Role of bcl 2 in metabolic and redox regulation via its effects on cytochrome c oxidase and mitochondrial functions in tumor cells

1. Introduction and literature review 1.1 Early discovery of Bcl-2 as an oncogene: Bcl-2, which stands for B-cell Lymphoma/Leukemia-2 gene, was first discovered in B-cell malignancies more than twenty years ago (Tsujimoto, Cossman et al. 1985). It was identified through a set of chromosomal translocations that resulted in its activation in the majority of non-Hodgkin’s B-cell and follicular lymphomas. More specifically, bcl-2 was found to translocate from its usual 18q21 chromosomal location to 14q32, where it fuses with the promoter and enhancer of the immunoglobulin heavy chain gene to result in its excessive and deregulated expression (Cleary, Smith et al. 1986). Also, this became known as the t(14,18) breakpoint. In terms of function, increased Bcl-2 expression has been demonstrated to confer a survival advantage in B-cells, thus promoting tumorigenesis (Reed, Cuddy et al. 1988). In a pilot study, mice injected with NIH3T3 cells containing constructs of bcl2 gene developed a greater number of tumors than their negative control counterparts (Reed, Cuddy et al. 1988). In separate studies, Bcl-2 transgenic mice demonstrated an uncontrolled expansion of B-cell lymphocytes, leading to lymphadenopathy whereas Bcl-2 knockout mice were more susceptible to irradiation-mediated apoptosis and displayed lower T-lymphocyte survival rates (McDonnell, Deane et al. 1989; Sentman, Shutter et al. 1991). These results point towards the ability of Bcl-2 to protect cells from apoptosis and promote survival. The deregulation of cellular life and death homeostasis is the key to the onset and maintenance of the transformed phenotype. 1.2 Bcl-2 and Bcl-2 family proteins: Since the discovery of Bcl-2, many other Bcl-2-like proteins were subsequently discovered and documented. These Bcl-2 family proteins were generally classified into two major groups, namely pro-apoptotic and anti-apoptotic. Some of the proapoptotic proteins include Bax and Bcl-xs (Boise, Gonzalez-Garcia et al. 1993; Oltvai, Milliman et al. 1993). An alternate form of Bcl-xs is Bcl-xL which exerts antiapoptotic characteristics (Boise, Gonzalez-Garcia et al. 1993). Sequence analysis of the Bcl-2 family of proteins revealed strong homology in several regions, commonly referred to as Bcl-2 homology (BH) domains. These domains were shown to be important for the heterodimerization of the Bcl-2 family proteins, such as the BH1 and BH2 domains necessary for Bcl-2 and Bax interaction (Yin, Oltvai et al. 1994). The ability of these proteins to heterodimerize suggests that their ratio in cellular abundance is critical in determining the life and death outcome of the cell. Currently, four BH domains have been elucidated and extensively studied. Today, Bcl-2 family proteins are divided into three classifications based on these domains. The first group of Bcl-2 family proteins is anti-apoptotic and contains BH1-4 domains. These include Bcl-2, Bcl-xL, Bcl-w and Mcl-1 (Strasser 2005). The second group of members is pro-apoptotic and contains BH1-3 domains. These include Bax, Bak and Bok. Indeed, deletion of Bax and Bak impaired the apoptotic pathway through the failure to induce mitochondrial outer membrane permeability, thus preventing the release of essential apoptotic factors such as cytochrome c (Wei, Zong et al. 2001; Kuwana and Newmeyer 2003). Furthermore, deletion of the BH3 domain obliterated the pro-apoptotic activity of Bax and Bak by preventing the binding of these proteins to anti-apoptotic Bcl-2, suggesting that these pro-apoptotic proteins kill by binding and inhibiting their anti-apoptotic counterparts through the crucial BH3 motif (Chittenden, Flemington et al. 1995; Sedlak, Oltvai et al. 1995). The third group of proteins is also pro-apoptotic in nature and consist only the BH3 domain. They are the BH3-only proteins and include Bad, Bid, Bim, Bmf, Noxa and PUMA (Youle and Strasser 2008). These small proteins act through either the direct binding and inhibition