**8. Autophagy**

78 Apoptosis and Medicine

*6.3.3. Lysosomes* 

*6.3.4. Cytosol* 

*6.3.5. Cytoskeleton* 

unfolded proteins in the lumen of the ER, triggering an evolutionary conserved signalling pathway known as the Unfolded Protein Response (UPR) which can culminate in cell death. UPR consists of global protein synthesis reduction, synthesis induction of chaperones and other proteins related to protein folding and retro-translocation of misfolded or unfolded proteins from the ER to the cytosol, where they will be degraded by the proteasome [59]. When the ER stress is sustained and ER function cannot be restored, UPR activates a specific apoptotic pathway. Caspase-12, which is localized in the ER, is activated by calciumdependent proteases known as calpains [60]. Once activated, caspase-12 activates caspase-9 without apoptosome intervention [61,62]. It has been postulated that ER stress can also activate caspase-8, which induces cytochrome c release through Bid processing [29,63]. In

Lysosomes are organelles that contain acidic hydrolases such as cathepsins. Rupture of lysosomes release cathepsins to the cytosol, where they can trigger apoptosis or necrosis. Cystatins, on the other hand, are cytosolic proteins that act as negative regulators of cathepsins when they are translocated from lysosomes to the cytosol. Apoptosis initiated in lysosomes follows a mitochondrion-dependent pathway associated to caspase activation. However, it has been shown that cathepsin D activates Bax and AIF release, triggering a caspase-independent apoptotic pathway [37]. Furthermore, some cathepsins induce Bid cleavage [38], interaction with Bcl-2 proteins and permeabilization of the mitochondrial membrane [64]. In addition, cathepsins can alter mitochondria functions by cleaving subunits of the oxidative phosphorylation complexes, inducing ROS generation [12].

Several signals coming from the cytosol can induce MMP. These include: metabolites such as glucose 6-phospate and palmitate, ROS and activation of certain kinases: GSK3β, protein kinase C (PKC) δ, [65] and members of the JNK signalling pathway [38,66]. On the other hand, other molecules inactivate PTP and protect mitochondrial membrane from permeabilization, inhibiting apoptosis. These include: metabolites (ATP, glucose, NADH, UTP, etc.), antiapoptotic Bcl-2 family members, antioxidant enzymes such as glutathiones S transferase and prosurvival kinases such as Akt. In this sense, Akt can inhibit apoptosis by several mechanisms: activation of NFkB [67], inactivation of GSK3β and caspases and

The cytoskeleton is composed of microtubules, microfilaments and intermediate filaments that play important roles in cell motility, polarity, attachment, shape maintenance, etc. Adherent cells can undergo a specific type of caspase-dependent cell death called **anoikis**

addition, ER stress can also initiate **autophagy,** a different type of cell death.

Lysosomes are essentially involved in **autophagic cell death** mechanisms.

through hexokinase II-dependent mechanisms [12].

**Autophagy** is a self-digestive physiological process that occurs in all eukaryotic cells, during which long-lived proteins and organelles are degraded by lysosomes to maintain cellular homeostasis [79]. To date, at least three types of autophagic pathways have been described: macroautophagy (simply called ''autophagy''), microautophagy and Chaperone-Mediated Autophagy (CMA). These forms differ in the mode the cargo is delivered to the lysosome.

**Macroautophagy** is a dynamic process in which portions of the cellular cytoplasm and organelles are sequestered in a double-membrane bound vesicle called autophagosome, which then fuses with the lysosome [80,81]. **Microautophagy** involves the sequestering by the lysosome itself of part of the cytoplasm. During microautophagy, the membrane of the lysosome invaginates, and then pinches off to form an internal vacuole that contains material derived from the cytoplasm [82]. The notable difference between macroautophagy and microautophagy is that in the latter, part of the cytoplasm is directly taken up into the lysosome. Both macroautophagy and microautophagy are basically nonselective degradation pathways in which bulk cytoplasm is randomly sequestered. However, in some cases, autophagy can selectively eliminate some organelles, such as damaged peroxisomes, mitochondria or ER. In contrast, **CMA** does not involve vesicular traffic and is specific for the degradation of proteins. During this process, proteins are delivered to lysosomes with the help of molecular chaperones and a lysosomal receptor. Cytosolic proteins with a specific peptide sequence motif (''KFERQ'' motif) are recognized by a complex of molecular chaperones (Hsc70) and then bind to a lysosomal receptor called lysosome associated membrane protein (LAMP) type 2a [83].

