. As the intrinsic mitochondrial pathway is initiated, Bax is translocated from cytosol to the outer mitochondrial membrane. The assembly of Bax proteins in mitochondrial outer membrane results in protein-lined channels or pore formation and intensely increase its permeability that cause a dramatic loss of electrical potential in mitochondria and cytochrome c release to cytoplasm. Subsequently, released cytochrome c binds to APAF-1 to facilitate the formation of the apoptosome, a wheel shaped heptametrical complex, which can then recruit and activate pro-caspase-9. Consequently, caspase-9 activates effector caspases (caspase-3/-7) that eventually lead to apoptosis (**Figure 1**) [39–41].

### *2.1.6 The extrinsic death receptor pathway*

The extrinsic pathway is activated through the interactions between the transmembrane death receptors of the tumor necrosis factor (TNF) superfamily and their related ligands. The TNF receptor family has common cysteine-rich extracellular domains and cytoplasmic death domains that involve in transmitting the death signal from the cell surface to the intracellular signaling pathways. Ligation of death receptors with death ligands causes conformational change in death domain and consequently recruits apoptosis-related adaptor proteins that associate with procaspase-8/-10. At this point, a death-inducing signaling complex (DISC) consisting of the death receptor, an adaptor molecule, and pro-caspase-8/−10 is formed, resulting in the auto-catalytic activation of procaspases (**Figure 1**). The activated form of the caspase-8/-10 enzyme, as an initiator caspase, subsequently cleaves and activates other downstream or executioner caspases [42, 43]. Finally, both apoptotic pathways result in the activation of effector caspases (caspase-3/-7) causing the cleavage of key cellular macromolecules which are required for normal cellular function. They cleave the structural proteins in the cytoskeleton and nuclear proteins such as DNA repair enzymes and activate degradative enzymes such as DNases, which together contribute to the typical morphological changes and promote cell death [44, 45].

#### **2.2 Dysregulation of apoptosis in carcinogenesis**

The pathogenesis of many diseases is most closely related to inappropriate apoptosis (either too little or too much) and cancer is one of the situations where too little apoptosis happens, leading to malignant cells that highly proliferate. Defects at any points along apoptotic pathways may lead to malignant transformation of the affected cells, tumor metastasis, and resistance to anti-cancer drugs [12, 46]. Defects in Bcl-2 family of proteins and caspases are well-known chief factors to be involved in the evasion of apoptosis by tumor cells.

#### **Figure 1.**

*Apoptosis signaling pathways. Abbreviations: TRADD, TNF receptor-associated death domain protein; FADD, Fas-associated death domain protein; Bid, BH3 interacting-domain death agonist; Bak, Bcl-2 homologous antagonist/killer; tBid, truncated BID; Bax, Bcl-2 associated X protein; APAF-1, apoptotic protease activating factor-1; Bcl-2, B-cell lymphoma 2, Bcl-xL; B-cell lymphoma-extra large.*

#### *2.2.1 Dysfunction of the Bcl-2 family of proteins in apoptosis*

The Bcl-2 family of proteins consist of pro-apoptotic and anti-apoptotic proteins that act as a master regulator of initiation of apoptosis through intrinsic pathway and function chiefly at the mitochondrial level. The first protein of this family, B-cell lymphoma 2 (Bcl-2), was recognized almost 30 years ago. Currently 25 members of the Bcl-2 family have been determined and based on the presence of conserved Bcl-2 homology (BH) domains and their role in mitochondrial-mediated apoptosis, they are categorized into the following three subfamilies [47, 48]. Anti-apoptotic subgroup consisting of Bcl-2, Bcl-w, Bcl-xL, A1/Bfl-1, Mcl-1and Bcl-B/Bcl2L10 proteins contain four BH domains designated as 1–4 and inhibit the

