In this study, we investigated how colonic epithelial cells maintained pyridine nucleotide (NADH/NAD+) redox homeostasis upon acute metabolic variation imposed by glucose deprivation or supplementation with mitochondrial substrates, succinate and malate/glutamate (M/G). Our results showed that low glucose caused cellular NADH/NAD+ redox imbalance that diminished lactate dehydrogenase (LDH) activity and resulted in lower lactate contents. The concurrent activation of malic enzyme (ME) suggested a role for malate in preserving cellular pyruvate that remained unchanged at low glucose. Mitochondrial substrates restored cellular NADH/NAD+ redox homeostasis at low glucose in association with specific compartmental catabolism of mitochondrial substrates. As compared with normal glucose, M/G and low glucose promoted glycolytic ATP production but inhibited mitochondrial-derived ATP generation in association with decreased glucose availability for mitochondrial respiration. At normal glucose, succinate and M/G enhanced mitochondrial respiratory activity, but had minimal impact on mitochondrial-derived ATP production. Collectively, these results are consistent with low glucose-induced NADH/NAD+ redox imbalance in association with decreased aerobic glycolysis that is reversed by supplementation with M/G but not succinate.

Previously we have shown that the redox cycler menadione (MQ) induced cellular pyridine nucleotide redox imbalance that was linked to a decrease in aerobic glycolysis and perturbation of the mitochondrial respiratory activity due to the redox cycling of the compound; these processes were potentiated by low glucose. In this study, we investigated how colonic epithelial cells maintained pyridine nucleotide (NAD+/NADH and NADP+/NADPH) redox homeostasis upon acute metabolic variation and exposure to the redox cycling diquat (DQ). Our results show that DQ challenge disrupted cellular NADH/NAD+ redox status and enhanced cellular NADPH generation. Notably, DQ-induced NADH decrease was associated with enhanced lactate production, a process that was potentiated by glucose availability, but not by the mitochondrial substrates, succinate or malate/glutamate. In addition, DQ increased glucose 6-phoshate dehydrogenase (G6PDH) activity consistent with glucose diversion towards pentose phosphate pathway. As a consequence, steady-state NADPH levels were maintained during MQ challenge at normal glucose. In contrast and despite increased G6PDH and malic enzyme (ME) activities, DQ induced cellular NADPH-to-NADP+ shift at low glucose, a situation that was reversed by mitochondrial substrates. Collectively, these results are consistent with increased aerobic glycolysis by DQ and specific metabolic changes leading to enhanced NADPH generation upon oxidative challenge.

