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

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

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

bcl-2 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 pro-apoptotic 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 anti-apoptotic 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 Bcl-

2 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, non-programmed 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 anti-autophagic 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 3 (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 3 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 pro-apoptotic members to the mitochondria Upon translocation, these pro-apoptotic members such as Bax and Bak have been postulated to oligomerize and form pore-like 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 pre-

condition 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 3 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 pro-apoptotic 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 anti-apoptotic 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 independent 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) Many other studies went on

transcriptional-to confirm this finding (Tyurina, Tyurin et al 1997) In addition, Bcl-2 was also shown to reduce NO2- production in response to oxidative stress and in contrast, mice lacking Bcl-2 were more susceptible to oxidative stress-mediated damage (Hochman,

Sternin et al 1998; Lee, Hyun et al 2001) Moreover, it was reported that the oxidative property of Bcl-2 was attributed to its ability to up-regulate cellular anti-oxidant defense mechanisms such as Cu/Zn SOD, catalases, glutathione peroxidases and GSH levels in tumor cells (Ellerby, Ellerby et al 1996; Jang and Surh 2003; Rudin, Yang et al 2003; Zimmermann, Loucks et al 2007)

anti-In spite of conventional acceptance of Bcl-2 as an anti-oxidant, another body of evidence has challenged this notion by demonstrating that under normal physiological conditions, overexpression of Bcl-2 in cells did not result in an initial anti-oxidative intracellular milieu but instead brought about increased oxidative damage (Steinman 1995) The consequential up-regulation of intracellular anti-oxidant defenses was postulated to be a compensatory response to the initial pro-oxidant activity of Bcl-2 (Steinman 1995) Still, other models employing mouse and bacteria corroborated our results demonstrating Bcl-2 as a pro-oxidant protein (Adams, Pierce et al 2001) Various experimental models established the anti-oxidant property of Bcl-2 by triggering cells with death-inducing stimuli or directly overwhelming the cells with oxidative stress before accruing the resultant anti-oxidant response to Bcl-2 expression At best, these studies accredit a redox regulatory role for Bcl-2 in countering oxidative stress, but do not suffice to confirm Bcl-2 as having innate anti-oxidant characteristics True to this aspect, pure Bcl-2 has been shown to be devoid of intrinsic anti-oxidant activity (Lee, Hyun et al 2001) Hence, it was suggested that the anti-oxidant function of Bcl-2 previously reported could be accrued to the physiological response of Bcl-2 as a modulator of ROS

1.7 Types of ROS:

Reactive oxygen species (ROS) refer to a set of molecules derived from molecular oxygen and consist of a combination of oxygen radicals including superoxide (O2-), and hydroxyl (OH-) as well as non-radical derivatives of molecular oxygen such as hydrogen peroxide (H2O2) Physiological processes such as the mitochondrial electron transport chain activities and membrane-bound enzymes such as NADPH oxidase, typically found in phagocytic cells for its microbicidal function, represent the two main producers of intracellular ROS (Bergstrand 1990; Nohl, Gille et al 2005) The leakage of electrons to spontaneously combine with molecular oxygen in the mitochondria and the enzymatic reduction of molecular oxygen with a single electron by NADPH oxidase forms O2-

Despite the notion that O2- and H2O2 are not highly reactive with other intracellular constituents, the end product of a reaction between these two species is able to elicit extensive detrimental effects within a cell and accounts for most of the intracellular oxidative damage This end product is known as the hydroxyl radical (Fridovich 1978) Hydroxyl radical interacts and damages intracellular components such as DNA, lipids and proteins in a non-specific fashion, eventually leading to necrotic cell death ROS is also widely implicated in a variety of oxidative stress-related clinical manifestations and applications such as neurodegeneration, inflammation, aging, atherosclerosis, ischemia-reperfusion injury in cardiac tissues and chemotherapeutic effects (Waris and Ahsan 2006) Involvement of ROS in an extensive network of

signaling pathways, governing a multitude of cellular processes that eventually determine cellular functions, survival and death necessitates and justifies the amount

of past and ongoing work that has been conducted in the field

1.8 Regulation of ROS – Producers of ROS and anti-oxidant mechanisms:

As mentioned in the previous section, the mitochondria constitute one of the largest ROS-producing organelles Being the powerhouse of the cell in generating valuable energy for various cellular processes and functions, the mitochondrial electron transport chain naturally and inevitably becomes one of the greatest sources of ROS This is due to the fact that the electron transport is not an entirely efficient process as

up to 1-3% of molecular oxygen can be converted to ROS in the mitochondria due to the leakage of electrons (Boveris and Chance 1973) Mitochondrial respiration results

in a continuous stream of electrons being transferred from one complex enzyme to another before eventually reducing molecular oxygen to produce water at the terminal enzyme, COX Some of these electrons are inevitably lost during electron transport and react with the surrounding oxygen molecules to give rise to O2- within the organelle

The main O2--producing complexes are complex I and III, where it was further shown that complex III predominantly produces O2- to the cytoplasmic side of the inner mitochondrial membrane, suggesting that O2- from complex III preferentially accumulates at the mitochondrial intermembrane space (Grigolava, Ksenzenko et al