Cell Death and Cancer, Novel Therapeutic Strategies 81

The cell death process that takes place when mitosis cannot be completed is called mitotic catastrophe. This phenomenon is triggered as a consequence of perturbations of the mitotic machinery that governs appropriate chromosome segregation. The main hallmark of mitotic catastrophe is the enlarged cell size accompanied by multinucleation [5]. Other features are chromatin condensation, DNA degradation, MMP, cyrochrome c release from the mitochondria and caspase activation [86]. Some types of mitotic catastrophe, however, take place without intervention of caspases, what has been named caspase-independent mitotic death [87]. Mitotic catastrophe results form the combination of deficient checkpoints (DNA and mitotic spindle) and DNA damage. Cells that evade the mitotic checkpoint and do not undergo apoptosis are prone to generate aneuploidy. Therefore, mitotic catastrophe is conceived as a device to avoid genomic instability. The players that take part in mitotic catastrophe are: cell cycle-dependent kinases (Cdk1, Aurora, Plk), cell cycle-check points proteins (Chk2, p53, p73), survivin, MCl-2, Blc-2 proteins, caspase-2, etc. [86]. Mitotic catastrophe is a poorly defined molecular signalling pathway that precedes apoptosis,

**Figure 7. Mitotic catastrophe. a)** In the absence of perturbations cells progress normally. A mitotic defect is detected and: **b)** cells die without exiting mitosis, **c)** cells undergo mitotic arrest, exit mitosis (mitotic slippage), reach the subsequent G1 and die or **d)** undergo senescence (taken from Galluzzi et al.

Necrosis is characterized by plasma membrane permeabilization, swelling and rupture. Necrosis can be accompanied in many instances by release of lysosomal hydrolases. Recently, a novel form of regulated necrosis has emerged and has been named **necroptosis**  [78]. This cell death modality presents morphological features of necrosis but is regulated by signalling pathways and catabolic mechanisms. The most studied necroptotic pathway is mediated by the death receptor TNFR1 and inhibited by necrostatin-1. Upon TNF binding, TNFR1 undergoes a conformational change and recruits TRADD, TRARF2, cIAP1, cIAP2

**9. Mitotic catastrophe** 

necrosis or senescence [88].

2012).

**10. Necroptosis** 

Macroautophagy (hereafter referred to as autophagy) is the most studied and prevalent form of autophagy in cells. This process begins with the formation of a "C" shaped doublemembrane structure in the cytosol, called "omegasome", which is formed from the ER (Initiation phase). Following this, the omegasome grows to form the "isolation membrane", which elongates to engulf cytoplasmic components (Elongation phase). Then, the "isolation membrane" curves and closes to form a vacuole called the autophagosome (Maturation phase). As a result, portions of the cell cytoplasm and some organelles are sequestered in this vacuole. Finally, the outer membrane of the autophagosome fuses with the lysosomal membrane and the inner membrane (the autophagic body) carrying the cytosolic constituents enters the lysosome. The autophagic body is degraded in the lysosome by hydrolases and the resulting free amino acids and macromolecules are transported back into the cytosol for reuse [84]. In this way, autophagy contributes to the maintenance of the cellular energy homeostasis, to the clearance of damaged organelles and to adaptation to environmental stresses [85]. Accordingly, autophagy defects have been linked to a wide range of human pathologies, including cancer.

**Figure 6. Autophagy.** Three different forms of autophagy are depicted: Macroautophagy, Microautophagy and Chaperone-mediated autophagy (CMA) (taken from Yen and Klionsky, 2008).
