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

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

#### *2.2.2. The Bcl-2 superfamily*

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].

**Figure 5.** Intrinsic pathway of apoptosis [28].

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

94 Cytotoxicity

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 killing action [38, 39].

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

BH3-only members function upstream of Bax and Bak. It is shown that members of the BH3 only subfamily are required for the activation of proapoptotic Bax/Bak function. But it should be noted that prosurvival members Bcl-2 and Bcl-XL have a role in this way [41].

can act as a molecular bodyguard or assassin during apoptosis [47]. Saxena et al. showed that Mcl-1 can play an important role in CLL, by insertion of small sequences in its promoter [47]. They showed the presence of specific insertions in 29% patients with CLL and while in none of the controls. They found that these insertions were correlated with rapid disease progression, with a poor response to chemotherapy and shorter disease-specific survival. By founding of insertions in CD38-negative patients, they suggest that a poor prognostic marker [47] can be present.

Cytotoxicity and Apoptosis Induction by Coumarins in CLL

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

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Finally, since specific genetic targets are not defined in CLL, Mcl-1 seems to be an appropriate biomolecule to therapeutically manipulate. Mcl-1 protein production and maintenance are dependent on several pathways. At the apical level, the microenvironment provides factors that dramatically increase this protein in CLL cells [48]. Hence, a strategy that interferes with the interaction of microenvironment and CLL cells is a logical approach. Production of Mcl-1 through these signals is carried via increased transcription of the Mcl-1 gene. Transcription and polyadenylation inhibition, albeit not selective, is an approach that works because of AU-rich elements in the transcript of Mcl-1, which leads to its rapid turnover [49]. The N-terminal region of Mcl-1 protein contains 2PEST domains that are rich in proline, glutamic acid, serine, and threonine residues, resulting in a short half-life of the protein [49] and making translation inhibition and rapid degradation of endogenous Mcl-1 via proteasome pathway a viable option to reduce the protein level [50]. Mcl-1 is also essential during early lymphoid development [51] and is abundantly expressed in the germinal center B-cell compartment. Pim kinase and Akt-PI3-kinase pathways and downstream of BLyS have been identified to maintain the Mcl-1 levels in B-cells [52]. The roles of these pathways and consequence of their perturbations need to be investigated in malignant lymphocytes. Similarly, work is needed on posttranslational modification leading to increased or decreased half-life of Mcl-1 protein. Finally, and probably most intriguingly, small molecule antagonists of Mcl-1 protein that bind to the BH3 domain releasing proapoptotic proteins provide a new avenue of research and therapeutics.

Coumarins (2H-1-benzopyran-2-one) consist of a large class of phenolic substances found in plants and all of which consist of a benzene ring joined to a pyrone ring. More than 1300 coumarins have been identified as secondary metabolites from plants, bacteria, and fungi. The prototypical compound is known as 1,2 benzopyrone or, less commonly, as -hydroxycinnamic acid and lactone. Coumarins were initially extracted in *tonka* bean (*Dipteryx odorata* Wild) and are reported in about 150 different species distributed over nearly 30 different families, of which a few important ones are *Rutaceae*, *Umbelliferae* (*Apiaceae*), *Clusiaceae*, *Guttiferae*, *Caprifoliaceae*, *Oleaceae*, and *Nyctaginaceae* [53]. They are found at high levels in some essential oils, particularly in *cinnamon* bark oil, *cassia* leaf oil, and *lavender* oil. Coumarin is also found in fruits (e.g., bilberry and cloudberry), green tea, and other foods such as chicory. The richest sources of most coumarins among the higher plants are *Rutaceae* and *Umbelliferone*. The coumarins occur at the highest levels in the fruits, followed by the roots, stems, and leaves although they are distributed throughout all parts of the plant. Environmental conditions and

seasonal changes can influence the occurrence in diverse parts of the plant [54].

**3. Coumarins**

In summary, as it is shown in **Figure 6**, in a viable cell antiapoptotic proteins like Bcl-2 antagonize Bax/Bak. In response to an apoptotic stimulus, BH3-only proteins are activated. Activated BH3-only proteins prevent antiapoptotic Bcl-2 members from inhibiting proapoptotic members. Therefore, Bax/Bak are activated and form pores in the mitochondrial membrane. In consequence, cytochrome C and other proapoptotic factors are released from the inner mitochondrial membrane into the cytosol. They cause the formation of the apoptosome and the subsequent activation of the caspase cascade [26].
