Metabolic control of cellular function is significant in the context of inflammation-induced metabolic dysregulation in immune cells. Generation of reactive oxygen species (ROS) such as hydrogen peroxide and superoxide are one of the critical events that modulate the immune response in neutrophils. When activated, neutrophil NADPH oxidases consume large quantities of oxygen to rapidly generate ROS, a process that is referred to as the oxidative burst. These ROS are required for the efficient removal of phagocytized cellular debris and pathogens. In chronic inflammatory diseases, neutrophils are exposed to increased levels of oxidants and pro-inflammatory cytokines that can further prime oxidative burst responses and generate lipid oxidation products such as 4-hydroxynonenal (4-HNE). In this study we hypothesized that since 4-HNE can target glycolysis then this could modify the oxidative burst. To address this the oxidative burst was determined in freshly isolated healthy subject neutrophils using 13-phorbol myristate acetate (PMA) and the extracellular flux analyzer. Neutrophils pretreated with 4-HNE exhibited a significant decrease in the oxidative burst response and phagocytosis. Mass spectrometric analysis of alkyne-HNE treated neutrophils followed by click chemistry detected modification of a number of cytoskeletal, metabolic, redox and signaling proteins that are critical for the NADPH oxidase mediated oxidative burst. These modifications were confirmed using a candidate immunoblot approach for critical proteins of the active NADPH oxidase enzyme complex (Nox2 gp91phox subunit and Rac1 of the NADPH oxidase) and glyceraldehyde phosphate dehydrogenase, a critical enzyme in the metabolic regulation of oxidative burst. Taken together, these data suggest that 4-HNE-induces a pleiotropic mechanism to inhibit neutrophil function. These mechanisms may contribute to the immune dysregulation associated with chronic pathological conditions where 4-HNE is generated.
This chapter contains sections titled: Introduction Cyclooxygenase and the Conversion of Nonreactive Lipids to Thiol Switching Molecules Lipid Peroxidation and the Nonenzymatic Formation of Lipid Adducts Capable of Modifying Proteins The Thiol Switch and Redox Cell Signaling Biological Responses to Endogenous Electrophilic Lipid Production A New Paradigm of Oxidized Lipid Signaling—The Covalent Advantage Implications for the Pathophysiology of Disease Summary
Temozolomide (TMZ) is an oral alkylating agent used for the treatment of high-grade gliomas. Acquired chemoresistance is a severe limitation to this therapy with more than 90% of recurrent gliomas showing no response to a second cycle of chemotherapy. Efforts to better understand the underlying mechanisms of acquired chemoresistance to TMZ and potential strategies to overcome chemoresistance are, therefore, critically needed. TMZ methylates nuclear DNA and induces cell death; however, the impact on mitochondria DNA (mtDNA) and mitochondrial bioenergetics is not known. Herein, we tested the hypothesis that TMZ-mediated alterations in mtDNA and respiratory function contribute to TMZ-dependent acquired chemoresistance. Using an in vitro model of TMZ-mediated acquired chemoresistance, we report 1) a decrease in mtDNA copy number and the presence of large heteroplasmic mtDNA deletions in TMZ-resistant glioma cells, 2) remodeling of the entire electron transport chain with significant decreases of complexes I and V and increases of complexes II/III and IV, and 3) pharmacologic and genetic manipulation of cytochrome c oxidase, which restores sensitivity to TMZ-dependent apoptosis in resistant glioma cells. Importantly, human primary and recurrent pairs of glioblastoma multiforme (GBM) biopsies as well as primary and TMZ-resistant GBM xenograft lines exhibit similar remodeling of the ETC. Overall these results suggest that TMZ-dependent acquired chemoresistance may be due to a mitochondrial adaptive response to TMZ genotoxic stress with a major contribution from cytochrome c oxidase. Thus, abrogation of this adaptive response may reverse chemoresistance and restore sensitivity to TMZ, providing a strategy for improved therapeutic outcomes in GBM patients.
Prototypical electrophiles such as the lipid 15-deoxy-Delta12,14-prostaglandin J2 (15d-PGJ2) are well recognized for their therapeutic potential. Electrophiles modify signalling proteins in both the cytosol and mitochondrion, which results in diverse cellular responses, including cytoprotective effects and, at high doses, cell death. These findings led us to the hypothesis that targeting electrophiles to specific compartments in the cell could fine-tune their biological effects. To examine this, we synthesized a novel mitochondrially targeted analogue of 15d-PGJ2 (mito-15d-PGJ2) and tested its effects on redox cell signalling. Mito-15d-PGJ2 caused profound defects in mitochondrial bioenergetics and mitochondrial membrane depolarization when compared with 15d-PGJ2. We also found that mito-15d-PGJ2 modified different members of the electrophile-responsive proteome, was more potent at initiating intrinsic apoptotic cell death and was less effective than 15d-PGJ2 at up-regulating the expression of HO-1 (haem oxygenase-1) and glutathione. These results demonstrate the feasibility of modulating the biological effects of electrophiles by targeting the pharmacophore to mitochondria.
Solid tumors are characterized by regions of low oxygen tension (OT), which play a central role in tumor progression and resistance to therapy. Low OT affects mitochondrial function and for the cells to survive, mitochondria must functionally adapt to low OT to maintain the cellular bioenergetics. In this study, a novel experimental approach was developed to examine the real-time bioenergetic changes in breast cancer cells (BCCs) during adaptation to OT (from 20% to <1% oxygen) using sensitive extracellular flux technology. Oxygen was gradually removed from the medium, and the bioenergetics of metastatic BCCs (MDA-MB-231 and MCF10CA clones) was compared with non-tumorigenic (MCF10A) cells. BCCs, but not MCF10A, rapidly responded to low OT by stabilizing HIF-1α and increasing HIF-1α responsive gene expression and glucose uptake. BCCs also increased extracellular acidification rate (ECAR), which was markedly lower in MCF10A. Interestingly, BCCs exhibited a biphasic response in basal respiration as the OT was reduced from 20% to <1%. The initial stimulation of oxygen consumption is found to be due to increased mitochondrial respiration. This effect was HIF-1α-dependent, as silencing HIF-1α abolished the biphasic response. During hypoxia and reoxygenation, BCCs also maintained oxygen consumption rates at specific OT; however, HIF-1α silenced BCC were less responsive to changes in OT. Our results suggest that HIF-1α provides a high degree of bioenergetic flexibility under different OT which may confer an adaptive advantage for BCC survival in the tumor microenvironment and during invasion and metastasis. This study thus provides direct evidence for the cross-talk between HIF-1α and mitochondria during adaptation to low OT by BCCs and may be useful in identifying novel therapeutic agents that target the bioenergetics of BCCs in response to low OT.
Supplementary Figure Legends 1- 4 from Homotypic Gap Junctional Communication Associated with Metastasis Suppression Increases with PKA Activity and Is Unaffected by PI3K Inhibition
<p>PDF file - 1136K, Supplemental Figure 1. Glycolytic enzyme expression in KISS1-expressing cells. Transcript level of glucose transporter (GLUT1) and several glycolytic enzymes (HKII, PFK1, PKM1, PKM2, LDHA and LDHB) were examined by qRT-PCR in C8161.9Vector (V), C8161.9KFMdeltaSS (D) and C8161.9KFM (K) as well as MelJuSoVector (V) and MelJuSoKFM (K) cells. Values are mean plus-minus standard deviation of three independent experiments. P values are based on a two-sided Student's T-test (*, p<0.05; **, p<0.01; ***, p<0.001).</p>
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