Faculty Bibliography

G6PD is required for embryonic development in animals, as severe G6PD deficiency is lethal to mice, zebrafish and nematode. Lipid peroxidation is linked to membrane-associated embryonic defects in Caenorhabditis elegans (C. elegans). However, the direct link between lipid peroxidation and embryonic lethality has not been established. The aim of this study was to delineate the role of lipid peroxidation in gspd-1-knockdown (ortholog of g6pd) C. elegans during reproduction. tert-butyl hydroperoxide (tBHP) was used as an exogenous inducer. Short-term tBHP administration reduced brood size and enhanced germ cell death in C. elegans. The altered phenotypes caused by tBHP resembled GSPD-1 deficiency in C. elegans. Mechanistically, tBHP-induced malondialdehyde (MDA) production and stimulated calcium-independent phospholipase A₂ (iPLA) activity, leading to disturbed oogenesis and embryogenesis. The current study provides strong evidence to support the notion that enhanced lipid peroxidation in G6PD deficiency promotes death of germ cells and impairs embryogenesis in C. elegans.

Glucose-6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme of the pentose phosphate pathway that modulates cellular redox homeostasis via the regeneration of NADPH. G6PD-deficient cells have a reduced ability to induce the innate immune response, thus increasing host susceptibility to pathogen infections. An important part of the immune response is the activation of the inflammasome. G6PD-deficient peripheral blood mononuclear cells (PBMCs) from patients and human monocytic (THP-1) cells were used as models to investigate whether G6PD modulates inflammasome activation. A decreased expression of IL-1β was observed in both G6PD-deficient PBMCs and PMA-primed G6PD-knockdown (G6PD-kd) THP-1 cells upon lipopolysaccharide (LPS)/adenosine triphosphate (ATP) or LPS/nigericin stimulation. The pro-IL-1β expression of THP-1 cells was decreased by G6PD knockdown at the transcriptional and translational levels in an investigation of the expression of the inflammasome subunits. The phosphorylation of p38 MAPK and downstream c-Fos expression were decreased upon G6PD knockdown, accompanied by decreased AP-1 translocation into the nucleus. Impaired inflammasome activation in G6PD-kd THP-1 cells was mediated by a decrease in the production of reactive oxygen species (ROS) by NOX signaling, while treatment with hydrogen peroxide (H₂O₂) enhanced inflammasome activation in G6PD-kd THP-1 cells. G6PD knockdown decreased Staphylococcus aureus and Escherichia coli clearance in G6PD-kd THP-1 cells and G6PD-deficient PBMCs following inflammasome activation. These findings support the notion that enhanced pathogen susceptibility in G6PD deficiency is, in part, due to an altered redox signaling, which adversely affects inflammasome activation and the bactericidal response.

The generation of reducing equivalent NADPH via glucose-6-phosphate dehydrogenase (G6PD) is critical for the maintenance of redox homeostasis and reductive biosynthesis in cells. NADPH also plays key roles in cellular processes mediated by redox signaling. Insufficient G6PD activity predisposes cells to growth retardation and demise. Severely lacking G6PD impairs embryonic development and delays organismal growth. Altered G6PD activity is associated with pathophysiology, such as autophagy, insulin resistance, infection, inflammation, as well as diabetes and hypertension. Aberrant activation of G6PD leads to enhanced cell proliferation and adaptation in many types of cancers. The present review aims to update the existing knowledge concerning G6PD and emphasizes how G6PD modulates redox signaling and affects cell survival and demise, particularly in diseases such as cancer. Exploiting G6PD as a potential drug target against cancer is also discussed.

