Mannose impairs tumour growth and enhances chemotherapy.

It is now well established that tumours undergo changes in cellular metabolism1. As this can reveal tumour cell vulnerabilities and because many tumours exhibit enhanced glucose uptake2, we have been interested in how tumour cells respond to different forms of sugar. Here we report that the monosaccharide mannose causes growth retardation in several tumour types in vitro, and enhances cell death in response to major forms of chemotherapy. We then show that these effects also occur in vivo in mice following the oral administration of mannose, without significantly affecting the weight and health of the animals. Mechanistically, mannose is taken up by the same transporter(s) as glucose3 but accumulates as mannose-6-phosphate in cells, and this impairs the further metabolism of glucose in glycolysis, the tricarboxylic acid cycle, the pentose phosphate pathway and glycan synthesis. As a result, the administration of mannose in combination with conventional chemotherapy affects levels of anti-apoptotic proteins of the Bcl-2 family, leading to sensitization to cell death. Finally we show that susceptibility to mannose is dependent on the levels of phosphomannose isomerase (PMI). Cells with low levels of PMI are sensitive to mannose, whereas cells with high levels are resistant, but can be made sensitive by RNA-interference-mediated depletion of the enzyme. In addition, we use tissue microarrays to show that PMI levels also vary greatly between different patients and different tumour types, indicating that PMI levels could be used as a biomarker to direct the successful administration of mannose. We consider that the administration of mannose could be a simple, safe and selective therapy in the treatment of cancer, and could be applicable to multiple tumour types.

Oncogene-induced maladaptive activation of trained immunity in the pathogenesis and treatment of Erdheim-Chester disease.

Trained immunity (TI) is a proinflammatory program induced in monocyte/macrophages upon sensing of specific pathogens and is characterized by immunometabolic and epigenetic changes that enhance cytokine production. Maladaptive activation of TI (ie, in the absence of infection) may result in detrimental inflammation and development of disease; however, the exact role and extent of inappropriate activation of TI in the pathogenesis of human diseases is undetermined. In this study, we uncovered the oncogene-induced, maladaptive induction of TI in the pathogenesis of a human inflammatory myeloid neoplasm (Erdheim-Chester disease, [ECD]), characterized by the BRAFV600E oncogenic mutation in monocyte/macrophages and excess cytokine production. Mechanistically, myeloid cells expressing BRAFV600E exhibit all molecular features of TI: activation of the AKT/mammalian target of rapamycin signaling axis; increased glycolysis, glutaminolysis, and cholesterol synthesis; epigenetic changes on promoters of genes encoding cytokines; and enhanced cytokine production leading to hyperinflammatory responses. In patients with ECD, effective therapeutic strategies combat this maladaptive TI phenotype; in addition, pharmacologic inhibition of immunometabolic changes underlying TI (ie, glycolysis) effectively dampens cytokine production by myeloid cells. This study revealed the deleterious potential of inappropriate activation of TI in the pathogenesis of human inflammatory myeloid neoplasms and the opportunity for inhibition of TI in conditions characterized by maladaptive myeloid-driven inflammation.

Fumarate is an epigenetic modifier that elicits epithelial-to-mesenchymal transition.

Mutations of the tricarboxylic acid cycle enzyme fumarate hydratase cause hereditary leiomyomatosis and renal cell cancer. Fumarate hydratase-deficient renal cancers are highly aggressive and metastasize even when small, leading to a very poor clinical outcome. Fumarate, a small molecule metabolite that accumulates in fumarate hydratase-deficient cells, plays a key role in cell transformation, making it a bona fide oncometabolite. Fumarate has been shown to inhibit α-ketoglutarate-dependent dioxygenases that are involved in DNA and histone demethylation. However, the link between fumarate accumulation, epigenetic changes, and tumorigenesis is unclear. Here we show that loss of fumarate hydratase and the subsequent accumulation of fumarate in mouse and human cells elicits an epithelial-to-mesenchymal-transition (EMT), a phenotypic switch associated with cancer initiation, invasion, and metastasis. We demonstrate that fumarate inhibits Tet-mediated demethylation of a regulatory region of the antimetastatic miRNA cluster mir-200ba429, leading to the expression of EMT-related transcription factors and enhanced migratory properties. These epigenetic and phenotypic changes are recapitulated by the incubation of fumarate hydratase-proficient cells with cell-permeable fumarate. Loss of fumarate hydratase is associated with suppression of miR-200 and the EMT signature in renal cancer and is associated with poor clinical outcome. These results imply that loss of fumarate hydratase and fumarate accumulation contribute to the aggressive features of fumarate hydratase-deficient tumours.

S-nitrosylation drives cell senescence and aging in mammals by controlling mitochondrial dynamics and mitophagy.

S-nitrosylation, a prototypic redox-based posttranslational modification, is frequently dysregulated in disease. S-nitrosoglutathione reductase (GSNOR) regulates protein S-nitrosylation by functioning as a protein denitrosylase. Deficiency of GSNOR results in tumorigenesis and disrupts cellular homeostasis broadly, including metabolic, cardiovascular, and immune function. Here, we demonstrate that GSNOR expression decreases in primary cells undergoing senescence, as well as in mice and humans during their life span. In stark contrast, exceptionally long-lived individuals maintain GSNOR levels. We also show that GSNOR deficiency promotes mitochondrial nitrosative stress, including excessive S-nitrosylation of Drp1 and Parkin, thereby impairing mitochondrial dynamics and mitophagy. Our findings implicate GSNOR in mammalian longevity, suggest a molecular link between protein S-nitrosylation and mitochondria quality control in aging, and provide a redox-based perspective on aging with direct therapeutic implications.