1980; Turrens and Boveris 1980; Turrens, Alexandre et al 1985; Muller, Liu et al 2004)

In addition to the mitochondrial electron transport chain, NADPH oxidase complex is another major source of ROS production The enzyme consists of subunits gp91phox, p22phox, p47phox, p67phox, p40phox (Babior 1999) Association of the small GTP-binding protein Rac1 is necessary for the membrane complex to be functional in order

to carry out its microbicidal role through the massive production of an oxidative burst

of O2-, typically observed in phagocytic cells (Babior 1999) Recent evidence has demonstrated that the role of NADPH oxidase observed in phagocytic cells extends into transformed cells as well and contributes towards oncogenesis Indeed, various forms of NADPH oxidases (Nox) have been reported in different cancers such as Nox4 in pancreatic cancer and Nox5 in melanoma and prostate cancer (Brar, Corbin

et al 2003; Mochizuki, Furuta et al 2006) In these studies, ROS produced from these Nox proteins contribute towards the survival signaling pathways observed in these cancers

Apart from NADPH oxidases, other ROS-producing enzymes include xanthine oxidase, flavoprotein dehydrogenase, aldehyde oxidase, tryptophan dioxygenase and dihydroorotate dehydrogenase (Freeman and Crapo 1982) Out of these, xanthine oxidase remains the most widely studied oxidase and its O2--producing function has

been exploited for in vitro studies to understand the effect of ROS on a variety of

cellular processes Over and above the enzymatic production of ROS, organelles such

as endoplasmic reticulum and nuclear membrane has also been reported to participate

in ROS production More specifically, endoplasmic reticulum-derived O2-, though yet

to be implicated in growth and survival signaling, has nonetheless been postulated to play a role in the some of the organelle’s main functions such as protein folding and secretion (Bauskin, Alkalay et al 1991; Hwang, Sinskey et al 1992; Bader, Muse et

al 1999) In relation to nuclear membrane, studies have demonstrated the existence of cytochrome oxidases and electron transport systems that may contribute to the formation of ROS through the leakage of electrons from these systems, leading to DNA damage, even though the exact function of these systems remain unclear (Droge 2002) Moreover, nuclear localization of Nox4 also implies redox regulation of nuclear-related or even cellular processes (Kuroda, Nakagawa et al 2005; Ushio-Fukai 2006)

With the presence of ROS-producing entities and the escalating threat of oxidative stress-induced damage if uncontrolled, the cell has evolved a set of anti-oxidant protective mechanisms to maintain a level of ROS that is sufficient for intracellular signaling functions but does not suffice to produce deleterious effects The anti-oxidant defense machinery comprises of several enzymes and non-enzymatic ROS scavengers These enzymes include MnSOD, Cu/Zn SOD, catalase, GSH peroxidase and GSH reductase ROS-scavenging compounds include alpha-tocopherol (vitamin E), β-carotene, ascorbate (vitamin C), glutathione and some free amino acids (Fridovich 1986; Halliwell 1999)

The function of MnSOD and Cu/Zn SOD is to dismutate radical O2- to non-radical

H2O2 (Fridovich 1986) In spite of H2O2 not being highly reactive with other cellular components, the iron-dependent, reductive homolytic cleavage of the compound is able to generate a more potently cytotoxic compound called hydroxyl radical in a reaction known as the Fenton reaction (Andreyev, Kushnareva et al 2005) The hydroxyl radical is responsible for the majority of oxidative damage leading up to necrotic cell death In order to prevent this detrimental route of H2O2 breakdown, the presence of catalase converts two molecules of H2O2 to form molecular oxygen and water Apart from catalase, which is a form of peroxidase, glutathione peroxidase also helps to transform H2O2 to molecular oxygen and water, albeit with the requirement

of the intracellular reducing agent, glutathione (Fridovich 1978; Furtmuller, Zederbauer et al 2006)

It has also been noted that the ability of free amino acids to act as ROS scavengers is more enhanced than proteins Thus, as far as oxidative proteolysis is concerned, the ability of free amino acids to scavenge ROS provides the basis for self-regulation and homeostasis of redox-dependent protein breakdown (Droge 2002) Thus, anti-oxidative defense mechanisms are essential to regulate the cellular redox state without incurring excessive and overwhelming levels of ROS that can bring about irreversible cellular damage However, such protective, self-regulatory mechanisms are only possible and functional under transient increases in ROS whereas, overwhelming levels of ROS would culminate in cell death and tissue damage, regardless whether the mechanisms are elicited On the other hand, deregulation in

ROS production and/or its regulatory machinery can lead to abnormal accumulation

of ROS, leading to species-dependent conditions that are favorable for the onset, maintenance and progression of cancer

1.9 ROS in cell death and survival:

Conventional dogma has long established ROS as agents of detriment Indeed, a great number of studies have documented the role of ROS as mediators of damage to cellular structures, nucleic acids, proteins and lipids Modification of the DNA molecule by ROS represents the first step of mutagenesis and if left unchecked, carcinogenesis ensues Lipid peroxidation and protein modification of Bax monomer

to promote oligomerization of Bax are common features of ROS-mediated damage, culminating in the compromise of the mitochondrial outer membrane integrity and the

subsequent release of cytochrome c to initiate the mitochondrial death pathway

(Buccellato, Tso et al 2004) These form the premise for the use of ROS-based chemotherapeutics in cancer intervention and management