Abstract Background: Niclosamide, an anthelminthic drug, has demonstrated anti-cancer potential in variety of malignancies. However only a limited number of studies have been performed in lymphoma models, therefore we hypothesized that niclosamide may also have anti-cancer potential on B-cell lymphomas. Materials and Methods: Established B lymphoma cell lines were exposed to different concentrations of niclosamide and IC50 was calculated using GraphPad Prism 6.0 software. Cell viability and proliferation were assessed by CellTiter-Blue and trypan blue exclusion assays. Apoptosis was assessed by flow cytometry following Annexin-V/ propidium iodide staining. Gene expression changes were studied using GeneChip Human Transcriptome Array 2.0. Colony forming assays were performed in methylcellulose. Ultrastructural cellular changes were studied with electron microscopy. Peripheral blood mononuclear cells (PBMCs) from individuals without active cancer and from patients with different hematologic disorders, were also exposed with niclosamide. Results: Treatment with niclosamide resulted in time-and dose- dependent apoptosis, cytotoxicity and inhibition of proliferation in different lymphoma cell lines including vincristine-refractory cell line. The IC50 of lymphoma cells lines is as follows: Daudi: 0.33 ìM; HBL-2: 0.57 ìM; KOPN-8: 0.72 ìM; Ramos: 0.53 ìM and SU-DHL4-VR: 0.45 ìM. Niclosamide also inhibited clonal growth in semi-solid media. Gene expression changes were studied in Daudi and KOPN-8 cells treated with 2.5 ìM Niclosamide for 3 and 6 hours. 96 genes were consistently overexpressed, 59 down-regulated. 10 genes involved in the tumor necrosis factor (TNF) pathway and 10 genes involving the DNA damage pathway were overexpressed. 13 out of the 59 down-regulated genes were involved in mitochondrial function. Electron microscopy showed that filopodia increased and lipid vacuoles developed whereas mitochondria were less numerous in KOPN-8 cells. The viability of PBMCs from 8 individuals without lymphoma was unchanged when incubated with niclosamide, whereas niclosamide showed significant cytotoxicity in a patient with mantle cell lymphoma (MCL). Conclusion: Niclosamide effectively inhibits the proliferation of B lymphoma cell lines, including vincristine-refractory lymphoma cells, and induces apoptosis at concentrations non-toxic to PBMCs. Interestingly, niclosamide exhibited cytotoxic activity against MCL cells - a finding worth testing further in this difficult-to-treat disease. The mechanism of action of Niclosamide may involve the TNF receptor pathway, mitochondrial function and DNA damage response pathway. We plan to elucidate further specific mechanism(s) of action, and evaluate synergistic effects with other antineoplastic agents, and perform in vivo studies. Citation Format: Junaid Ansari, Hazem El-Osta, Paula Polk, Jeffrey J. Aufman, Guillermo A. Herrera, James Cardelli, Rodney E. Shackelford, Glenn M. Mills, Magdalena L. Circu, Felicity N. E. Gavins, Reinhold Munker. Potent inhibition of the cell proliferation and induction of apoptosis in lymphoma cells by the anthelminthic drug niclosamide: in vitro data. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4816.

Lysosome trafficking plays a significant role in tumor invasion, a key event for the development of metastasis. Previous studies from our laboratory have demonstrated that the anterograde (outward) movement of lysosomes to the cell surface in response to certain tumor microenvironment stimulus, such as hepatocyte growth factor (HGF) or acidic extracellular pH (pHe), increases cathepsin B secretion and tumor cell invasion. Anterograde lysosome trafficking depends on sodium-proton exchanger activity and can be reversed by blocking these ion pumps with Troglitazone or EIPA. Since these drugs cannot be advanced into the clinic due to toxicity, we have designed a high-content assay to discover drugs that block peripheral lysosome trafficking with the goal of identifying novel drugs that inhibit tumor cell invasion. An automated high-content imaging system (Cellomics) was used to measure the position of lysosomes relative to the nucleus. Among a total of 2210 repurposed and natural product drugs screened, 18 "hits" were identified. One of the compounds identified as an anterograde lysosome trafficking inhibitor was niclosamide, a marketed human anti-helminthic drug. Further studies revealed that niclosamide blocked acidic pHe, HGF, and epidermal growth factor (EGF)-induced anterograde lysosome redistribution, protease secretion, motility, and invasion of DU145 castrate resistant prostate cancer cells at clinically relevant concentrations. In an effort to identify the mechanism by which niclosamide prevented anterograde lysosome movement, we found that this drug exhibited no significant effect on the level of ATP, microtubules or actin filaments, and had minimal effect on the PI3K and MAPK pathways. Niclosamide collapsed intralysosomal pH without disruption of the lysosome membrane, while bafilomycin, an agent that impairs lysosome acidification, was also found to induce JLA in our model. Taken together, these data suggest that niclosamide promotes juxtanuclear lysosome aggregation (JLA) via modulation of pathways involved in lysosome acidification. In conclusion, we have designed a validated reproducible high-content assay to screen for drugs that inhibit lysosome trafficking and reduce tumor invasion and we summarize the action of one of these drugs.