of anti-apoptotic Bcl-2 proteins or the direct activation of Bax and Bak. They also exhibit varying specificities in their binding to other Bcl-2 family members (Willis and Adams 2005). Apart from their BH domains, Bcl-2 family proteins also consist of a carboxyl terminal hydrophobic transmembrane domain, which is critical for membrane localization and insertion (Goping, Gross et al. 1998). Through various imaging and biochemical techniques, Bcl-2 was found localized to various sub-cellular membranous compartments, namely the nuclear envelope, endoplasmic reticulum and outer mitochondrial membrane (Krajewski, Tanaka et al. 1993). Interestingly, structural studies of Bcl-xL revealed the importance of BH1-3 domains in defining the top of the hydrophobic groove, which is part of an essential region that interacts with pro-apoptotic members such as Bax and Bak (Muchmore, Sattler et al. 1996; Sattler, Liang et al. 1997). Analogous observation was also made in Bcl-2, differing only by amino acid sequences and size of the hydrophobic groove, possibly accounting for the different binding affinities for pro-apoptotic proteins between Bcl2 and Bcl-xL. 1.3 Role of Bcl-2 in non-apoptotic cell death: Oncogenesis is typically characterized by an imbalance between life and death, whereby an excessive signal for proliferation is further aggravated by an inability to respond to physiological death triggers, eventually leading to a buildup of cell mass. Thus, the ability to avoid various forms of cell death must certainly be a hallmark of cancer. Cell death is classified into programmed and non-programmed. Programmed cell death consists of apoptosis and autophagy, which are organized and sequential processes involved in the orderly removal of unwanted cells. In contrast, nonprogrammed cell death consists of a series of random events that lead to the disorderly disruption of cellular components, often leading to inflammation. This is known as necrotic cell death. Necrosis-associated loss of mitochondrial functions resulting in ROS formation and leakage, leading to downstream deleterious events can be modulated and altered by the action of Bcl-2 at the outer mitochondrial membrane, regulating the organelle’s membrane integrity and permeability (Kane, Ord et al. 1995; Bredesen, Rao et al. 2006). In normal cells, the physiological function of autophagy seems to promote survival in order to protect cells from starvation and nutrient-deprived conditions (Levine and Klionsky 2004). However, in tumor cells, excessive breakdown of cellular components may lead to cell death (Otsuka and Moskowitz 1978; Kisen, Tessitore et al. 1993). In this respect, nutrient-deprived cancer cells often generate a lower autophagic response than normal cells. This protective down-regulation may perhaps be associated with Bcl-2. Indeed, a key autophagic and tumor suppressive protein known as Beclin 1, was shown to physically interact with Bcl-2 and Bcl-xL using its BH3 domain, thus neutralizing its autophagic activity (Shimizu, Kanaseki et al. 2004; Pattingre, Tassa et al. 2005; Maiuri, Le Toumelin et al. 2007). Disruption of this interaction restored the autophagic function of Beclin 1, suggesting an antiautophagic role for Bcl-2 and Bcl-xL (Maiuri, Le Toumelin et al. 2007). 1.4 Classical mechanisms of Bcl-2 in apoptotic cell death: Although apoptosis was discovered in 1972, the first detailed illustration of the apoptotic cell death pathway was elegantly conducted in by following the development of Caenorhabditis elegans (Kerr, Wyllie et al. 1972; Sulston and Brenner 1974). In mammals, apoptosis can be separated into two forms, the extrinsic and intrinsic pathways (Danial and Korsmeyer 2004). Both pathways lead to the downstream processing of unique proteases, known as initiator and executioner caspases. The extrinsic pathway is signaled through the activation of a surface receptor such as Fas receptor, leading to the activation of initiator caspase 8, triggering the cleavage and activation of downstream effector caspases such as caspase (Hengartner 2000). Cells that are