While it is true that overwhelming ROS are harmful to cells, an emergent growing body of evidence indicates the importance of low levels intracellular ROS in physiological signaling, tumor promotion/ initiation and its subsequent maintenance and progression There is sufficient evidence to strongly support a paradigm shift from the convention of ROS as only a mediator of cell damage/death to that of survival/proliferation Mild elevation in O2- or H2O2 has been demonstrated to promote growth responses in a variety of cell types through activation of growth-

related genes such as c-fos and c-jun, alterations in protein kinase activities, oxidative

modifications to phosphatases and activation of transcription factors (Burdon 1995; Sauer, Wartenberg et al 2001) Some of these include ROS stimulatory effect on the PI3-kinase/AKT survival pathway through the oxidative inactivation of PTEN, redox regulation of MAPKs such as c-Jun N-terminal kinase, p38MAPK and ERK, as well

as activation of transcription factors such as AP-1 and NF-κB (Heffetz, Bushkin et al 1990; Droge 2002; Leslie, Bennett et al 2003; Qin and Chock 2003; Bubici, Papa et

al 2006) More importantly, NADPH-dependent generation of ROS has been reported upon growth factor stimulation or cytokine receptor activation such as PDGF, TNF-α, IL6, IL3, FGF-2, TGF-α and insulin, implicating ROS as secondary messengers of survival and proliferative signaling (Sauer, Wartenberg et al 2001; Droge 2002)

Intracellular ROS functions in a diverse fashion, implicating different cellular components, to promote cell growth and survival Indeed, ROS has been shown to positively modulate a variety of ion channels such as IP3 receptor-dependent Ca2+transport, Na+/Ca2+ and Na+/H+ exchangers at the plasma membrane, leading to growth stimulation (Burdon 1995; Sauer, Wartenberg et al 2001) One of these studies recently implicate ROS in the species-dependent activation and inactivation of

Na+/H+ exchanger to promote cell division or death via the creation of an alkaline or acidic intracellular environment respectively (Shibanuma, Kuroki et al 1988; Akram, Teong et al 2006) Moreover, species-specific regulation of various processes in different lineages of cells has also been widely investigated It has been shown that

O2- in the presence of an intracellular 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 1992; Droge 2002)

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, coupled with the enhanced production and reduced removal of ROS to create a pro-oxidant intracellular milieu, has been linked to promote the survival of cancer cells (Cerutti 1985; Burdon, Gill et al 1989; Burdon, Gill et al 1990) This causal effect of ROS to promote cellular transformation is backed by evidence provided from various studies such as the ability of O2- to cause DNA oxidation and promote the transformed cell type, which can be reversed by the expression of MnSOD Expectedly, a pro-oxidant milieu is also able to suppress MnSOD expression and activity, leading to further accumulation of the species This not only emphasizes the role of MnSOD as a tumor suppressor but also the importance of O2- in tumorigenesis (Church, Grant et al 1993; St Clair, Oberley et al 1994; Oberley 2001) In addition, 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

O2- and abolished the resistance conferred against apoptosis (Irani and Clermont 1998; Pervaiz, Cao et al 2001)

Goldschmidt-Further studies have gone on to show that the intricate balance between the different species of free oxygen radicals is important in determining the decision of the cell fate A tilt in favor of O2- levels such as the inhibition of Cu/Zn SOD using DDC, with no appreciable increase in H2O2 renders the cancer cell refractory to death execution and preserves viability, irrespective of the trigger (Clement, Ponton et al 1998; Clement and Pervaiz 1999; Pervaiz, Ramalingam et al 1999; Pervaiz, Seyed et

al 1999; Clement and Pervaiz 2001) In contrast, increased levels of H2O2 and a corresponding drop in O2- levels via an overexpression of Cu/Zn SOD create a reduced and acidified environment conducive for the apoptotic signal to filter through (Clement, Ponton et al 1998; Pervaiz, Seyed et al 1999) This notion was further reinforced by a particular study showing a direct determination of cell death or survival by non-necrotic and non-overwhelming fluctuation in levels of O2- (Lin, Pasinelli et al 1999) Correspondence between increased activity of O2- producing systems and proliferation networks such as the capability of Nox 1 to produce O2- and induce cell growthas well as the fact that several anti-cancer drugs exert their effects via H2O2 mediated killing further lends weight to the pro-oxidant theory of carcinogenesis, prevalent in many tumor types, depending on theintracellular levels and species involved