We recently demonstrated that methylglyoxal (MG) induced apoptosis of brain microvascular endothelial cells (IHECs) that was preceded by glutathione (GSH) depletion. Here, we test the hypothesis that MG induces occludin glycation and disrupts IHEC barrier function, which is prevented by GSH-dependent MG metabolism. Exposure of IHECs to MG decreased transendothelial electrical resistance (TEER) in association with MG-adduct formation. A 65-kDa MG-glycated protein corresponded to occludin, which was confirmed by immunoprecipitation. Moreover, immunofluorescence staining showed that MG disrupted the architectural organization of ZO-1. Occludin glycation and ZO-1 disruption were prevented by N-acetylcysteine (NAC). Accordingly, TEER loss was abrogated by NAC (via GSH synthesis) and exacerbated by buthionine sulfoximine (BSO; GSH synthesis inhibitor). BSO treatment attenuated D-lactate production, consistent with a role for GSH in glyoxalase I-catalyzed MG elimination. Although MG increased reactive oxygen species (ROS) generation, the ROS scavengers tempol and tiron did not block barrier disruption. This suggests that endogenously generated ROS were unlikely to be a major cause of or did not reach a threshold to elicit barrier failure as elicited by exogenous hydrogen peroxide (300-400 μM). Immunohistochemistry revealed a lower percentage of microvessels stained with anti-occludin, but a higher percentage stained with anti-MG in diabetic rat brain compared to controls. Western analyses confirmed the decrease in diabetic brain occludin expression, but an increase in glycated occludin levels. These results provide novel evidence that reactive carbonyl species can mediate occludin glycation in cerebral microvessels and in microvascular endothelial cells that contribute to barrier dysfunction, a process that was prevented by GSH through enhanced MG catabolism.

The intestinal epithelium sits at the interface between an organism and its luminal environment, and as such is prone to oxidative damage induced by luminal oxidants. Mucosal integrity is maintained by the luminal redox status of the glutathione/glutathione disulfide (GSH/GSSG) and cysteine/cystine (Cys/CySS) couples which also support luminal nutrient absorption, mucus fluidity, and a diverse microbiota. The epithelial layer is uniquely organized for rapid self-renewal that is achieved by the well-regulated processes of crypt stem cell proliferation and crypt-to-villus cell differentiation. The GSH/GSSG and Cys/CySS redox couples, known to modulate intestinal cell transition through proliferation, differentiation or apoptosis, could govern the regenerative potential of the mucosa. These two couples, together with that of the thioredoxin/thioredoxin disulfide (Trx/TrxSS) couple are the major intracellular redox systems, and it is proposed that they each function as distinctive redox control nodes or circuitry in the control of metabolic processes and networks of enzymatic reactions. Specificity of redox signaling is accomplished in part by subcellular compartmentation of the individual redox systems within the mitochondria, nucleus, endoplasmic reticulum, and cytosol wherein each defined redox environment is suited to the specific metabolic function within that compartment. Mucosal oxidative stress would result from the disruption of these unique redox control nodes, and the subsequent alteration in redox signaling can contribute to the development of degenerative pathologies of the intestine, such as inflammation and cancer.

Isocitrate dehydrogenase 1 (IDH1), a cytosolic enzyme that converts isocitrate to alpha‐ketoglutarate, has been shown to be dysregulated during tumorigenesis. However, at what stage of cancer development IDH1 is dysregulated and how IDH1 may affect cell transformation and tumor promotion during early stages of cancer development are unclear. We used a skin cell transformation model and mouse skin epidermal tissues to study the role of IDH1 in early skin tumorigenesis. Our studies demonstrate that both the tumor promoter TPA and UVC irradiation decreased expression and activity levels of IDH1, not IDH2, in the tumor promotable JB6 P+ cell model. Skin epidermal tissues treated with dimethylbenz[α]anthracene/TPA also showed decreases in IDH1 expression and activity. In non‐promotable JB6 P‐cells, IDH1 was upregulated upon TPA treatment, whereas IDH2 was maintained at similar levels with TPA treatment. Interestingly, IDH1 knockdown enhanced, whereas IDH1 overexpression suppressed, TPA‐induced cell transformation. Finally, manganese superoxide dismutase overexpression suppressed tumor promoter induced decreases in IDH1 expression and mitochondrial respiration, while intracellular alpha‐ketoglutarate levels were unchanged. These results suggest that decreased IDH1 expression in early stage skin tumorigenesis is highly correlated with tumor promotion. In addition, oxidative stress might contribute to IDH1 inactivation, because manganese superoxide dismutase, a mitochondrial antioxidant enzyme, blocked decreases in IDH1 expression and activity. (Cancer Sci, doi: 10.1111/j.1349‐7006.2012.02317.x, 2012)