BACKGROUND:The nematodeCaenorhabditis elegans is an emerging invertebrate animal model for investigating neuronal functions in behavioral assays. C. elegans mechanosensation was characterized by the use of a constant mechanical stimulation transmitter followed by quantitative imaging. NEW METHOD/UNASSIGNED:C. elegans reflex and habituation behaviors were characterized by mechanical vibration followed by image analysis. A custom-designed system consists of an aluminum alloy Petri dish holder frame coupled with a mechanical vibration buzzer delivering adjustable pulsed vibration to an agar plate. The basal and evoked movements of C. elegans were recorded by a microscopic digital camera followed by quantitative analysis using microscopic imaging software. RESULTS:Application of the platform inC. elegans was demonstrated with three proof-of-concept experiments: (1) Evaluation of the reflex response stimulated by tapping and mechanical vibration with a mechano-sensation defective mutant. (2) Comparison of the reflex response stimulated by mechanical vibration between wild type and aging mutants. (3) Assessment of the efficacy of the mechanical vibration system on long-term memory for habituation. COMPARISON WITH EXISTING METHODS/UNASSIGNED:Conventional C. elegans mechanosensation techniques depend on stimulation either by manually touching a single animal or tapping the Petri dish followed by scoring via visual observation from the examiner. The mechanical vibration method has greater capacity compared to conventional methods which are labor-intensive, have low throughput and lack quantifiable parameters. CONCLUSIONS:The mechanical vibration system followed by image analysis is a convenient and integrated platform for investigatingC. elegans reflex and habituation in aging and neural behavioral assays.

Cancer development is closely related to chronic inflammation, which is associated with identifiable markers of tumor progression, such as uncontrolled cell proliferation, angiogenesis, genomic instability, chemotherapeutic resistance, and metastases. Redox processes mediated by reactive oxygen species (ROS) and nitric oxide (NO) within the inflammatory tumor microenvironment play an essential role in directly influencing intercellular and intracellular signaling. These reactive species originating in the cancer cell or its microenvironment, mediate the epithelial-mesenchymal transition (EMT) and the mesenchymal-epithelial transition (MET). However, intracellular interactions between NO and ROS must be controlled to prevent cell death. Melanoma, breast, and colon cancer cells have developed a mechanism to survive and adapt to oxidative and nitrosative stress. The mechanism involves a spatial-temporal fine adjustment of the intracellular concentrations of NO and ROS, thereby guaranteeing the successful development of cancer cells. Physiological concentrations of NO and supra physiological concentrations of ROS are prevalent in cancer cells at the primary site. The situation reverses in cancer cells undergoing the EMT prior to being released into the blood stream. Intracellular supra physiological concentrations of NO found in circulating cancer cells endow them with anoikis resistance. When the anoikis-resistant cancer cells arrive at a metastatic site they undergo the MET. Endogenous supra physiological concentrations of ROS and physiological NO concentrations are prevalent in these cells. Understanding tumor progression from the perspective of redox signaling permits the characterization of new markers and approaches to therapy. The synthesis and use of compounds with the capacity of modifying intracellular concentrations of NO and ROS may prove effective in disrupting a redox homeostasis operative in cancer cells.

Nitric Oxide (NO) and Hydrogen Sulfide (H₂S) are components of an "interactome", which is defined as a redox system involving the interactions of RSS, RNS and ROS. Chemical interaction by these species is common and is characterized by one and two electron oxidation, nitrosylation, nitration and sulfuration/polysulfidation reactions. NO and H₂S are gases that penetrate cell membranes, are synthesized by specific enzymes, are ubiquitous, regulate protein activities through post-translational modifications and participate in cell signaling. The two molecules at high concentrations compared to physiological concentrations may result in cellular damage particularly through their interaction with other reactive species. NO and H₂S can interact with each other and form a variety of molecular species which may have constructive or destructive behavior depending on the cell type, the cellular environment (ex. oxygen tension, pH, redox state), where the products are produced and in what concentrations. Cross talk exists between NO and H₂S, whereby they can influence the generation and signaling behavior of each other. Given the above mentioned properties of NO and H₂S and studies in cancer cells and animal models employing NO and H₂S donors that generate higher than physiological concentrations of NO and H₂S and are effective in killing cancer cells but not normal cells, lend credence to the possibility of the utility of these donors in an approach to the treatment of cancer.