On the other hand, the requirement of a reduced intracellular environment for apoptotic conditions confounds the essential role of O2- in apoptotic signaling, thus throwing the involvement of H2O2 into the spotlight (Halliwell and Gutteridge 1990; Jacobson, Burne et al 1993) Despite being relatively non-reactive and a poor redox agent, H2O2 has been shown to trigger apoptosis or necrosis depending on its concentration (Hampton and Orrenius 1997; Clement, Hirpara et al 1998) H2O2-mediated apoptosis is typified by an environment permissive for death signaling through a corresponding drop in O2- levels and intracellular pH whereas H2O2-induced necrosis brings about characteristics reminiscent of oxidative stress and damage (Clement, Ponton et al 1998; Ahmad, Clement et al 2004; Ahmad, Iskandar

pro-et al 2004) Generally, treatment with anti-cancer therapeutics triggers the intracellular production of H2O2, leading to MOMP and release of apoptogenic

factors such as cytochrome c and AIF (Reed and Kroemer 2000) Nonetheless, of

interest to this thesis is to pinpoint the mechanisms leading to the emergence of the early O2- that is generated in creating the pro-oxidant intracellular state, favorable for the survival of tumor cells, with respect to Bcl-2

1.10 Paradigm shift on ROS implicating a pro-oxidant role of Bcl-2:

Recent work has unraveled ROS as being more than just indiscriminate mediators of cell and tissue damage through oxidative stress Although it may be true that overwhelming levels of ROS may induce non-specific and random deleterious events detrimental to the cell, moderate levels of ROS may determine specific signaling pathways governing cell death and survival that are both species- and threshold-

dependent Specifically, when the ratio of O2- over H2O2 is increased, survival is favored through the enhancement of proliferative signals (Clement, Hirpara et al 1998; Pervaiz, Cao et al 2001) Conversely, when the concentration of H2O2 is greater than O2-, the intracellular microenvironment becomes conducive for the apoptotic machinery to function through an acidification of the cytosolic compartment (Hirpara, Clement et al 2001; Pervaiz and Clement 2002; Ahmad, Iskandar et al 2004; Akram, Teong et al 2006) Thus, low levels of ROS are essential for the homeostatic regulation of various cellular processes by acting as signaling messengers, thus ensuring a normal cellular turnover, necessary for tissue survival (Sauer, Wartenberg et al 2001) In the tumor context, moderate levels of ROS are tightly regulated by both 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

This paradigm shift in the perspective on ROS was illustrated by a growing body of evidence implicating Bcl-2 and the production of O2- to result in a pro-oxidant state that is responsible in conferring a survival advantage in tumor cells Recent work indicate that Bcl-2 did not operate as an anti-oxidant on its own but rather, its expression levels was directly associated with a pro-oxidant intracellular milieu that triggered the reinforcement of the endogenous anti-oxidant defense machinery

(Pervaiz and Clement 2007) In turn, this mild pro-oxidant state was connected to the death inhibitory activity of Bcl-2 as shown in leukemia cells (Clement, Hirpara et al 2003) Moreover, inhibition of NADPH oxidase activity by DPI and dominant-negative form of Rac1 decreased intracellular O2− and rendered Bcl-2 overexpressing cells more sensitive to apoptosis, suggesting specificity for O2− in Bcl-2 mediated pro-oxidant state (Clement, Hirpara et al 2003) These reports firmly established the significance of a mild pro-oxidant milieu in tumor progression/initiation through enhanced survival, with Bcl-2 at the heart of this phenomenon This slight pro-oxidant state has been shown to favor a cascade of survival signaling pathways in cancer cells (Steinman 1995; Clement and Stamenkovic 1996; Ahmad, Clement et al 2003; Clement, Hirpara et al 2003; Pervaiz and Clement 2007)

Thus, mitochondria being a major site of O2- production throughits electron transport chain activities are crucial organelles for thestudy on the potential impact of onco-proteins such as Bcl-2 on itsphysiology in order to validate the pro-oxidant state, necessary for thetransformed phenotype The pro-oxidant role for mitochondria is further supported by evidence demonstrating that upstream events such as increased mitochondrial biogenesis or mutant mitochondrial proteins can lead to increased production of ROS via increased mitochondrial respiration (Zhang, Gao et al 2007; Dasgupta, Hoque et al 2008) Hence, increased activity of the terminal, rate-limiting COX enzyme will inadvertently enhance the overall rate of electron transport across the mitochondrial respiratory chain and increase the propensity for leakage of electrons along the chain to form O2- upon reaction with molecular oxygen,

particularly at complex I and III However, one confounding factor is thenotion that cancer cells generally exhibit reduced oxidative phosphorylationand the next section seeks to address this issue

1.11 Altered tumor metabolism and cell fate:

Early studies on tumor metabolism proposed a unique, signature characteristic in the way these rogue cells obtained their source of ATP to meet the rigorous demands of proliferation, invasion and adaptations to the harsh tumor microenvironment Back in

1924, Otto Warburg proposed that cancer cells preferentially utilize the glycolytic pathway over oxidative phosphorylation to provide for the majority of the energy supply This is in stark contrast to normal cells where the reliance on oxidative phosphorylation is far greater than glycolysis for the generation of ATP Warburg attributed this phenomenon to the dysfunction of the mitochondria whereby these metabolic differences were regarded as an adaptation to the hypoxic environment within the solid tumor (Gatenby and Gillies 2004)