Apoptosis is a highly organized form of cell death that is important for tissue homeostasis, organ development and senescence. To date, the extrinsic (death receptor mediated) and intrinsic (mitochondria derived) apoptotic pathways have been characterized in mammalian cells. Reduced glutathione, is the most prevalent cellular thiol that plays an essential role in preserving a reduced intracellular environment. glutathione protection of cellular macromolecules like deoxyribose nucleic acid proteins and lipids against oxidizing, environmental and cytotoxic agents, underscores its central anti-apoptotic function. Reactive oxygen and nitrogen species can oxidize cellular glutathione or induce its extracellular export leading to the loss of intracellular redox homeostasis and activation of the apoptotic signaling cascade. Recent evidence uncovered a novel role for glutathione involvement in apoptotic signaling pathways wherein post-translational S-glutathiolation of protein redox active cysteines is implicated in the potentiation of apoptosis. In the present review we focus on the key aspects of glutathione redox mechanisms associated with apoptotic signaling that includes: (a) changes in cellular glutathione redox homeostasis through glutathione oxidation or GSH transport in relation to the initiation or propagation of the apoptotic cascade, and (b) evidence for S-glutathiolation in protein modulation and apoptotic initiation.

The integrity of the vascular endothelium of the blood-brain barrier (BBB) is central to cerebrovascular homeostasis. Given the function of the BBB as a physical and metabolic barrier that buffers the systemic environment, oxidative damage to the endothelial monolayer will have significant deleterious impact on the metabolic, immunological, and neurological functions of the brain. Glutathione (GSH) is a ubiquitous major thiol within mammalian cells that plays important roles in antioxidant defense, oxidation-reduction reactions in metabolic pathways, and redox signaling. The existence of distinct GSH pools within the subcellular organelles supports an elegant mode for independent redox regulation of metabolic processes, including those that control cell fate. GSH-dependent homeostatic control of neurovascular function is relatively unexplored. Significantly, GSH regulation of two aspects of endothelial function is paramount to barrier preservation, namely, GSH protection against oxidative endothelial cell injury and GSH control of postdamage cell proliferation in endothelial repair and/or wound healing. This paper highlights our current insights and hypotheses into the role of GSH in cerebral microvascular biology and pathobiology with special focus on endothelial GSH and vascular integrity, oxidative disruption of endothelial barrier function, GSH regulation of endothelial cell proliferation, and the pathological implications of GSH disruption in oxidative stress-associated neurovascular disorders, such as diabetes and stroke.

