Oxidative stress by targeted agents promotes cytotoxicity in hematologic malignancies.

The term oxidative stress refers to an imbalance in the antioxidant-to-prooxidant ratio within a cell. This ratio is constantly negotiated in cells because homeostatic cellular function generates oxidative species that are continuously inactivated by antioxidant systems. Endogenous sources of oxidant stress, such as mitochondrial electron transport and activation of oxidases, generate free radicals as byproducts of their function (Fig. 1). It has been estimated that ∼1–2% of the total oxygen consumption of mitochondria generates reactive oxygen species (ROS); therefore, oxidative phosphorylation is the major endogenous source of oxidative stress (41). Cellular oxidases are another source of ROS. A prototypical example of such an oxidase is the NADPH oxidase complex, which functions to deliver a superoxide burst as a defense against bacteria. Similarly, oxidases like the xanthine oxidases, monoamine oxidases, and other flavoenzymes are also endogenous sources of oxidants (33). FIG. 1. Endogenous sources of oxidative stress. Four sources of oxidative stress that represent normal metabolic pathways are represented. The NADPH oxidase is a membrane-bound enzyme complex that generates superoxide. Xanthine oxidase is an enzyme important ... Superoxide is the specific byproduct of both mitochondrial respiration and of the aforementioned oxidases and is one example of a ROS. ROS refers to oxygen-containing breakdown products of molecular oxygen that are highly reactive and are able to damage lipid membranes, proteins, and DNA when present in high amounts. This damage is not necessarily perpetuated by superoxide itself but by further breakdown products of molecular oxygen. For example, superoxide is inactivated primarily by the superoxide dismutase (SOD) enzymes. The reaction by which superoxide is broken down actually generates hydrogen peroxide, another ROS entity. Unlike superoxide, hydrogen peroxide can traverse biologic membranes, thereby expanding its range of reactivity because it can travel from outside the cell to inside the cell and from one subcellular organelle to another. Hydrogen peroxide can be further inactivated by an array of antioxidants. However, in the presence of transition metals such as Fe and Cu, the Fenton reaction catalyzes the generation of hydroxyl radical, the most highly reactive and damaging ROS species. Therefore, overt damage to macromolecules is most often promoted by the hydroxyl radical. Again, endogenous oxidative stress rarely leads to damage, because a healthy cell generally possesses an armory of antioxidants to inactivate and dispel ROS, thereby obviating any harm to the cell. However, when cellular antioxidants are overwhelmed to a great degree, which occurs in the context of external environmental challenges like toxic insults or radiation, cell death is the expected outcome. The type of cell death triggered by oxidative stress is dependent on the dose and duration of the exposure. Necrotic cell death is thought to result from a higher amount and exposure to oxidant stress than the amount necessary to elicit apoptotic cell death. The examples of cancer therapies to be discussed in this article focus on oxidative stress–induced apoptosis, although we cannot rule out the possibility that some of these agents cause other types of cell death, such as autophagy and necrosis. A defining feature of apoptotic cell death is activation of cysteine proteases called caspases that function to activate one another and ultimately dismantle the cell (65). A multimember family of proteins, caspases that initiate the cell-death cascade are caspase-8, caspase-9, caspase-2, and caspase-4. Caspase-8 is the initiator for death signals stemming from outside the cell, whereas caspase-9 is triggered by signals within the cell. Mechanisms of caspase-2 and -4 activation are less well understood, but are also internal to the cell. Caspase-4 is thought to be activated by stress initiated in the endoplasmic reticulum (ER), also referred to as ER stress (45). Mitochondria represent a convergence point for oxidative stress–induced apoptosis. This organelle can serve as a starting point of oxidative stress or a transducer of oxidative stress–induced apoptotic signaling (116). A large percentage of endogenously produced oxidants are generated continuously during mitochondrial oxidative phosphorylation, with superoxide being the predominant entity. A higher rate of oxidative phosphorylation could initiate an oxidative stress if mitochondrial antioxidants are not adequately protective (42). In line with this concept, a loss of mitochondrial antioxidant capacity could also initiate an oxidative stress from within mitochondria, because the normal