More than 80 years on, the Warburg effect is widely applied as the de rigueur

metabolic phenomenon to distinguish cancer cells from non-cancerous ones The idea that cancer cells predominantly utilize glycolysis for energy production is being employed as a parameter in positron emission tomography to assess the prevalence of tumors in the clinical setting Indeed, extensive studies have shown that fast-growing and highly de-differentiated cancer cell types demonstrated highly modified metabolic patterns compared to their normal counterparts (Pedersen 1978; Dastidar

and Sharma 1989; Mazurek, Michel et al 1997; Rodriguez-Enriquez, Torres-Marquez

et al 2000; Ziegler, von Kienlin et al 2001; Griguer, Oliva et al 2005) In agreement with these observations, a large body of evidence has emerged suggesting significant up-regulation in the expression of glycolytic genes in aggressive malignancies (Bustamante and Pedersen 1977; Oskam, Rijksen et al 1985; Vora, Halper et al 1985; Nakashima, Paggi et al 1988; Atsumi, Chesney et al 2002; Medina and Owen 2002; Wood and Trayhurn 2003; Macheda, Rogers et al 2005; Marin-Hernandez, Rodriguez-Enriquez et al 2006)

With these compelling evidence, the concept that enhanced glycolysis is always induced or accompanied by near-defunct oxidative phosphorylation has been ubiquitously and indiscriminately applied to all types of cancer The universal acceptance of the Warburg effect thus formed the central metabolic dogma that come

to characterize all cancer cells However, it is important to note that fundamental genetic, biochemical and morphological heterogeneity of tumor cells may render the convenient application of the Warburg effect to define all types of cancer as generalization Tumor cell types such as glioblastoma multiforme, astrocytoma, MCF7 and certain forms of hepatoma utilize both glycolysis and oxidative phosphorylation to an equal extent for energy production (Elwood, Lin et al 1963; Lowry, Berger et al 1983; Liu, Hu et al 2001; Zu and Guppy 2004) More importantly, tumors such as bone sarcoma, lung carcinoma, breast cancer, skin melanoma, cervical, ovarian and uterus carcinomas all primarily make use of oxidative phosphorylation for the generation of ATP (Elwood, Lin et al 1963;

Balaban and Bader 1984; Kallinowski, Schlenger et al 1989; Rodriguez-Enriquez, Torres-Marquez et al 2000; Zu and Guppy 2004; Rodriguez-Enriquez, Vital-Gonzalez et al 2006) Moreover, hypoxia could not entirely justify for the assumption of compromised oxidative phosphorylation because the concentration of

oxygen in the hypoxic regions of most human tumors is way above the KM O2 of cytochrome c oxidase (COX) (Moreno-Sanchez, Rodriguez-Enriquez et al 2007)

Therefore, it is likely that tumor oxidative metabolism remains unaffected by the level of hypoxia within the tumor microenvironment (Moreno-Sanchez, Rodriguez-Enriquez et al 2007) Compounded by evidence taken from fast-growing tumors showing large increase in glycolytic flux even in the presence of high oxygen concentration, it is indisputable that all tumor cells possess an increase in glycolytic capacity but not necessarily in response to a defective oxidative phosphorylation system (Pedersen 1978; Mazurek, Michel et al 1997; Rodriguez-Enriquez, Torres-Marquez et al 2000; Ziegler, von Kienlin et al 2001; Gatenby and Gillies 2004; Griguer, Oliva et al 2005; Marin-Hernandez, Rodriguez-Enriquez et al 2006; Moreno-Sanchez, Rodriguez-Enriquez et al 2007) In fact, the demanding energetic needs of the tumor brought on by its highly proliferative nature may be the main driving force behind the increase in glycolytic flux to boost the ATP supply, together with an intact oxidative metabolic pathway (Moreno-Sanchez, Rodriguez-Enriquez et

al 2007) Enhanced tumor glycolysis may be operating in tandem with oxidative phosphorylation or in some cases; the latter may even predominate to meet the energy requirements of the highly invasive tumor (Moreno-Sanchez, Rodriguez-Enriquez et

al 2007)

1.12 Implication of COX and mitochondrial respiration in cancer cells:

As mitochondria is the central organelle for the provision of cellular ATP as well as the main producer of ROS, an intricate balance exists between meeting the energetic needs and maintaining the redox states of the cell for normal homeostasis, deregulation of which may lead to the promotion of tumorigenesis or cell death COX plays a paramount role in regulating these mitochondrial functions Alterations in COX activity or perturbations in the final assembled enzyme may significantly impact cellular performance and survival to transform normal cells into diseased entities and have serious implication on human health

Indeed, several groundbreaking findings from recent work have highlighted the impact of COX on cancer by linking the mitochondrial respiratory complex to various established tumorigenic signaling pathways One landmark study indicated that p53 regulates mitochondrial respiration through SCO2, which is critical for the regulation

of COX (Matoba, Kang et al 2006) Thus, it was postulated that mutations of p53 in cancers might be responsible for the metabolic switch from respiratory to glycolytic pathway for energy production, accounting for the Warburg effect as well as suggesting a transcriptional-independent role for p53 (Matoba, Kang et al 2006)

