OTHER FORMS OF CELL DEATH: NECROSIS AND AUTOPHAGY

Although knowledge of alterations in the genetically programmed apoptotic cell death pathway is crucial for understanding cell survival in cancer and cell death in degenerative disease, many pathological forms of cell death involve nonapoptotic mechanisms.

Necrosis

Necrosis is morphologically characterized by a loss of plasma membrane integrity and leakage of cellular contents into the intercellular space, which stimulates an inflammatory response. This form of cell death can be initiated by both external stimuli and internal cellular damage. Although the mechanisms behind death-receptor-induced necrosis are not completely understood,^176-180 an impor­tant mediator common to both signaling sources appears to be the accumulation of reactive oxygen species (ROS).

Reactive Oxygen Species

Oxidative stress results from an imbalance between production of ROS (such as superoxide anion, hydrogen peroxide, and the hydroxyl radical) and reduction of ROS by endogenous antioxidant enzymes (such as superoxide dismutase, glutathione peroxidase, catalase, and thioredoxin). When the antioxidant capacity of the cell is exceeded, ROS accumulation damages DNA, lipids, and protein. Free radicals induce DNA single-strand breaks and can initiate the DNA damage response to induce cell death or repair, depending on the extent of damage. Lipid peroxidation products include F₂-isoprostanes, free-radical catalyzed isomers of prostaglandins. Lipid peroxidation can have profound effects on structural and metabolic properties of cell membranes and ultimately might lead to disruption of Ca²⁺ homeostasis and subsequent apoptotic cell death. Protein oxidation by free radicals can cause dysfunction due to cross-linking between oxidized cysteine residues or, when combined with nitric oxide, can lead to tyrosine nitration or S-nitrosylation to affect protein function.¹⁸¹ Cell death induced by ROS was shown to occur through both mitochondria-dependent and mitochondria-independent pathways.

ROS in Alzheimer's Disease

Alzheimer’s disease (AD) is characterized by progressive accumulation of the amyloid-β peptide and subsequent neuronal degeneration in regions of the brain affecting learning and memory. In addition to the presence of abundant senile plaques and neurofibrillary tangles caused by protein aggregation seen in the AD brain, factors thought to contribute to disease pathogenesis include increased oxidative stress, inflammation, and alterations in lipid metabolism. Animal models and human studies have suggested that oxidative damage is an early functional event in AD patho- genesis,¹⁸²-¹⁸³ although the initiating events that cause increased ROS in AD remain controversial. Some studies have suggested a role for enzymes involved in arachidonic acid metabolism, such as 12/15-lipoxygenase,¹⁸⁴ and found increased expression and activity of this enzyme in affected areas of AD brains when compared to unaffected regions.¹⁸⁵,¹⁸⁶ Regulation of expression and activity of this enzyme is controlled by inflammatory cytokines,¹⁸⁷ which are known to mediate inflammation in AD.

Downstream of ROS production, multiple studies have shown that increased oxidative stress and ceramide synthesis can lead to accumulation of cholesterol in cells, and this process was observed in brains of AD patients compared with age-matched controls.¹⁸⁸ Furthermore, a role for increased cholesterol levels in AD is supported by the increased risk of AD in people with an apolipoprotein E4 allele¹⁸⁹ and decreased risk in people prescribed cholesterol-lowering drugs.¹⁹⁰ The cellular toxicity of high levels of cholesterol was shown by the ability of inhibitors of cholesterol-metabolizing enzymes to induce apoptosis¹⁹¹ and by the ability of statins to protect neurons against oxidative injury.¹⁹² Cell culture experiments showed that antioxidants (i.e., toco­pherol) or inhibitors of ceramide synthesis (ISP-1) can protect neurons from amyloid-β-induced cell death in vitro.¹⁸⁸

ROS in Ischemia-Reperfusion Injury

Ischemic-reperfusion injury occurs after restoration of oxygen and metabolic substrates to energet­ically deprived tissue.

Protection against Reactive Oxygen Species

Protection against ROS can be achieved by enhancing the reducing capacity of the cell. Small molecules were designed that have the catalytic activity of both superoxide dismutase and catalase and, therefore, remove both superoxide anions and hydrogen peroxide. These agents, EUK-8 and EUK-134, showed protective effects in rodent models of ischemia-reperfusion of the brain,¹⁹⁸ kidney,¹⁹⁹ and liver,²⁰⁰ in a murine model of amyotrophic lateral sclerosis²⁰¹ and in a rat model of endotoxic shock.²⁰² Additional antioxidant chemicals that can prevent cell death induced by ROS include V-acetyl-cysteine and tocopherol, among others.

