Apoptosis Pathways and New Anticancer Agents

Apoptosis or programmed cell death is a physiologic process that determines tissue homeostasis and provides an effective way to remove unwanted cells, such as those that have accumulated oncogenic mutations. Inhibition of apoptosis disrupts the balance between cell proliferation and cell death and has been recognized as one of six key mechanisms that are essential for the generation of fully transformed malignant cells [1]. Cells that undergo apoptosis are characterized by morphologic changes that include cytoplasmic shrinkage, plasma membrane blebbing, and chromatin condensation in the nucleus, which facilitate the efficient inflammation-free removal of apoptotic cells by macrophages [2]. At the molecular level, proteolytic enzymes such as caspases play an important role as the executors of apoptosis leading to cell death. Apoptosis is distinct from passive nonregulated cell death (necrosis), and, in general, is referred to as caspase-dependent cell death. The term “classical apoptosis” has been coined to distinguish it from other forms of programmed cell death that display a mixture of morphology or molecular features or both representing caspaseor noncaspase-dependent cell death. We focus on the therapeutic exploitation of the core apoptotic machinery that regulates caspase-dependent cell death. Two main caspase activation pathways have been identified (see also Fig. 12-1). One route, known as the intrinsic or mitochondrial pathway, is triggered upon disruption of mitochondria, e.g., because of DNA damage induced by cytotoxic agents, and causes the release of cytochrome c into the cytoplasm [3, 4]. Together with dATP, cytochrome c is a cofactor for the assembly of the apoptosome, which contains Apaf-1 and procaspase-9, and leads to the processing and activation of caspase-9. The second route, the so-called extrinsic or death receptor pathway, is initiated through specific cell membrane receptors, such as Fas/CD95 and tumor necrosis factor (TNF) family receptors, that upon ligand binding recruit the cytosolic death-domain-containing protein FADD (Fas-associated protein with death domain), which is able to bind and activate procaspase-8 in a complex named the death-inducing signaling complex (DISC). Both caspase-8 and -9 can activate the effector caspases-3, -6, and -7 provided that the caspase inhibitory effect of the inhibitor of apoptosis proteins (IAP) is relieved by Smac/Diablo, a proapoptotic protein that is also released from the mitochondria. The IAP family comprises proteins that contain one or more baculovirus IAP repeat (BIR) domains, which mediate in some IAP the interaction with caspases [5, 6]. The most potent caspase-inhibitory IAP is X-linked IAP (XIAP). When released in the cytosol, Smac binds to XIAP facilitating caspase activation. The activation of the effector caspases leads to the cleavage of various substrates, which results in the characteristic morphologic features of apoptotic cell death. An important class of regulators of apoptosis are the BCL-2 family proteins [7–9], comprising both antiapoptotic members, such as BCL-2, BCL-X L , and MCL-1, as well as proapoptotic members such as BAX and BAK, that share homology throughout four or three BCL-2 homology domains, respectively. Their primary mode of action has been assumed to be the regulation of mitochondrial integrity; however, they also appear to be involved in maintaining the integrity of other intracellular membrane structures, such as the endoplasmatic reticulum. Upon apoptosis activation, BAX and BAK translocate from the cytoplasm to the mitochondrial membrane where they oligomerize to form porelike structures, thereby causing mitochondrial outer membrane permealization (MOMP) and the release of apoptogenic factors, such as cytochrome c and Smac. The BH3-only proteins constitute a third class of proapoptotic BCL-2 proteins, which includes BID, BAD, BIK, PUMA, NOXA, BMF, and HRK. These proteins share homology in only one region, the BH3 domain. The BH3-only proteins appear to function as sentinels for the detection of cellular damage or aberrations; for example, BIM is activated by microtubule disarray, whereas