More notably, 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

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 isoforms is an optimized efficiency in mitochondrial respiration, COX activity, ATP production, oxygen consumption and a control on ROS production from the initial burst generated by hypoxia (Fukuda, Zhang et al 2007)

1.13 Structure, assembly and regulation of COX:

COX which stands for cytochrome c oxidase, otherwise known as complex IV, is the

terminal enzyme of the mitochondrial electron transport chain It is responsible for the reduction of molecular oxygen to water by receiving electrons from the upstream complexes and in turn, pumping protons into the mitochondrial intermembrane space

to generate an electrochemical gradient necessary for the production of ATP The mammalian COX enzyme functions as a dimer and each monomer consists of 13 subunits, of which 3 are encoded by the mitochondrial genome and the rest of the subunits are nuclear-encoded The mitochondrial-encoded subunits I to III are transmembrane proteins embedded in the mitochondrial inner membrane and form the catalytic core of the enzyme (Fontanesi, Soto et al 2006) Subunit III has also been postulated to regulate the assembly and stability of subunit I and II as well as modulating the access of oxygen to the binuclear center and the proton transfer process through subunit I and II (Brunori, Antonini et al 1987; Riistama, Puustinen et

al 1996; Hosler 2004) COX assembly and catalytic activity also requires co-factors

such as copper and heme a (Fontanesi, Soto et al 2006) Briefly, in bacterial and

mammalian COX enzymes, electrons are transferred from ferrocytochrome c via the

two-copper center CuA in subunit II to the low-spin heme a and then to binding high-spin heme a3-CuB center, both located in subunit I (Kadenbach, Ramzan

oxygen-et al 2009) In mammalian cells, it appears that proton translocation from the matrix

to the intermembrane space is mediated by three channels within the COX enzyme, namely K-, D- and H-channel (Yoshikawa, Shinzawa-Itoh et al 1998; Muramoto, Hirata et al 2007; Sharpe, Ferguson et al 2008) In particular, H-channel was observed only in mammalian cells and served exclusively as a channel for proton transfer (Salje, Ludwig et al 2005; Shimokata, Katayama et al 2007)

The nuclear-encoded subunits which are synthesized in the cytoplasm and localized

to the mitochondria are neither essential for the reduction of oxygen nor the pumping

of protons into the intermembrane space Nonetheless, they are involved in the assembly, stability, 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 2006) In mammalian cells, these nuclear-encoded subunits occur in tissue-specific isoforms Different isoforms for subunits IV, VIa, VIb, VIIa and VIII are expressed in lung tissue, heart, testes, skeletal muscles and non-muscle tissues respectively (Huttemann, Kadenbach et al 2001; Lee, Salomon et

al 2005) Of particular interest to this thesis are subunits COX Va and Vb, where their yeast homologues have been shown to be critical for the assembly/stability of COX (Dowhan, Bibus et al 1985; Trueblood and Poyton 1987)

Assembly of COX occurs in a sequential and coordinated process where subunits are added one after another to form the complete enzyme (Villani and Attardi 2000) The first assembly intermediate in mammalian cells corresponds to COX I biogenesis and subsequent recruitment of COX II and III The second intermediate involves the amalgamation of COX IV and COX Va (Williams, Valnot et al 2004) Finally, the third intermediate contains all of the subunits including COX Vb with the exception

of COX VIa and VIIa or VIIb, suggesting that COX Va may be critical for the stable assembly of the early COX intermediate and subsequent addition of COX Vb may play a vital role in its enzymatic activity (Nijtmans, de Jong et al 2000; Williams, Valnot et al 2004) As nuclear-encoded subunits are often hydrophobic due to the requirement for membrane insertion and extensive interaction with other subunits to form COX, the expression and maturation of these subunits in the cytoplasm may require chaperones to ensure their efficient localization and import into the mitochondria in order to prevent mis-localization or non-specific aggregation with other proteins or subunits, thus hindering the assembly process (Fontanesi, Soto et al 2006)

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 includes availability of the

subunits and assembly factors regulated at the transcriptional and translational levels, presence of cofactors, mitochondrial protein import and membrane integration and the overall coordination of the different steps in the assembly process (Fontanesi, Soto et al 2006) In this respect, of particular interest to this thesis is the transcriptional and translational regulation of COX Va and COX Vb in response to oxygen and glucose availability Using the yeast model, the transcriptional regulation

of COX Va and Vb homologues is tightly linked to the concentration of oxygen (Poyton and Burke 1992; Bunn and Poyton 1996) Both COX Va and Vb subunits showed a marked decrease in expression as oxygen concentration falls from 200µM

to 100µM, with the greatest decline observed at 1µM oxygen (Burke, Raitt et al 1997) Increasing concentrations of oxygen was unable to completely recover the expression of these subunits back to the atmospheric steady-state levels (Burke, Raitt

et al 1997) The amount of fully assembled COX also correspondingly decreases with decreasing oxygen concentration, in line with the repression of the aerobically-regulated nuclear-encoded subunits (Poyton and McEwen 1996) In addition, COX