S-glutathionylation (Pr–SSG) is a specific post-translational modification of cysteine residues by the addition of glutathione. S-Glutathionylated proteins induced by oxidative or nitrosative stress play an essential role in understanding the pathogenesis of the aging and age-related disorder, such as Alzheimer’s disease (AD). The purpose of this research is to develop a novel and ultrasensitive method to accurately and rapidly quantify the Pr–SSG by using capillary gel electrophoresis with laser-induced fluorescence detection (CGE-LIF). The derivatization method is based on the specific reduction of protein-bound S-glutathionylation with glutaredoxin (Grx) and labeling with thiol-reactive fluorescent dye (Dylight 488 maleimide). The experiments were performed by coupling the derivatization method with CGE-LIF to study electrophoretic profiling in in vitro oxidative stress model–S-glutathionylated bovine serum albumin (BSA-SSG), oxidant-induced human colon adenocarcinoma (HT-29) cells, brain tissues, and whole blood samples from an AD transgenic (Tg) mouse model. The results showed almost an eightfold increase in S-glutathionyl abundance when subjecting HT-29 cells in an oxidant environment, resulting in Pr–SSG at 232 ± 10.64 (average ±SD; n = 3) nmol/mg. In the AD–Tg mouse model, an initial quantitative measurement demonstrated the extent of protein S-glutathionylation in three brain regions (hippocampus, cerebellum, and cerebrum), ranging from 1 to 10 nmol/mg. Additionally, we described our developed method to potentially serve as a highly desirable diagnostic tool for monitoring S-glutathionylated protein profile in minuscule amount of whole blood. The whole blood samples for S-glutathionyl expression of 5-month-old AD–Tg mice are quantified as 16.3 μmol/L (=7.2 nmol/mg protein). Altogether, this is a fast, easy, and accurate method, reaching the lowest limit of Pr–SSG detection at 1.8 attomole (amol) level, reported to date.

S-nitrosylated proteins are biomarkers of oxidative damage in aging and Alzheimer's disease (AD). Here, we report a new method for detecting and quantifying nitrosylated proteins by capillary gel electrophoresis with laser induced fluorescence detection (CGE-LIF). Dylight 488 maleimide was used to specifically label thiol group (SH) after switching the S-nitrosothiol (S-NO) to SH in cysteine using the "fluorescence switch" assay. In vitro nitrosylation model-BSA subjected to S-nitrosoglutathione (GSNO) optimized the labeling reactions and characterized the response of the LIF detector. The method proves to be highly sensitive, detecting 1.3 picomolar (pM) concentration of nitrosothiols in nanograms of proteins, which is the lowest limit of detection of nitrosothiols reported to date. We further demonstrated the direct application of this method in monitoring protein nitrosylation damage in MQ mediated human colon adenocarcinoma cells. The nitrosothiol amounts in MQ treated and untreated cells are 14.8±0.2 and 10.4±0.5 pmol/mg of proteins, respectively. We also depicted nitrosylated protein electrophoretic profiles of brain cerebrum of 5-month-old AD transgenic (Tg) mice model. In Tg mice brain, 15.5±0.4 pmol of nitrosothiols/mg of proteins was quantified while wild type contained 11.7±0.3 pmol/mg proteins. The methodology is validated to quantify low levels of S-nitrosylated protein in complex protein mixtures from both physiological and pathological conditions.