defenses would be reduced. A buildup of oxidants outside mitochondria can also promote apoptosis. The mitochondrial permeability transition, a pore complex that allows the exchange of solutes across mitochondrial membranes, is triggered by oxidants (59). A major player in promoting activation of caspases is the mitochondrial resident, cytochrome c. On exposure to an apoptotic trigger perturbing mitochondria, cytochrome c leaves the intermembrane space of mitochondria and enters the cytosol. Once in the cytosol, it forms a complex with dATP, an adaptor molecule called Apaf-1, and caspase-9. This conglomerate, called the apoptosome, functions to activate caspase-9, which then activates caspase-3 (26, 58). Other proapoptotic molecules are known to be released from mitochondria to promote caspase activation, but of these, only the exit of cytochrome c from mitochondria has been linked to redox (43). In the intermembrane space of mitochondria, cardiolipin, a mitochondrial phospholipids, tethers cytochrome c in place. Oxidation of cardiolipin, under conditions of mitochondrial oxidative stress, loosens cytochrome c and promotes its release. Release of cytochrome c, in addition to the other various proteins released when mitochondria are disrupted, is thought to require permeabilization of the mitochondrial outer membrane. This permeabilization exposes the rest of the cell to various oxidant species normally housed (and neutralized) within mitochondria. The way in which oxidative stress triggers apoptosis can loosely be characterized as a direct effect on apoptotic machinery or via an indirect effect on apoptosis (Fig. 2). Adaphostin, a kinase inhibitor, which is discussed in greater detail later, is an agent that acts in a direct manner because the active drug accumulates in mitochondria, thereby compromising oxidative phosphorylation (58). Because oxidative phosphorylation is a major generator of ATP, the cellular energy currency, this causes an ATP-depletion/energy crisis and initiates apoptosis. Another example of chemotherapeutic agents that directly modulate oxidative stress and cell death are the Bcl-2 inhibitors (88, 114). In contrast, an indirect effect on apoptosis induction would be a proteasome inhibitor blocking proteasome function and then causing accumulation of an ubiquitinated proapoptotic protein. Similarly, epigenetically targeted agents also induce cell death indirectly because the proximal targets for those agents are gene expression. All four of these classes of agents are discussed in greater detail later. FIG. 2. Direct versus indirect effects of new agents on oxidative stress–induced apoptosis. The tyrphostin inhibitor, adaphostin, induces cell death by a more direct route than do proteasome inhibitors. Adaphostin (chemical structure is shown) accumulates ... Several leukemia and lymphoma model systems have been associated with heightened levels of oxidative stress. Instead of triggering death pathways, this increased oxidative stress in transformed cells, as compared with nontransformed cells, appears to be part of the milieu in which the cancer cell thrives. Consistent with this notion, numerous reports document the ability of a low level of oxidative stress to stimulate signaling, via activation of kinases or inactivation of phosphatases or both, that ultimately transduces a mitogenic signal (27, 44, 52). The source of the increased oxidative stress seen in hematologic malignancies is likely not attributable to a single entity. Hyperactivation of endogenous sources of ROS, such as electron transport or the NADPH oxidase, has been documented in leukemia cells, potentially accounting for the observed increased levels of ROS. Furthermore, specific oncogenes are known to cause an increase in oxidative stress in addition to exerting effects on downstream signaling molecules. Relevant to leukemia, the BCR/ABL oncogene, which is expressed in chronic myeloid leukemia (CML), increases levels of intracellular peroxides and superoxides (100, 108). Electron transport and PI3-kinase signaling have been fingered as responsible for ROS in BCR/ABL-expressing cells; however, inhibitors of these processes yielded only a partial reduction in ROS levels (48). In AML, mutation of the Flt3 receptor is seen in a subset of poor-prognosis patients and was recently shown to increase ROS levels via a NADPH oxidase–dependent pathway (99). Yet another explanation for heightened ROS in leukemia cells invokes mitochondrial DNA mutations that may alter electron transport (84). As observed in other cancer cells, leukemia cells appear to use glycolytic pathways more than do normal lymphocytes, harkening back to the 1920s when Otto Warburg (14) proposed that fundamental differences in metabolism exist between normal cells and cancer cells.