NADPH is a reducing equivalent that maintains redox homeostasis and supports reductive biosynthesis. Lack of major NADPH-producing enzymes predisposes cells to growth retardation and demise. It was hypothesized that double deficiency of the NADPH-generating enzymes, GSPD-1 (Glucose-6-phosphate 1-dehydrogenase), a functional homolog of human glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway, and IDH-1 (isocitrate dehydrogenase-1) affect growth and development in the nematode, Caenorhabditis elegans (C. elegans). The idh-1;gspd-1(RNAi) double-deficient C. elegans model displayed shrinkage of body size, growth retardation, slowed locomotion, and impaired molting. Global metabolomic analysis was employed to address whether or not metabolic pathways were altered by severe NADPH insufficiency by the idh-1;gspd-1(RNAi) double-deficiency. The principal component analysis (PCA) points to a distinct metabolomic profile of idh-1;gspd-1(RNAi) double-deficiency. Further metabolomic analysis revealed that NADPH-dependent and glutamate-dependent amino acid biosynthesis were significantly affected. The reduced pool of amino acids may affect protein synthesis, as indicated by the absence of NAS-37 expression during the molting process. In short, double deficiency of GSPD-1 and IDH-1 causes growth retardation and molting defects, which are, in part, attributed to defective protein synthesis, possibly mediated by altered amino acid biosynthesis and metabolism in C. elegans.

Tumour progression involves the establishment of tumour metastases at distant sites. Resistance to anoikis, a form of cell death that occurs when cells lose contact with the extracellular matrix and with neighbouring cells, is essential for metastases. NO has been associated with anoikis. NO treated HeLa cells and murine melanoma cells in suspension triggered a nitric oxide (NO)-Src kinase signalling circuitry that enabled resistance to anoikis. Two NO donors, sodium nitroprusside (SNP) (500 µM) and DETANO (125 µM), protected against cell death derived from detachment of a growth permissive surface (experimental anoikis). Under conditions of NO-mediated Src activation the following were observed: (a) down-regulation of the pro-apoptotic proteins Bim and cleaved caspase-3 and the cell surface protein, E-cadherin, (b) up-regulation of caveolin-1, and (c) the dissociation of cell aggregates formed when cells are detached from a growth permissive surface. Efficiency of reattachment of tumour cells in suspension and treated with different concentrations of an NO donor, was dependent on the NO concentration. These findings indicate that NO-activated Src kinase triggers a signalling circuitry that provides resistance to anoikis, and allows for metastases.

Accumulating mutations may drive cells into the acquisition of abnormal phenotypes that are characteristic of cancer cells. Cancer cells feature profound alterations in proliferation programs that result in a new population of cells that overrides normal tissue construction and maintenance programs. To achieve this goal, cancer cells are endowed with up regulated survival signaling pathways. They also must counteract the cytotoxic effects of high levels of nitric oxide (NO) and of reactive oxygen species (ROS), which are by products of cancer cell growth. Accumulating experimental evidence associates cancer cell survival with their capacity to up-regulate antioxidant systems. Elevated expression of the antioxidant protein thioredoxin-1 (Trx1) has been correlated with cancer development. Trx1 has been characterized as a multifunctional protein, playing different roles in different cell compartments. Trx1 migrates to the nucleus in cells exposed to nitrosative/oxidative stress conditions. Trx1 nuclear migration has been related to the activation of transcription factors associated with cell survival and cell proliferation. There is a direct association between the p21Ras-ERK1/2 MAP Kinases survival signaling pathway and Trx1 nuclear migration under nitrosative stress. The expression of the cytoplasmic protein, the thioredoxin-interacting protein (Txnip), determines the change in Trx1 cellular compartmentalization. The anti-apoptotic actions of Trx1 and its denitrosylase activity occur in the cytoplasm and serve as important regulators of cell survival. Within this context, this review focuses on the participation of Trx1 in cells under nitrosative/oxidative stress in survival signaling pathways associated with cancer development.