Cancer cells distinguish themselves from normal cells in multiple ways. One such distinction is that cancer cells predominantly produce energy by a high rate of glycolysis followed by lactic acid fermentation even in the presence of oxygen, known as the “Warburg Effect”. Although glycolysis is inefficient to produce ATP compared with oxidative phosphorylation, metabolic intermediates produced during glycolysis may provide building blocks for cancer cells. Associated with this metabolic switch is up- or down-regulation of important metabolic enzymes, such as the M1 and M2 isoforms of pyruvate kinase (PKM1; PKM2) and isocitrate dehydrogenase 1 and 2 (IDH1; IDH2). PKM2 is highly expressed and critical for proliferation of tumor cells. Our initial studies showed that both UV irradiation and tumor promoters altered cellular metabolism via upregulating the activity and expression levels of PKM2 and downregulating PKM1. However, this M1/M2 shift has not been reported in early stage of cancer development. IDH1, a cytosolic enzyme which converts isocitrate to alpha-ketoglutarate, was recently discovered to be mutated in human brain cancer and leukemia. Interestingly, wild-type IDH1 produces alpha-ketoglutarate; however, mutant IDH1 generates 2-hydroxyglutarate, a known “oncometabolite.” We have found that decreased expression and activity levels of IDH1, not IDH2 were seen both in vitro in the tumor promotable JB6 P+ cell model and in vivo using mouse skin epidermal tissues, following UV and 12–0-tetradecanoylphorbol 13-acetate (TPA) treatment. More importantly, knockdown of IDH1 enhanced whereas overexpression of IDH1 suppressed cell transformation induced by tumor promoter TPA. During tumorigenesis, oncogenic activation can also generate oxidative stress. Since metabolism is the major source for ROS (reactive oxygen species) production, metabolic changes often observed in cancer cells may also be regulated by oxidative stress. Our initial studies revealed that PKM2 was upregulated and IDH1 downregulated in promotable mouse skin epidermal JB6 P+ cells, not in non-promotable JB6 P-cells. We have observed in previous studies that MnSOD expression/activity is lower in P+ cells, compared to P-cells, thus providing evidence that PKM2 activation and IDH1 inactivation may be regulated by oxidative stress. Lastly, overexpression of manganese superoxide dismutase (MnSOD) suppressed tumor promoter-induced PKM2 activation and decreased IDH1 expression. These results suggest that oxidative stress may contribute to PKM2 activation and IDH1 inactivation during early stage tumor promotion, suggesting mediation with MnSOD, a mitochondrial antioxidant enzyme, may play a cancer preventative role in regulating cellular metabolism. Citation Information: Cancer Prev Res 2011;4(10 Suppl):B59.

The intestinal tract, known for its capability for self-renew, represents the first barrier of defence between the organism and its luminal environment. The thiol/disulfide redox systems comprising the glutathione/glutathione disulfide (GSH/GSSG), cysteine/cystine (Cys/CySS) and reduced and oxidized thioredoxin (Trx/TrxSS) redox couples play important roles in preserving tissue redox homeostasis, metabolic functions, and cellular integrity. Control of the thiol-disulfide status at the luminal surface is essential for maintaining mucus fluidity and absorption of nutrients, and protection against chemical-induced oxidant injury. Within intestinal cells, these redox couples preserve an environment that supports physiological processes and orchestrates networks of enzymatic reactions against oxidative stress. In this review, we focus on the intestinal redox and antioxidant systems, their subcellular compartmentation, redox signalling and epithelial turnover, and contribution of luminal microbiota, key aspects that are relevant to understanding redox-dependent processes in gut biology with implications for degenerative digestive disorders, such as inflammation and cancer.

Stationary-phase Saccharomyces cerevisiae cells transferred from spent rich media into water live for weeks, whereas the same cells die within hours if transferred into water with 2% glucose in a process called sugar-induced cell death (SICD). Our hypothesis is that SICD is due to a dysregulated Crabtree effect, which is the phenomenon whereby glucose transiently inhibits respiration and ATP synthesis. We found that stationary-phase cells in glucose/water consume 21 times more O(2) per cell than exponential-phase cells in rich media, and such excessive O(2) consumption causes reactive oxygen species to accumulate. We also found that inorganic phosphate and succinate protect against SICD but by different mechanisms. Phosphate protects by triggering the synthesis of Fru-1,6-P(2), which inhibits respiration in isolated mitochondria. Succinate protects in wild-type cells but fails to protect in dic1Δ cells. DIC1 codes for a mitochondrial inner membrane protein that exchanges cytosolic succinate for matrix phosphate. We propose that succinate depletes matrix phosphate, which in turn inhibits respiration and ATP synthesis. In sum, restoring the Crabtree effect, whether with phosphate or succinate, protects cells from SICD.