Va and Vb yeast homologues have been shown to be repressed by glucose and transcriptionally regulated by mitochondrial cardiolipin respectively (Wright, Rosenzweig et al 1989; Wright and Poyton 1990; Su and Dowhan 2006)

post-Given the complexity in the biogenesis of COX, where the coordinated expression of the gene products from the nuclear and mitochondrial genomes precedes the ordered amalgamation of the subunits involving the cytoplasm and mitochondria, a series of bidirectional signaling between the nucleus and mitochondria is essential to maintain

mitochondrial functions Retrograde communication from the mitochondria to the nucleus in response to functional changes of the former organelle also enables cellular adjustment of metabolism at the various levels, including the mitochondria (Fontanesi, Soto et al 2006) Furthermore, recent data indicate that the coordinated expression of all 13 subunits is mediated by NRF-1, which binds to the promoters of all nuclear-encoded subunits as well as mitochondrial transcription factors A and B, which are responsible for the expression of the mitochondrial-encoded subunits, thus indirectly regulating the parallel expression from the mitochondrial genome (Dhar, Ongwijitwat et al 2008)

Interestingly, two models exist to illustrate the involvement of COX in the arrangement of the electron transport chain The first is the ‘solid-state’ linear model where the components of the chain form a fixed single unit as electrons are passed from one complex to another (Chance and Williams 1955) The second is the ‘random collision’ model where the complexes are distributed randomly across the inner mitochondrial membrane and electron transfer occurs during transient and chance encounter of one complex with another (Chazotte and Hackenbrock 1989) In this respect, COX was found to be part of a supercomplex in association with complex III and I to provide evidence in support of the first model, although both models are thought to co-exist in yeast and mammalian systems (Bianchi, Genova et al 2004; Fontanesi, Soto et al 2006)

1.14 COX Va and COX Vb as cancer markers:

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 2000) Moreover, protection against apoptosis by precursor peptide progastrin in intestinal epithelial cells correlated with increased expression of COX Vb and enhanced ATP synthesis (Wu, Owlia et al 2003) The expression of COX Va has been shown to correlate with tumorigenesis in squamous cell cancer of the larynx, intra-ductal carcinoma of the breast and colorectal cancer whereas COX Vb is implicated in prostate cancer (Bini, Magi et al 1997; Melis and White 1999; Herrmann, Gillespie et al 2003; Fu, Shang et al 2006)

In addition, up-regulation of COX Vb has been observed in energy-demanding cell types and healthy tissues such as heart, kidney and brain as compared to liver Increase in expression of COX Vb is also associated with an increase in COX activity

in cervical (HeLa) and lung carcinoma cells (A549) (Vijayasarathy, Biunno et al 1998; Campian, Gao et al 2007)

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

2 Aim of the study

Our group has previously established a pro-oxidant role for Bcl-2 in executing its protective effects against apoptotic stimuli, implicating O2- as the determinant species (Clement, Hirpara et al 2003) With respect to the mitochondrial localization of Bcl-2 and mitochondria being the major producer of ROS, this study aims to advance the field’s understanding of Bcl-2 on mitochondrial bioenergetics and the role its plays in O2- production from the organelle 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 by-production of O2- due to increased leakage of electrons from enhanced electron transport In conclusion, this is a detailed study of the mechanism governing the ability of Bcl-2 to generate a pro-oxidant state with respect to mitochondrial functions and COX activity and the possible regulatory machinery that may be involved

3 Extra details on methods

3.1 Alternative protocol for isolation of mitochondria:

Remove the medium from the cells and wash the cells once with PBS Remove PBS and detach the cells using a cell scraper Transfer the cell suspension to a 50 ml polypropylene Falcon tube Wash the plate once with PBS and scrape the dish to detach the remaining cells Transfer the cells to the same polypropylene Falcon tube

as defined before Centrifuge cells at 600g at 4°C for 10 min Discard the supernatant and resuspend cells in 3 ml of ice-cold extraction buffer for mitochondria Homogenize the cells using a Teflon pestle operated at 1,600 r.p.m or stroke the cell suspension placed in a glass potter 30–40 times Precool the glassware in an ice-bath

5 min before starting the procedure Homogenization as well as the following steps must be performed at 4°C to minimize the activation of damaging phospholipases and proteases Transfer the homogenate to a 50 ml polypropylene Falcon tube and centrifuge at 600g for 10 min at 4°C Collect the supernatant, transfer it to a glass centrifuge tube and centrifuge it at 7,000g for 10 min at 4°C Discard the supernatant and wash the pellet with 200 ml of ice-cold extraction buffer Resuspend the pellet in

200 ml of ice-cold extraction buffer and transfer the suspension to a 1.5 ml microfuge tube Centrifuge the homogenate at 7,000g for 10 min at 4°C Discard the supernatant and resuspend the pellet containing mitochondria Resuspend the mitochondrial pellet

in minimal volume of buffer required, avoiding formation of air bubbles Transfer the mitochondrial suspension to a microfuge and store it on ice