deficient in the extrinsic pathway are often compensated by a robust intrinsic pathway, where the mitochondria play a central role in the induction of apoptosis. The intrinsic pathway usually involves the translocation of cleaved Bid to the mitochondria, which in turn drives the activation of Bax to induce cytochrome c release via the disruption of the mitochondrial outer membrane permeability, leading to downstream events including the formation of the apoptosome, activation of caspase 9, cleavage of caspase and the downstream degradation of cellular components such as lamin and PARP (Hengartner 2000). Indeed, overexpression of Bcl-2 in Caenorhabditis elegans was shown to rescue the cells from programmed cell death (Vaux, Weissman et al. 1992). Furthering this, many other studies went on to demonstrate the involvement of various other Bcl-2 family proteins in the regulation of apoptosis (Horvitz 1999). With respect to Bcl-2, given its localization to the outer mitochondrial membrane, overexpression of Bcl-2 would block the intrinsic apoptotic pathway and not the extrinsic pathway (Krajewski, Tanaka et al. 1993; Nguyen, Millar et al. 1993). Mitochondria, the powerhouse of the cell, essential for providing the main source energy, is also a crucial regulator of the intrinsic apoptotic pathway as it contains a plethora of apoptogenic factors that can trigger apoptosis upon release (Green and Reed 1998; Kroemer, Dallaporta et al. 1998). Death-inducing stimuli such as irradiation, cytokine deprivation and chemotherapeutic compounds can all trigger mitochondrial-dependent apoptosis, characterized by the depolarization of mitochondrial transmembrane potential leading to the permeabilization of the mitochondrial outer membrane (MOMP) (Hail 2005). In this respect, overexpression of Bcl-2 in tumor cells can inhibit MOMP and bring about chemoresistance (Vander Heiden and Thompson 1999). Upon exposure to apoptotic triggers, MOMP is induced by pro-apoptotic cytosolic Bid and Bax, which undergo a conformational change caused by mechanisms such as dephosphorylation and proteolytic cleavage in order to expose the pro-apoptotic BH3 domain of these proteins (Zha, Harada et al. 1996; Desagher, Osen-Sand et al. 1999; Li, Boehm et al. 2007). This conformational change brings about the translocation of these proapoptotic members to the mitochondria. Upon translocation, these pro-apoptotic members such as Bax and Bak have been postulated to oligomerize and form porelike channels to permeabilize the outer mitochondrial membrane or regulate mitochondrial membrane channels such as ANT and VDAC in a fashion that causes mitochondrial matrix swelling and outer membrane disruption, with MOMP being the end result (Brenner, Cadiou et al. 2000; Wei, Zong et al. 2001; Zamzami and Kroemer 2001). The onset of MOMP leads to the release of several apoptogenic factors resident within the mitochondrial intermembrane space and these include cytochrome c and Apoptosis Inducing Factor (AIF). Cytochrome c released into the cytosol is a precondition for the downstream induction of Apaf-1 oligomerization as well as activation of caspase 9. These components associate together to form a complex called the apoptosome that triggers the activation of executioner caspases and 7, leading to protein degradation and overall breakdown of the cell (Gross, McDonnell et al. 1999; Slee, Harte et al. 1999; Hengartner 2000). Contrary to the actions of Bax and Bak, Bcl-2 and Bcl-xL are able to inhibit MOMP through the direct interaction with the outer mitochondrial membrane channel, VDAC, preventing its closure induced by Bax and Bak (Shimizu, Narita et al. 1999; Vander Heiden, Li et al. 2001; Shi, Chen et al. 2003). On the other hand, Bcl-2 has also been proposed to function as an ionophore to dissipate the transmembrane potential that is responsible for the closure of VDAC (Vander Heiden and Thompson 1999). Nonetheless, both mechanisms of action result in the maintenance of the ATP/ADP exchange and prevent hyperpolarization of the mitochondrial transmembrane potential, leading to organelle swelling, rupture and eventual collapse of the transmembrane potential. 