apoptosis occurrence so named pro-survival proteins. However, second group, known as apoptosis effectors, belongs to pro-apoptotic members of this family containing BH 1–3 and missing the BH4 domain. Some example of this group are Bak, Bax, and Bok/Mtd. The last group that can be considered as subdivision of pro-apoptotic proteins including Bik, Bid, Bim, Bmf, Puma, Bad, Hrk and Noxa are named "BH3-only"' proteins as they contain only the BH3 domain. The members of this group function as initiators of apoptosis and the major mediators of the interaction with anti-apoptotic proteins [47, 49, 50]. Structural studies have determined that BH1, BH2 and BH3 areas together form a hydrophobic pocket that can be filled by the amphipathic a-helical BH3 domain of pro-apoptotic Bcl-2 proteins. Consequently, Bcl-2 family interactions regulate mitochondrial outer membrane (MOM) integrity and function and eventually onset of mitochondrial-mediate apoptosis [37, 51].

The balance disturbance of anti-apoptotic and pro-apoptotic proteins cause to dysregulated apoptosis in the affected cells. Altered expression of these proteins frequently occurs in cancers. Overexpression of anti-apoptotic proteins such as Bcl-2 or Bcl-xL occurs in a huge number of human cancers [52–55]. In one study, targeted proteomic analysis have revealed the contribution of Bcl-2 overexpression to cell survival of laryngeal carcinoma (LC) though its interaction with Hsp90β and formation of Bcl-2 Hsp90β complex involving in the anti-apoptotic progression of LC [56]. In cervical cancer SiHa cells, overexpressing Bcl-2 gene, the suppression of down-regulation of HPV16 E7 and c-myc gene expression may inhibit the induction of apoptosis [57]. Besides, high levels of Bcl-2 have been reported in hematological malignancies. Various mechanisms such as gene amplification, chromosomal translocations and dysregulation of miRNAs involved in Bcl-2 RNA degradation may cause to Bcl-2 upregulation [58–60]. Furthermore, there have been a number of studies reporting the involvement of Bcl-xL anti-apoptotic protein in tumorigenesis. The increased level of Bcl-xL gene expression determined in human cancers such as colorectal cancer, breast cancer, gastric adenomas and carcinomas, hepatocellular carcinoma and prostate cancer promotes cancer cell survival [61–65]. In addition, several attempts have revealed the association of enhanced levels of Bcl-xL and MCL1 with the malfunction of miRNAs that usually diminish their expression such as miR-29, miR-125b, miR-193 [66–68]. Furthermore, overexpression of anti-apoptotic Bcl-2 and its close relatives have been recognized as chief components of chemoresistance [69–72].

Deficiency in pro-apoptotic members of the Bcl-2 family has also been extensively studied in tumorigenesis and cancers. Pro-apoptotic gene Bim is frequently silenced in human Burkitt's lymphoma [73, 74]. Homozygous deletion and the loss of mRNA and protein expression have also been determined in mantle cell lymphoma and renal cell carcinoma. Hence, blocking Bim expression caused by gene deletion or epigenetic silencing is mainly contributed to pathogenesis of these tumors [75, 76]. Furthermore, a number of researchers have reported that down-regulation and mutation of Bax plays a significant role in tumor resistance to apoptosis. Reduced Bax expression was reported to be correlated with acquiring resistance to 5-FU in colorectal cancer cell line and cisplatin in ovarian carcinoma [77, 78]. Sensitivity of non-small cell lung cancer to Zoledronic was also found to be Bax dependent [79]. Suppressed Bax activity is one of the major reasons of TRAIL resistance in melanoma [80–82]. Besides, inactivated mutation in gene Bax such as frameshift mutations, loss of function mutations and point mutations has been reported in colon cancers, certain hematopoietic malignancies and acquired resistance to antineoplastic drugs [83–85]. Additionally, cells lacking both Bax and Bak have confirmed to be completely resistant to truncated Bid (t-Bid)-induced cytochrome c release and apoptosis [86]. Therefore, all these abnormalities regarding

Bcl-2 family protein members affect the ratio and equilibrium of pro-apoptotic to anti-apoptotic proteins which result in apoptosis dysfunction and resistance to cell death.

