**2. Apoptosis**

Apoptosis means cell suicide. It is a normal component of the development and health of multicellular organisms. Cells perform in a controlled, regulated fashion by apoptosis. Apoptosis is different from another form of cell death called necrosis [22]. Cancer is often characterized by too little apoptosis. In the case of cancer, damaged cells, which should undergo apoptosis, have mutations that prevent them from undergoing apoptosis [22]. Apoptotic cells can be recognized by stereotypical morphological changes (**Figure 3**).

### **2.1. Pathways of apoptosis**

Apoptosis consists of two major pathways: extrinsic pathway and intrinsic (mitochondrial) pathway.

**Figure 3.** Hallmarks of the apoptotic and necrotic cell-death process. Modified from [23].

#### *2.1.1. Extrinsic pathway*

targeting is currently under development. CD40 signaling may also have a positive effect on conventional therapy. It has been shown that CD154 (CD40L) application was able to induce the p53-related transcription factor p73, leading to a sensitization of p53-deficient CLL cells to

A number of approaches have been taken to directly modulate the core components of the Bcl-2 cell-death machinery. The Bcl-2 antisense molecule oblimersen is the most advanced agent in clinical testing. "BH3-mimetics" and "pan-Bcl-2 family antagonists" can mimic the BH3 domain of BH3-only death-inducing proteins and are thought to liberate BH3-only proteins from the inhibition by antiapoptotic Bcl-2 proteins, thus making them effective killers.

Apoptosis means cell suicide. It is a normal component of the development and health of multicellular organisms. Cells perform in a controlled, regulated fashion by apoptosis. Apoptosis is different from another form of cell death called necrosis [22]. Cancer is often characterized by too little apoptosis. In the case of cancer, damaged cells, which should undergo apoptosis, have mutations that prevent them from undergoing apoptosis [22]. Apoptotic cells can be

Apoptosis consists of two major pathways: extrinsic pathway and intrinsic (mitochondrial)

conventional therapeutics such as fludarabine [21].

**Figure 2.** The interaction between Bcl-2 family member proteins [15].

recognized by stereotypical morphological changes (**Figure 3**).

**2. Apoptosis**

92 Cytotoxicity

**2.1. Pathways of apoptosis**

pathway.

"Death receptors" transmit apoptotic signals after ligation with specific ligands in extrinsic pathway. Death receptors belong to a superfamily, including TNFR-1, Fas/CD95, and the TRAIL receptors DR-4 and DR-5 [24]. Caspase-8 is the hallmark of this pathway. It is activated by a complex named death-inducing-signaling complex (DISC). Activated death receptor recruited adapter molecules like FADD (Fas-associated protein with death domain) or TRADD (tumor necrosis factor receptor type 1-associated DEATH domain). These adapter molecules form the DISC (**Figure 4**). Caspase-8 then cleave and activate other caspases resulting in cell death. These types of cells, which have the capacity to induce such direct and mainly caspase-dependent apoptosis pathways, were classified to type I cells [25].

### *2.1.2. Intrinsic pathway*

In this pathway, the signal does not come from death receptors. In this case, the signal amplified via mitochondria-dependent apoptotic pathways. Bcl-2 family member, Bid, is cleaved by caspase-8 (tBid) and translocates to the mitochondria. tBid in concert with the proapoptotic Bcl-2 family members Bax (Bcl-2-associated x) and Bak (Bcl-2 homologous antagonist/ killer) induces the release of cytochrome C and other mitochondrial proapoptotic factors into the cytosol [27].

Cytosolic cytochrome C binds to monomeric Apaf-1 (apoptotic protease-activating factor 1) which then oligomerizes to assemble the apoptosome that triggers the activation of the

**2.2. Apoptotic pathway proteins**

*2.2.2. The Bcl-2 superfamily*

killing action [38, 39].

lating in the hematopoietic system and in the brain [26].

*2.2.1. Caspases are central initiators and executioners of apoptosis*

The term caspase is derived from cysteine-dependent aspartate-specific proteases. So far, seven different caspases have been identified in *Drosophila*, and 14 different members of the caspase-family have been described in mammals, with caspase-11 and caspase-12 only identified in the mouse [30, 31]. According to a unified nomenclature, the caspases are referred to in the order of their publication: caspase-1 is ICE (interleukin-1β-converting enzyme), the first mammalian caspase [32, 33]. There are many documents about the importance of caspases in apoptosis phenomenon. For example, it has been shown that caspase-1, -4, -5, -11, and -12 are involved in the maturation of pro-inflammatory cytokines such as pro-IL-1β and pro-IL-18

Cytotoxicity and Apoptosis Induction by Coumarins in CLL

http://dx.doi.org/10.5772/intechopen.72446

95

[31] or studies show that caspase-3 and -9 have a role in brain development [34, 35].

fication of the death signal and eventually in the execution of cell death [36].

Caspases are synthesized as inactive zymogens, the so-called procaspases. Upon maturation, the procaspases are proteolytically processed. The proapoptotic caspases can be divided into the group of initiator caspases including procaspases-2, -8, -9, and -10, and into the group of executioner caspases including procaspases-3, -6, and -7 [26]. As mentioned earlier, in extrinsic apoptosis pathways procaspase-8 is the hallmark of this pathway. In return of caspase-8, caspase-9 is the hallmark of intrinsic pathway. Once the initiator caspases have been activated, they can proteolytically activate the effector procaspases-3, -6, and -7. Effector caspases subsequently cleave a specific set of protein substrates, resulting in the mediation and ampli-

Bcl-2 is an oncogene which was the first example of an oncogene that inhibits cell death rather than promoting proliferation. Bcl-2 family of proteins can be defined by the presence of conserved sequence motifs known as Bcl-2 homology domains (BH1 to BH4). Bcl-2 proteins divided to a group of prosurvival members and others to a group of proapoptotic members [37]. Prosurvival proteins include Bcl-2 itself, Bcl-XL, Bcl-w, A1, and Mcl-1, all of which possess the domains BH1, BH2, BH3, and BH4. The proapoptotic group of Bcl-2 members can be divided into two subgroups: the Bax-subfamily consists of Bax, Bak, and Bok, all of which possess the domains BH1, BH2, and BH3. There is another group of proteins named the BH3 only proteins (Bid, Bim, Bik, Bad, Bmf, Hrk, Noxa, Puma, Blk, BNIP3, and Spike) that have only the short BH3 motif, an interaction domain that is both necessary and sufficient for their

Despite the existence of two hypotheses regarding how the Bcl-2 family controls apoptosis, it seems that the central function of mammalian Bcl-2 family members is to guard mitochondrial integrity and control the release of mitochondrial proteins into the cytoplasm [39]. Another hypothesis is that Bcl-2 members might directly control caspase activation [40]. The question is how mitochondrial integrity is affected by proapoptotic Bcl-2 family members? Central to this question are Bax and Bak. The double knockout of Bax and Bak resulted in dramatic impairment of apoptosis during development in many tissues with superfluous cells accumu-

**Figure 4.** Receptor-mediated caspase activation at the DISC [26].

initiator procaspase-9 [28]. Caspase-9 is the hallmark of intrinsic pathway. Activated caspase-9 ultimately results in cell death by subsequently initiating a caspase cascade involving downstream effector caspases such as caspase-3, caspase-7, and caspase-6 (**Figure 5**) [29].

## **2.2. Apoptotic pathway proteins**