Cancer Therapeutics That Induce ROS and Cell Death

In cancer treatment, many currently used therapeutics function by promoting oxidative stress. For example, photodynamic therapy (PDT) uses a photosensitizing compound, such as porphyrin or chlorin, that selectively accumulates in target cancer cells and induces cell death only locally after exposure to laser light with a specific wavelength.²⁰³ Absorption of light by the photosensitizing drug excites it to an extremely unstable state, which generates reactive free radical intermediates upon interaction with endogenous oxygen.

Modulation of PARP Activity in Necrotic Cell Death

The fact that DNA-alkylating agents are effective at killing cancer cells that have defective apoptotic machinery suggests that they can initiate another form of cell death. For example, follicular lymphoma cells are sensitive to DNA-alkylating agents, despite overexpression of Bcl-2.²⁰6 Many studies have shown that DNA damage can induce a form of cell death that depends on the activity of poly(ADP)-ribose polymerase (PARP). PARP is a nuclear enzyme that senses DNA strand breaks and catalyzes poly(ADP)-ribosylation of a variety of proteins, using NAD⁺ as a substrate. When activated by DNA damage, PARP activity contributes to the loosening of chromatin structure and recruitment of DNA repair enzymes. However, after excessive DNA damage, prolonged PARP activity consumes cellular NAD⁺ and leads to a necrotic cell death due to ATP depletion. It has been shown that alkylating agents selectively kill proliferating cells and not quiescent cells, due to their differential dependence on cytosolic NAD⁺ to generate ATP.²⁰⁷ During apoptosis, PARP is one of the first targets cleaved by caspase-3 and is a key determinant in the decision to die by necrosis or apoptosis. Therefore, PARP activation may represent a therapeutic target relevant for the treat­ment of cancer.

On the other hand, PARP inhibition shows protective effects in disorders characterized by necrotic cell death. Many PARP inhibitors were developed based on nicotinamide analogues and function by blocking catalytic activity of all PARP family members. These inhibitors have been protective in animal models of septic shock, cardiac ischemia, traumatic brain injury, and NMDA- mediated excitotoxicity, among others.²⁰⁸

Autophagy

Autophagy is increasingly being recognized as an important alternative mechanism for cell death during development and in response to nutrient deprivation.²⁰⁹ Autophagy controls protein and organelle turnover through rearrangement of subcellular membranes to deliver cargo to the lysosome or autophagic vacuole for degradation.

Proteins that are involved in the formation of the autophagosome, such as Beclin 1, were identified as tumor suppressors. Beclin 1 is the human homologue of yeast autophagy gene Apg6 and was found to interact with Bcl-2.²¹¹ Cells deficient in Beclin 1 can undergo apoptosis but not autophagy. The fact that Beclin 1 is a dose-dependent tumor suppressor found to be monoallelically deleted in breast and ovarian cancers suggests that the process of autophagy is important in tumor suppression.²¹² Transfection of Beclin 1 into breast cancer cell lines expressing low levels of it reduced tumorigenicity in nude mice. Therefore, Beclin 1 represents a potential therapeutic target to modulate autophagic activity.

The link between autophagy and cancer is further supported by the involvement of known tumor suppressors in autophagic pathways, including PTEN and DAP-kinase. During cancer pro­gression, activation of the Akt pathway, mTOR signaling, and loss of PTEN may promote trans­formation by simultaneously blocking apoptosis and autophagy.²¹³ Furthermore, an increasing number of studies suggest that the mechanism of action of some anticancer agents involves induction of autophagic cell death.

Potential cross talk between the pathways of apoptosis and autophagy has been reported, as inhibition of caspase-8 activity induces autophagic cell death.²¹⁴ This is relevant to the host response to viral pathogens, which have caspase inhibitors. Thus, the induction of autophagic death following caspase inhibition might provide a security mechanism that ensures destruction of an infected cell via nonapoptotic cell death.

Paradoxically, although autophagy is often considered an alternative form of cell death, in some situations, increased autophagic activity can promote cell survival. For example, in diseases char­acterized by the accumulation of misfolded proteins, autophagy may be an important mechanism for aggregate clearance and cell survival. This model was recently supported in a transgenic fly model of Huntington’s disease, in which rapamycin treatment increased autophagy, improved clearance of huntingtin aggregates, and enhanced cell survival.²¹⁵