#### *2.2.2 Dysfunction of caspases in apoptosis*

Caspases are a family of cysteine proteases that play crucial role in initiation and execution of apoptosis signaling pathway. During tumorigenesis, altered caspase activity or deficiency in their functions may lead to impairing apoptosis induction resulting in intense misbalance in the growth dynamics that eventually cause to decreased apoptosis, irregular growth of cancer cells and carcinogenesis [17, 87]. Human cancer cells dysregulate caspase activity through a different mechanism such as inactivated mutation, down-regulation and epigenetic alteration blocking their apoptotic activity [88–90].

Caspase-3/-7 is a critical executioner molecule in apoptotic mechanism through cleaving a variety of key cellular proteins. Many studies have demonstrated the close association of altered caspase-3 expression and various cancers such as cervical adenocarcinoma, colon cancer, glioma and breast cancer [91–97]. However, the role of caspase-3 in breast cancer patients has been an area of controversy. Metaanalysis study of 3091 cases have revealed that enhanced expression of caspase 3 is related to poor overall survival in patients [98].

As mentioned earlier, the activation of executioner caspases involves their proteolytic cleavage through mature and functioning initiator caspases. Therefore, deficiency in initiator caspases activity has been determined in cancer development and progression [99, 100]. Caspase-9 plays a critical role in the initiation phase of the intrinsic apoptosis pathway. Decreased levels of caspase-9 was reported in patients with stage II colorectal cancer associated with poor clinical outcome [90, 101]. Inhibition of caspase 9 activity has been reported to be involved in acquired cisplatin resistance in head and neck squamous cell carcinoma cells [102]. Several functional polymorphism of caspase-9 has also been determined which may influence its expression or activity and therefore alter susceptibility to cancer [103–106].

Since extrinsic signaling of apoptosis mechanism after external stimulation of the death receptors is mediated through initiators caspase-8 and caspase-10, their deregulated expression or function can block death receptor signaling pathway contributing to cancer development. Expression of caspase-10 was found to be reduced in rectal cancer [107]. The cDNA array analysis has also detected the reduced co-expression of initiator caspases of extrinsic pathway, caspase 8 and 10, that might contribute to the pathogenesis of choriocarcinoma [108]. In previous investigations, expression analysis of caspase-8 has shown its down regulation in breast cancer cell lines and tumor tissues of breast cancer and revealed significant association between altered caspase-8 expression and status of HR in breast cancer patients [109]. Some studies also revealed that loss of caspase-8 expression not only cause to reduced apoptosis, but also involved in enhanced cell migration, tumor microenvironment and amplification of MYCN oncogene which highlight its contribution in carcinogenesis. The lack of caspase-8 expression happens very commonly in neuroendocrine cancers such as glioblastoma, medulloblastoma, neuroblastoma [110–112]. Furthermore, the correlation between caspase-8 with cancer prognosis, cancer stage and therapy resistance has been reported [109, 110]. Loss of initiator caspase-8 protein expression has been shown to be related with undesirable survival outcome in medulloblastoma and gynecological tumors such as ovarian and breast cancers and stage of head and neck squamous cell carcinoma (HNSCC) [113, 114].

## **2.3 Targeting cellular apoptosis machinery in cancer treatment**

Since inhibition of apoptosis lies at the heart of all abnormal malignant growth, metastasis and conferring therapeutic failure, targeting the apoptosis mechanism players is of vital importance in cancer therapy. In this regard, Bcl-2 family of proteins as gate-keepers of intrinsic apoptotic pathway mediating the pro- and anti-apoptotic function at the mitochondrial level and caspases as the central player in the initiation and execution of apoptotic cell death have been the center of attraction for drug discovery studies and development of anticancer agents [10, 115, 116]. Here, various therapeutic strategies designed to target them have been reviewed.