3.2 Complex II activity assay:

Mitochondrial complex II activity was measured spectrophotometrically in a 96-well plate format Each reaction contained 25µg of mitochondrial protein, 0.1mM EDTA, 0.1% BSA (w/v), 3mM sodium azide and 100µM DCIP in 50mM potassium phosphate buffer at pH 7.4 in a final volume of 0.25ml After 5min of equilibration, reactions were started by adding 20mM succinate and the decrease in absorbance at 600nm was monitored for 10min at 30°C The reaction was terminated by the addition of 1mM TTFA and the change in absorbance was measured for another 5min

3.3 Complex II-III activity assay:

Mitochondrial complex II-III activity was measured spectrophotometrically in a

96-well plate reader based on the antimycin-sensitive rate of cytochrome c reduction,

using succinate as the substrate and measured at 550nm at 30°C Each reaction contained 20µg of mitochondrial protein, 0.1mM EDTA, 3mM sodium azide, 100µM

cytochrome c in 50mM potassium phosphate buffer at pH 7.4 in a final volume of

0.25ml

4 Discussion and conclusion

The present study was aimed at delineating the mechanism behind the induction of a survival-promoting pro-oxidant state by Bcl-2 in tumor cells, from the perspective of

an involvement in mitochondrial functions and COX activity Interestingly, during our study, a novel, unexpected role of Bcl-2 in redox homeostasis by modulating COX activity and mitochondrial respiration during oxidative stress emerged Together, the study presented evidence in support of a role for Bcl-2 in redox and metabolic regulation through an adaptation in COX activity and mitochondrial respiration and thus, contributing to a new and potentially death-evasive mechanism

of Bcl-2

4.1 Increased COX activity and mitochondrial respiration is responsible for the induction of pro-oxidant state by Bcl-2:

Our study revealed that tumor cells stably overexpressing Bcl-2 have marked increase

in oxygen consumption and COX activity, which primarily accounts for the higher mitochondrial O2- produced and in turn, contributing at least in part, to the slight pro-oxidant state in CEM/Bcl-2 cells (Clement, Hirpara et al 2003) This slight pro-oxidant state has been shown to favor a cascade of survival signaling pathways in cancer cells

Bcl-2 which is almost ubiquitous at the mitochondria has rarely been linked to the bioenergetics aspect of the mitochondria For the first time, by measuring the

enzymatic activity of COX which is the rate-limiting complex of the electron transport chain, leukemia cells (CEM) overexpressing Bcl-2 displayed an increase in COX activity and oxygen consumption, which associated with an increase in mitochondrial O2- production Similar observations were obtained in a cervical carcinoma (HeLa) model as well as other cancer types with varying levels of endogenous Bcl-2 expression such as nasopharyngeal cancers and colon carcinoma Bcl-2 overexpressingtumor cells not only displayed elevated oxygen consumptionbut also better coupled mitochondrial respiration, suggesting that the increase in O2-

generation is indeed a function of amplifiedmitochondrial respiration and not due to uncoupling Despitetreatment of mock-transfected and Bcl-2 overexpressing cells with theuncoupler FCCP, cells with Bcl-2 overexpression continued to displayhigher oxygen consumption Increased mitochondrial respiration suggests an increased tendency to leak electrons for the generation ofO2- as a by-product

Transient overexpression and silencing of Bcl-2 correlated with the presence and absence of the pro-oxidant effect respectively Similarly, reduction of electron transport activities through the partial inhibition of COX was able to abrogate O2-

levels in Bcl-2 expressingcells to that of non-transfected cells, further validating the impact ofBcl-2 on the pro-oxidant state through mitochondrial respiration.Although HA14-1, which is a well-known Bcl-2 inhibitor, has beenshown to induce oxidative stress in treated cells, functional inhibition of Bcl-2 led to a reduction in oxygen consumption

It has been reported that HA14-1 increased mitochondrial respiration due to uncoupling and led to ROS production in CEM cells (Hao, Yu et al 2004) Indeed,

we reported increased oxygen consumption in CEM/Neo cells treated in similar fashion However, we reason two possible alternative scenarios for our observation in CEM/Bcl-2 cells Firstly, it may be likely that the reduction in oxygen consumption caused by the inhibition of Bcl-2 could more than compensate for the intrinsic capability of HA14-1 to raise uncoupled mitochondrial respiration in CEM/Bcl-2 cells, with the outcome being reduced ROS generation and overall decreased oxygen consumption On the other hand, the result of increased uncoupled mitochondrial respiration and ROS production in CEM cells as reported previously was observed in the absence of Bcl-2 overexpression Thus, it may be conceivable that the overexpression of Bcl-2 abrogates the effect of HA14-1 by sensing ROS status and down-regulating mitochondrial respiration accordingly to maintain ROS at a manageable level Indeed, if Bcl-2 inhibitor HA14-1 uncouples mitochondrial respiration in CEM cells, it might be plausible to imply that Bcl-2 would make an unlikely uncoupling candidate (Hao, Yu et al 2004) This notion is confirmed by the results of this thesis showing that mitochondrial respiration in CEM/Bcl-2 cells displayed better respiratory coupling ratio, suggesting that increased mitochondrial respiration and O2- production brought about by Bcl-2 is not due to the effects of uncoupling

4.2 Modulation of COX activity and mitochondrial respiration during oxidative stress by Bcl-2: