Ivan Cherh Chiet Low

Regulation of mitochondrial metabolism: Yet another facet in the biology of the oncoprotein Bcl-2

The Bcl-2 (Bcl is B-cell lymphocytic-leukaemia proto-oncogene) family comprises two groups of proteins with distinct functional biology in cell-fate signalling. Bcl-2 protein was the first member to be discovered and associated with drug resistance in human lymphomas. Since then a host of other proteins such as Bcl-xL, Bcl-2A1 and Mcl-1 with similar anti-apoptotic functions have been identified. In contrast, the pro-apoptotic Bcl-2 proteins contain prototypic effector proteins such as Bax and Bak, and the BH3 (Bcl-2 homology)-only proteins comprising Bak, Bid, Bim, Puma and Noxa. A complex interplay between the association of pro-apoptotic and anti-apoptotic proteins with each other determines the sensitivity of cancer cells to drug-induced apoptosis. The canonical functional of Bcl-2 in terms of apoptosis inhibition is its ability to prevent mitochondrial permeabilization via inhibiting the translocation and oligomerization of pro-apoptotic proteins such as Bax; however, more recent evidence points to a novel mechanism of the anti-apoptotic activity of Bcl-2. Overexpression of Bcl-2 increases mitochondrial oxygen consumption and in doing so generates a slight pro-oxidant intracellular milieu, which promotes genomic instability and blocks death signalling. However, in the wake of overt oxidative stress, Bcl-2 regulates cellular redox status thereby preventing excessive build-up of ROS (reactive oxygen species), which is detrimental to cells and tissues. Taken together, the canonical and non-canonical activities of Bcl-2 imply a critical involvement of this protein in the processes of tumour initiation and progression. In the present paper we review these functionally distinct outcomes of Bcl-2 expression with implications for the chemotherapeutic management of cancers.

Bcl-2 Modulates Resveratrol-Induced ROS Production by Regulating Mitochondrial Respiration in Tumor Cells

Resveratrol is a naturally occurring flavanoid with potent apoptosis-inducing activity against human tumor cells. We investigated the effect of resveratrol on human leukemia cell lines, in particular its ability to induce intracellular reactive oxygen species production and the effect of Bcl-2 overexpression on this model. Exposure of CEM cells to increasing concentrations of resveratrol (0-50 microM) resulted in an increase in mitochondrial superoxide production, decrease in transmembrane potential, and a concomitant decrease in cell viability. Whereas overexpression of Bcl-2 increased mitochondrial oxygen consumption and complex IV activity, CEM/Bcl-2 cells responded to the increased mitochondrial oxidative stress induced by resveratrol by significantly reducing mitochondrial respiration, complex IV activity, and O(2)(-) production, and promoted cell survival. The inhibitory effect of Bcl-2 on resveratrol-induced mitochondrial O(2)(-) production is further corroborated by the neutralization of this regulatory effect upon siRNA-mediated gene silencing of Bcl-2. These data provide evidence implicating mitochondrial metabolism in the anticancer activity of resveratrol, and underscore a novel regulatory role of Bcl-2 against exogenous oxidative stress through its ability to fine tune mitochondrial respiration, and by doing so maintaining mitochondrial O(2)(-) at a level optimal for survival.

Bcl-2: A prime regulator of mitochondrial redox metabolism in cancer cells

SIGNIFICANCE Mitochondria play a critical role as death amplifiers during drug-induced apoptosis in cancer cells by providing pro-apoptotic factors that are released from the mitochondrial inter-membranous space upon the induction of mitochondrial outer membrane permeabilization. This intrinsic death signaling pathway is the preferred mechanism employed by most anticancer compounds, and as such, resistance to drug-induced apoptosis is invariably associated with inhibition of mitochondrial death signaling network. The latter is a function of a balance between the pro- and the anti-apoptotic members of the Bcl-2 family. Bcl-2 is the prototype anti-apoptotic protein that localizes to the mitochondria and blocks the recruitment and activation of pro-apoptotic proteins, such as Bax, to the mitochondria. RECENT ADVANCES AND CRITICAL ISSUES Recent evidence has highlighted a novel mechanism of anti-apoptotic activity of Bcl-2 in addition to its canonical activity in regulating mitochondrial outer membrane permeabilization. This novel activity is a function of cellular redox regulation, in particular, mitochondrial metabolism in cancer cells. FUTURE DIRECTIONS Here we review the current state of our understanding of the death inhibitory activity of Bcl-2 and provide insight into the novel functional biology of this remarkable protein, which could have implications for designing innovative strategies to overcome the problem of drug resistance in the clinical settings.

Modulation of Wnt/β-catenin signaling and proliferation by a ferrous iron chelator with therapeutic efficacy in genetically engineered mouse models of cancer

Using a screen for Wnt/β-catenin inhibitors, a family of 8-hydroxyquinolone derivatives with in vivo anti-cancer properties was identified. Analysis of microarray data for the lead compound N-((8-hydroxy-7-quinolinyl) (4-methylphenyl)methyl)benzamide (HQBA) using the Connectivity Map database suggested that it is an iron chelator that mimics the hypoxic response. HQBA chelates Fe2+ with a dissociation constant of ∼10−19 M, with much weaker binding to Fe3+ and other transition metals. HQBA inhibited proliferation of multiple cell lines in culture, and blocked the progression of established spontaneous cancers in two distinct genetically engineered mouse models of mammary cancer, MMTV-Wnt1 and MMTV-PyMT mice, without overt toxicity. HQBA may inhibit an iron-dependent factor that regulates cell-type-specific β-catenin-driven transcription. It inhibits cancer cell proliferation independently of its effect on β-catenin signaling, as it works equally well in MMTV-PyMT tumors and diverse β-catenin-independent cell lines. HQBA is a promising specific intracellular Fe2+ chelator with activity against spontaneous mouse mammary cancers.

Mitochondrial ROS and involvement of Bcl-2 as a mitochondrial ROS regulator

Mitochondria are the major intracellular source of reactive oxygen species (ROS). While excessive mitochondrial ROS (mitoROS) production induces cell injury and death, there is accumulating evidence that non-toxic low levels of mitoROS could serve as important signaling molecules. Therefore, maintenance of mitoROS at physiological levels is crucial for cell homeostasis as well as for survival and proliferation. This review describes the various mechanisms that keep mitoROS in check, with particular focus on the role of the onco-protein Bcl-2 in redox regulation. In addition to its canonical anti-apoptotic activity, Bcl-2 has been implicated in mitoROS regulation by its effect on mitochondrial complex IV activity, facilitating the mitochondrial incorporation of GSH and interaction with the small GTPase-Rac1 at the mitochondria. We also discuss some of the plausible mechanism(s) which allows Bcl-2 to sense and respond to the fluctuations in mitoROS.

Ser70 phosphorylation of Bcl-2 by selective tyrosine nitration of PP2A-B56δ stabilizes its antiapoptotic activity

Bcl-2 is frequently overexpressed in hematopoietic malignancies, and selective phosphorylation at ser70 enhances its antiapoptotic activity. Phospho-ser70 is dephosphorylated by specific heterotrimers of protein phosphatase 2A (PP2A). We report here that a mild pro-oxidant intracellular milieu induced by either pharmacological inhibition or genetic knockdown of superoxide dismutase 1 (SOD1) inhibits the functional holoenzyme assembly of PP2A and prevents Bcl-2 ser70 dephosphorylation. This redox-dependent regulation of Bcl-2 phosphorylation is due to nitrosative modification of B56δ, which we identify as the regulatory subunit mediating PP2A-dependent Bcl-2 dephosphorylation. Redox inhibition of PP2A results from peroxynitrite-mediated nitration of a conserved tyrosine residue within B56δ (B56δ(Y289)). Although nitrated B56δ(Y289) binds efficiently to ser70-phosphorylated Bcl-2, this specific modification inhibits the recruitment of the PP2A catalytic core (A and C subunits). Furthermore, inhibition of B56δ(Y289) nitration restores PP2A holoenzyme assembly, thereby permitting S70 dephosphorylation of Bcl-2 and inhibiting its antiapoptotic activity. More important, in primary cells derived from clinical lymphomas, Bcl-2 phosphorylation at S70 directly correlates with B56δ nitration and repression of SOD1, but inversely correlates with B56δ interaction with the PP2A-C catalytic subunit. These data underscore the role of a pro-oxidant milieu in chemoresistance of hematopoietic and other cancers via selective targeting of tumor suppressors such as PP2A.

Neural Basis of Exertional Fatigue in the Heat: A Review of Magnetic Resonance Imaging Methods

The central nervous system, specifically the brain, is implicated in the development of exertional fatigue under a hot environment. Diverse neuroimaging techniques have been used to visualize the brain activity during or after exercise. Notably, the use of magnetic resonance imaging (MRI) has become prevalent due to its excellent spatial resolution and versatility. This review evaluates the significance and limitations of various brain MRI techniques in exercise studies—brain volumetric analysis, functional MRI, functional connectivity MRI, and arterial spin labeling. The review aims to provide a summary on the neural basis of exertional fatigue and proposes future directions for brain MRI studies. A systematic literature search was performed where a total of thirty‐seven brain MRI studies associated with exercise, fatigue, or related physiological factors were reviewed. The findings suggest that with moderate dehydration, there is a decrease in total brain volume accompanied with expansion of ventricular volume. With exercise fatigue, there is increased activation of sensorimotor and cognitive brain areas, increased thalamo‐insular activation and decreased interhemispheric connectivity in motor cortex. Under passive hyperthermia, there are regional changes in cerebral perfusion, a reduction in local connectivity in functional brain networks and an impairment to executive function. Current literature suggests that the brain structure and function are influenced by exercise, fatigue, and related physiological perturbations. However, there is still a dearth of knowledge and it is hoped that through understanding of MRI advantages and limitations, future studies will shed light on the central origin of exertional fatigue in the heat.

Abstract 5278: Superoxide mediated selective tyrosine nitration of protein phosphatase 2A-B56δ stabilizes Bcl-2 phosphorylation and its anti-apoptotic activity

Our work over the years has provided strong evidence that an increase in intracellular superoxide anion provides cancer cells with a survival advantage and promotes tumor chemoresistance. The mechanisms underlying superoxide-mediated pro-survival signaling are poorly understood. Here we report that an increase in intracellular superoxide stabilizes the anti-apoptotic activity of Bcl-2 by mechanisms that involve sustained phosphorylation of Bcl-2 at serine 70. Of note, this effect on Bcl-2 S70 phosphorylation is a function of redox-dependent inactivation of Protein Phosphatase 2A (PP2A), which is targeted to Bcl-2 via its B56 subunit; superoxide induces the release of PP2A-AC catalytic core from Bcl-2-bound B56δ, resulting in sustained phosphorylation of Bcl-2 at S70. Dissociation of the PP2A heterotrimer results from the selective nitration (by peroxynitrite derived from the reaction of superoxide with NO) of a conserved tyrosine residue, which we identify as Y289 of B56δ (B56 δY289). Inhibition of B56δY289 nitration blocked the pro-survival effects of superoxide. Furthermore, we provide evidence for clinical relevance of these findings using primary tissue derived from human lymphoma biopsies. Taken together, these data provide evidence for a novel mechanism in which an increase in intracellular superoxide regulates PP2A function via specific B56 subunit tyrosine nitration, and that PP2A-B56 nitration may be a general mechanism for redox-regulated cellular signaling. Citation Format: Ivan C. C. Low, Thomas Loh, David Virshup, Shazib Pervaiz. Superoxide mediated selective tyrosine nitration of protein phosphatase 2A-B56δ stabilizes Bcl-2 phosphorylation and its anti-apoptotic activity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5278. doi:10.1158/1538-7445.AM2014-5278