We recently demonstrated that menadione (MQ), a redox cycling quinone, mediated the loss of mitochondrial glutathione/glutathione disulfide redox balance. In this study, we showed that MQ significantly disrupted cellular pyridine nucleotide (NAD(+)/NADH, NADP(+)/NADPH) redox balance that compromised cellular ATP, mitochondrial respiratory activity, and NADPH-dependent reducing capacity in colonic epithelial cells, a scenario that was exaggerated by low glucose. In the cytosol, MQ induced NAD(+) loss concurrent with increased NADP(+) and NAD kinase activity, but decreased NADPH. In the mitochondria, NADH loss occurred in conjunction with increased nicotinamide nucleotide transhydrogenase activity and NADP(+), and decreased NADPH. These results are consistent with cytosolic NAD(+)-to-NADP(+) and mitochondrial NADH-to-NADPH shifts, but compromised NADPH availability. Thus, despite the sacrifice of NAD(+)/NADH in favor of NADPH generation, steady-state NADPH levels were not maintained during MQ challenge. Impairments of cellular bioenergetics were evidenced by ATP losses and increased mitochondrial O(2) dependence of pyridine nucleotide oxidation-reduction; half-maximal oxidation (P(50)) was 10-fold higher in low glucose, which was lowered by glutamate or succinate supplementation. This exaggerated O(2) dependence is consistent with increased O(2) diversion to nonmitochondrial O(2) consumption by MQ-semiquinone redox cycling secondary to decreased NADPH-dependent MQ detoxication at low glucose, a situation that was corrected by glucose-sparing mitochondrial substrates.

Reactive oxygen species (ROS) are products of normal metabolism and xenobiotic exposure, and depending on their concentration, ROS can be beneficial or harmful to cells and tissues. At physiological low levels, ROS function as “redox messengers” in intracellular signaling and regulation, whereas excess ROS induce oxidative modification of cellular macromolecules, inhibit protein function, and promote cell death. Additionally, various redox systems, such as the glutathione, thioredoxin, and pyridine nucleotide redox couples, participate in cell signaling and modulation of cell function, including apoptotic cell death. Cell apoptosis is initiated by extracellular and intracellular signals via two main pathways, the death receptor- and the mitochondria-mediated pathways. Various pathologies can result from oxidative stress-induced apoptotic signaling that is consequent to ROS increases and/or antioxidant decreases, disruption of intracellular redox homeostasis, and irreversible oxidative modifications of lipid, protein, or DNA. In this review, we focus on several key aspects of ROS and redox mechanisms in apoptotic signaling and highlight the gaps in knowledge and potential avenues for further investigation. A full understanding of the redox control of apoptotic initiation and execution could underpin the development of therapeutic interventions targeted at oxidative stress-associated disorders.

The major cellular antioxidant glutathione is depleted during HIV infection and in obesity. Although the consequence of glutathione depletion on immune function is starting to emerge, it is currently not known whether glutathione dysregulation influences the differentiation and maturation of dendritic cells (DCs). Moreover, the effect of glutathione depletion on DC effector functions, such as Ag presentation, is poorly understood. Glutathione synthesis depends on the cystine/glutamate antiporter, which transports the rate-limiting precursor cystine into the cell in exchange for glutamate. In this paper, we present a detailed study of antiporter function in DCs and demonstrate a role for the antiporter in DC differentiation and cross-presentation. We show that the antiporter is the major mechanism for transport of cystine and glutamate and modulates the intracellular glutathione content and glutathione efflux from DCs. Blocking antiporter-dependent cystine transport decreases intracellular glutathione levels, and these effects correlate with reduced transcription of the functional subunit of the antiporter. We further demonstrate that blocking antiporter activity interferes with DC differentiation from monocyte precursors, but antiporter activity is not required for LPS-induced phenotypic maturation. Finally, we show that inhibiting antiporter uptake of cystine interferes with presentation of exogenous Ag to class II MHC-restricted T cells and blocks cross-presentation on MHC class I. We conclude that aberrant antiporter function disrupts glutathione homeostasis in DCs and may contribute to impaired immunity in the diseased host.