1.5 Bcl-2 and its network of interacting partners: It is well-established that Bcl-2 is able to recognize and bind to their pro-apoptotic counterparts, thus leading to their sequestration and inability to carry out their proapoptotic function. The ‘addiction’ of Bcl-2 family proteins to seek out and bind to one another in tumor cells suggests that the ratio of proteins from the various classes of Bcl-2 family can tilt the cell either towards life or death. This implicates a major chemotherapeutic advantage considering that tumor cells often overexpress antiapoptotic Bcl-2 and introducing pro-apoptotic Bcl-2 family mimetics can specifically target and neutralize Bcl-2 in tumor cells, without affecting or killing normal cells. More importantly, given that Bcl-2 has also been shown to localize to the nuclear envelope and endoplasmic reticulum, many studies have demonstrated the ability of Bcl-2 to bind and interact with proteins outside of the Bcl-2 family as well as beyond the mitochondria. The interactions with these non-homologous proteins bear significance in the capability of Bcl-2 to integrate into a larger signaling network, incorporating components and organelles outside of the mitochondria to govern cell death. Recently, p53 was shown to be able to localize to the mitochondria and directly induce apoptosis by inducing mitochondrial permeabilization and cytochrome c release (Marchenko, Zaika et al. 2000). Upon apoptotic stimuli such as irradiation, the ability of p53 to directly induce apoptosis via the mitochondrial-dependent pathway was attributed to its direct binding of Bcl-2 and Bcl-xL, displacing sequestered Bax and triggering the downstream oligomerization of Bax, leading to cytochrome c release (Mihara, Erster et al. 2003). Interestingly, this was achieved in the absence of a BH3 domain in p53, instead p53 binds to Bcl-2 using its proline-rich domain (Mihara, Erster et al. 2003). The results of these studies suggest that an overexpression of Bcl-2 could inhibit the transcriptional-independent, death-inducing role of p53 through the direct binding and sequestration of p53. Apart from p53, Bcl-2 can also bind to oncogenic Ras and orphan nuclear receptor Nur77 (Fernandez-Sarabia and Bischoff 1993; Lin, Kolluri et al. 2004). In the former interaction, although Ras is usually known to promote survival in tumor cells through the PI3-kinase/Akt pathway, it has also been demonstrated to possess pro-apoptotic activity by up-regulating Fas ligand and bringing about Fas receptor-mediated apoptosis. In this aspect, overexpression of Bcl-2 rescued cells from Fas-mediated apoptosis by interacting and blocking the apoptotic activity of mitochondrial Ras (Downward 1998; Denis, Yu et al. 2003). With regard to Bcl-2 interaction with Nur77, a highly novel function of Bcl-2 was reported. Interaction of Bcl-2 with Nur77 led to a conformational change in Bcl-2, exposing its BH3 domain, converting Bcl-2 from anti-apoptotic to pro-apoptotic (Lin, Kolluri et al. 2004). 1.6 Non-canonical role of Bcl-2 in redox regulation: Just as p53 has been portrayed to display a non-conventional transcriptionalindependent role in cell death regulation, the role of onco-protein Bcl-2 in promoting tumor cell survival has been designated for further investigation from another perspective, that of ROS and mitochondrial bioenergetics. Given the mitochondrial localization of Bcl-2, can Bcl-2 possibly preserve or optimize oxidative phosphorylation to tailor to the survival instincts of the tumor cell from a ROS perspective? Traditionally, Bcl-2 has been portrayed as an anti-oxidant due to its ability to suppress oxidative stress-induced lipid peroxidation when overexpressed in murine lymphoma cells (Hockenbery, Oltvai et al. 1993). 