Oxidative Stress as a Mediator and Inducer of Apoptosis

Unpaired electrons in atomic or molecular orbitals are the characteristic that makes free radicals harmful and oxidative stress a potential mediator of biological damage [34, 35]. Oxidative stress may interact generally with cellular macromole-cules, activating either survival pathways or, more often, apoptosis in cells [36].

In diseases where a redox imbalance is combined with a deregulation of apoptosis, we observe a large number of possible actions of oxidative stress on apoptotic pathways. For example, in neurodegenerative diseases, oxidative stress leads to an apoptotic early phase, which could involve the plasma membrane externalization of phosphatidylserine, and a later phase characterized by a loss of DNA integrity [37]. In the meantime, in the same cells, oxidative stress may also activate protein kinase C delta (PKC8), able to promote and amplify apoptosis signals by caspases [38]. Thus, the same disease can be targeted differentially by oxidative stress.

We can generalize the effect of oxidative stress as a result of four possible modifications [39]: nitrosation [40], carbonylation [41], disulfide bond formation [42] and glutathiolation [43] (Figure 16.2).

The involvement of oxidative stress in the extrinsic apoptotic pathway has not yet been clarified [44, 45]. In contrast, its effects on the mitochondrial apoptotic pathway are well described. Therefore, generally, oxidative stress could have common effects in different cell types: induction of the mitochondrial permeability transition pore [46], release of mitochondrial death amplification factors (i.e. cytochrome c, SMAC, AIF) [47-49] and alteration of constitutive mitochondrial proteins (i.e. VDAC) [50]. Moreover, ROS-mediated damage is probably the main source of instability of the mitochondrial genome, which leads to respiratory dysfunction, an important factor in aging, which will be discussed in more detail in Chapter 19 [20].

A deeper study of the literature underlines not only the involvement of oxidative stress in the mitochondrial apoptotic pathway, but a more important role because of its necessary presence for the induction of a functional apoptosis [57]; this makes oxidative stress the backbone of the intrinsic apoptotic pathway [18].

Figure 16.2 Effect of oxidative stress on biological functions related to apoptosis.

Box 16.2: Therapeutic Approaches Based on Modulating Levels of Reactive Oxygen Species

As mentioned before, various and often complicated relationships exist between intracellular levels of ROS and cancer. In some instances, where ROS kill cancer cells, this relationship may be used as part of cancer therapy. In principle, several avenues are available to increase intracellular levels of oxidative stress: ROS generation, inhibition of antioxidant enzymes, targeted redox catalysis.

Compounds able to generate ROS, such as arsenic trioxide (As2O3), which leads to increased levels ofsuperoxide radical anions, therefore have been considered as potential anticancer agents able to kill cancer cells via ROS production. Unfortunately, such simple "radical generators" are fairly nonspecific, and various attempts have been made to develop cancer-cell specific redox modulators.

One promising approach employs superoxide dismutase (SOD) inhibitors, such as 2-methoxyestradiol (2-ME). These compounds prevent SOD from detoxifying superoxide radicals. The subsequent build-up of these radicals may kill the affected cell. Since some cancer cells possess higher levels of oxidative stress compared to normal cells, inhibiting such antioxidant enzymes may even provide cancer cell selective effects. While SOD is not the most potent of antioxidants -after all, it generates H2O2 as part of O2"~ detoxification - other antioxidant enzymes, such as glutathione peroxidase (GPx), provide rather interesting targets for intervention into intracellular redox control.

Alternatively, SOD mimics, that is, small synthetic compounds with a SOD-like catalytic activity, have also been studied. These compounds catalytically convert superoxide to H2O2 and HO" radicals, and hence use the increased levels of superoxide radicals (and H2O2) in certain cancer cells to selectively and effectively kill these cells. Similar approaches are based on mimics of the enzyme GPx.

At the mitochondrial level the "main actors" of the apoptotic program are the proteins of the Bcl-2 family that are acting on different steps of apoptotic signaling as a modulator. The role of Bcl-2 as an antioxidant protein has been well documented for many years [51, 52], even though the antioxidant role of Bcl-2 is still controversial and a mechanism has not yet been established. In particular, Kane et al. demonstrated that Bcl-2 overexpression protects from several oxidative stress injuries, including glucose withdrawal, membrane peroxidation and glutathione depletion [53]. Interestingly, Bcl-2 and glutathione (GSH) are both antiapoptotic molecules and both exert their antiapoptotic effect via a radical scavenging mechanism; thus Bcl-2 and GSH may "exchange" their functions. Consistent with this view, pharmacological GSH depletion leads to the breakdown of Bcl-2 protein and sensitization to apoptosis [54]. The regulated decrease of one of these two important cellular antioxidant defenses, in response to an exogenous decrease in one or the other, indicates the existence of a signaling mechanism crossregulating Bcl-2 and GSH.