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Genova et al 20 04; Fontanesi, Soto et al 20 06) 1.14 COX Va and COX Vb as cancer markers: 32 Interestingly, recent studies have demonstrated up -regulation and increased involvement of COX Va and Vb in a variety of cancers Autocrine gastrins-induced up -regulation in COX Vb resulted in decreased cytochrome c release and caspase 3 activation in colon cells (Wu, Rao et al 20 00) Moreover, protection against apoptosis... through electron transport chain activities In particular, this study focuses the investigation on the terminal, rate-limiting COX enzyme Though not an indigenous electron-leaking, ROS-producing complex, COX is nonetheless crucial in determining the rate of electron transport across the complexes Increased mitochondrial respiration may consequentially increase the byproduction of O2- due to increased... Campian, Gao et al 20 07) 1.15 Concluding remarks: Existing literature continued to expound on the complexity of ROS regulation and the role it plays in tumorigenesis Concurrently, the emergence of COX facilitates the mechanistic study on the impact of ROS in cancer from a metabolic perspective as well as challenging the established role of Bcl- 2, thus forming the scope of this thesis 33 2 Aim of the study... dimerization and regulation of catalytic activity of the COX enzyme as well as protecting the catalytic core from ROS The importance of these subunits is best demonstrated by the loss of COX activity and mitochondrial respiration in yeast strains encoding null mutations of the various nuclear-encoded subunits (Fontanesi, Soto et al 20 06) In mammalian cells, these nuclear-encoded subunits occur in tissue-specific... in mammalian cells undergoing hypoxia, HIF-1 has been demonstrated to play a central role in regulating the efficiency of mitochondrial respiration via its effect on altering the composition of COX4 subunit isoforms by reinforcing the 27 expression of COX-4 -2 and LON protease, whereby the latter is responsible for COX-4-1 degradation The outcome of an oxygen-sensitive regulation in composition of COX4... ROS-producing systems and anti-oxidant defense mechanisms, maintaining a species-specific preference for cancer cell survival and death pathways The deregulation of mechanisms controlling ROS production and turnover can lead to serious consequences in upsetting the balance in concentration between the different species of ROS, leading to a more pronounced and aggressive malignancy or a regression in tumorigenesis... reduction in H2O2 results in an enhanced T-cell activation during an immune response via an increase in the promoter activity, transcription and expression of IL2 and its receptor (Droge, Eck et al 19 92; Droge 20 02) The imbalance between ROS production and breakdown has been postulated to be responsible for various disorders, more prominently, carcinogenesis The increased metabolic rate of tumor cells, ... oncoprotein p21Ras activation of Rac1 led to an increase in cell proliferation via a concomitant increase in levels of O2- (Irani, Xia et al 1997) This was corroborated by the ability of constitutively active Ras to maintain an elevated level of O2-, contributing to the resistance upon drug-induced apoptosis Conversely, the expression of a dominant-negative form of Rac1 reduced the levels of 19 O2- and. .. (Fontanesi, Soto et al 20 06) Regulation of COX biogenesis and activity in response to changing environment or physiological conditions plays a defining role in cellular metabolic adaptation Regulation in COX biogenesis enables the modulation of its enzymatic activity in response to substrate availability and oxygen concentration Various parameters defined the formation of the final COX enzyme, which . advantage considering that tumor cells often overexpress anti- apoptotic Bcl- 2 and introducing pro-apoptotic Bcl- 2 family mimetics can specifically target and neutralize Bcl- 2 in tumor cells, without. Interaction of Bcl- 2 with Nur77 led to a conformational change in Bcl- 2, exposing its BH3 domain, converting Bcl- 2 from anti-apoptotic to pro-apoptotic (Lin, Kolluri et al. 20 04). 1.6 Non-canonical. Non-canonical role of Bcl- 2 in redox regulation: Just as p53 has been portrayed to display a non-conventional transcriptional- independent role in cell death regulation, the role of onco-protein Bcl- 2 in