The latter may be viewed as the basis of a cell suicide strategy, to avoid survival of cells bearing oxidation-dependent genetic damage [54]. Interestingly, a careful analysis of the literature provides evidence that other members of the Bcl-2 family are also capable of responding to an oxidative intracellular environment. It is known,

16.3 Oxidative Stress as a Mediator and Inducer of Apoptosis | 381 Box 16.3: Reactive Oxygen Species, Checkpoint Proteins and DNA Repair

Chapters 16 and 17 deal with (redox) processes leading to apoptosis. Importantly, one must emphasize that such processes are not strictly unidirectional, and that apoptosis does not necessarily nor swiftly have to follow oxidative damage to DNA, for instance caused by ROS. Within this context, the human cell contains several checkpoint and repair systems which monitor the state of a cell, in particular damage to DNA. Such checkpoints for DNA occur, for instance, at the G1/S and G2/M boundaries and involve proteins such as cyclin D and cyclin E (at the G1/S phase transition) and Cdc25 phosphatase (at the G2/M phase transition). These checkpoints incorporate mechanisms that can stall the cell cycle until a repair of the damaged DNA has been carried out, or, if this is not possible, can initiate apoptosis.

Once DNA damage has been detected, DNA repair may involve a range of proteins and enzymes. For instance, damage leading to thymine dimers and methylated DNA bases can be reversed by chemical modification of DNA (e.g. by employing the enzyme guanine methyltransferase). In other cases, it may be more economical to by-pass the damaged DNA strand and use the sister strand for future purposes instead. As for single-strand damage, repair may include base excision, nucleotide excision and mismatch repair. Base excision repair, for instance, is a process removing a damaged single nucleotide (e.g. an alkylated, oxidized, hydrolyzed or deaminated nucleotide) and replacing it with an intact one.

for instance, that Bak, in order to achieve an active conformational state, requires a specific modulation of the thiol groups present at the active site [55]. Moreover, Bax, which requires a homodimerization in order to be converted to the active form, is activated by disulfide bond formation [42, 56]. In this view, oxidative alteration could be the trigger for the activation of the Bcl-2 family.

Downstream of the mitochondrial effects, ROS can act directly at the level of caspase activation; indeed, the cysteine within the active site renders caspases susceptible to oxidation. Considering that caspases have optimal activity under a reducing environment, it is not surprising that oxidative stress induced by H2O2 could suppress activation and activity of caspases by modulating the oxidation state of the cysteine and the redox status of the cellular environment [58].

Regardless ofthe activation ofpre-existing pro- or antiapoptotic proteins, oxidative stress is able to regulate and induce the transcription ofseveral genes responsible for survival and apoptosis through its activity on localization and activation of the main redox sensitive transcription factors: p53 [59], NF-kB (nuclear factor-KB) [60], AP-1 (activator protein-1) [61]. Once these factors have been activated, it is possible to observe either the upregulation of death proteins (i.e. Bax and Puma) [62] or the production of survival proteins (i.e. Xiap, Bcl-2) [63].

Transcription factor activity is modulated by oxidative stress through two different actions that may happen together or separately: the degradation of specific parts of the DNA or/and the activation of a signal transduction cascade acting as a secondary messenger. The most significant effect of redox imbalance on the signaling pathways has been observed in the MAP (mitogen-activated protein) kinase pathways [35], these may be activated by a huge variety of stimuli, but in particular JNK (Jun N-terminal kinase) and p38 kinase appear to be more responsive to various stress stimuli [64].

To conclude, we can assert that oxidative stress induces apoptosis once a cell fails to maintain the appropriate intracellular reducing environment. This process leads to activation at different levels of the apoptotic machinery, which ultimately ensures that the cells die in an orderly fashion (Figure 16.2). The next chapter will consider these apoptotic processes in more detail, focusing in part on the apoptosis of immune cells.

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