S-Nitrosoglutathione Reductase Plays Opposite Roles in SH-SY5Y Models of Parkinson's Disease and Amyotrophic Lateral Sclerosis.

Oxidative and nitrosative stresses have been reported as detrimental phenomena concurring to the onset of several neurodegenerative diseases. Here we reported that the ectopic modulation of the denitrosylating enzyme S-nitrosoglutathione reductase (GSNOR) differently impinges on the phenotype of two SH-SY5Y-based in vitro models of neurodegeneration, namely, Parkinson's disease (PD) and familial amyotrophic lateral sclerosis (fALS). In particular, we provide evidence that GSNOR-knocking down protects SH-SY5Y against PD toxins, while, by contrast, its upregulation is required for G93A-SOD1 expressing cells resistance to NO-releasing drugs. Although completely opposite, both conditions are characterized by Nrf2 localization in the nuclear compartment: in the first case induced by GSNOR silencing, while in the second one underlying the antinitrosative response. Overall, our results demonstrate that GSNOR expression has different effect on neuronal viability in dependence on the stimulus applied and suggest that GSNOR could be a responsive gene downstream of Nrf2 activation.

Redox implications of AMPK-mediated signal transduction beyond energetic clues.

Since the discovery of AMP-dependent protein kinase (AMPK), its fundamental role in regulating metabolic pathways and the molecular mechanism underlying the regulation of its activity by adenine nucleotides has been widely studied. AMPK is not only an energy-responsive enzyme, but it also senses redox signals. This review aims at recapitulating the recent lines of evidence that demonstrate the responsiveness of this kinase to metabolic and nitroxidative imbalance, thus providing new insights into the intimate networks of redox-based signals upstream of AMPK. In particular, we discuss its well-recognized activation downstream of mitochondrial dysfunction, debate the recent findings that AMPK is directly targeted by pro-oxidant species, and question alternative redox pathways that allow AMPK to be included into the large class of redox-sensing proteins. The possible therapeutic implications of the role of AMPK in redox-associated pathologies, such as cancer and neurodegeneration, are also discussed in light of recent advances that suggest a role for AMPK in the tuning of redox-dependent processes, such as apoptosis and autophagy.

ACOD1 deficiency offers protection in a mouse model of diet-induced obesity by maintaining a healthy gut microbiota.

Aconitate decarboxylase 1 (ACOD1) is the enzyme synthesizing itaconate, an immuno-regulatory metabolite tuning host-pathogen interactions. Such functions are achieved by affecting metabolic pathways regulating inflammation and microbe survival. However, at the whole-body level, metabolic roles of itaconate remain largely unresolved. By using multiomics-integrated approaches, here we show that ACOD1 responds to high-fat diet consumption in mice by promoting gut microbiota alterations supporting metabolic disease. Genetic disruption of itaconate biosynthesis protects mice against obesity, alterations in glucose homeostasis and liver metabolic dysfunctions by decreasing meta-inflammatory responses to dietary lipid overload. Mechanistically, fecal metagenomics and microbiota transplantation experiments demonstrate such effects are dependent on an amelioration of the intestinal ecosystem composition, skewed by high-fat diet feeding towards obesogenic phenotype. In particular, unbiased fecal microbiota profiling and axenic culture experiments point towards a primary role for itaconate in inhibiting growth of Bacteroidaceae and Bacteroides, family and genus of Bacteroidetes phylum, the major gut microbial taxon associated with metabolic health. Specularly to the effects imposed by Acod1 deficiency on fecal microbiota, oral itaconate consumption enhances diet-induced gut dysbiosis and associated obesogenic responses in mice. Unveiling an unrecognized role of itaconate, either endogenously produced or exogenously administered, in supporting microbiota alterations underlying diet-induced obesity in mice, our study points ACOD1 as a target against inflammatory consequences of overnutrition.

Nitric oxide is the primary mediator of cytotoxicity induced by GSH depletion in neuronal cells.

Glutathione (GSH) levels progressively decline during aging and in neurodegenerative disorders. However, the contribution of such event in mediating neuronal cell death is still uncertain. In this report, we show that, in neuroblastoma cells as well as in primary mouse cortical neurons, GSH decrease, induced by buthionine sulfoximine (BSO), causes protein nitration, S-nitrosylation and DNA strand breaks. Such alterations are also associated with inhibition of cytochrome c oxidase activity and microtubule network disassembly, which are considered hallmarks of nitric oxide (NO) toxicity. In neuroblastoma cells, BSO treatment also induces cell proliferation arrest through the ERK1/2-p53 pathway that finally results in caspase-independent apoptosis, as evident from the translocation of apoptosis-inducing factor from mitochondria towards nuclei. A deeper analysis of the signaling processes indicates that the NO-cGMP pathway is involved in cell proliferation arrest and death. In fact, these events are completely reversed by L-NAME, a specific NO synthase inhibitor, indicating that NO, rather than the depletion of GSH per se, is the primary mediator of cell damage. In addition, the guanylate cyclase (GC) inhibitor LY83583 is able to completely block activation of ERK1/2 and counteract BSO toxicity. In cortical neurons, NMDA (N-methyl-D-aspartic acid) treatment results in GSH decrease and BSO-mediated NO cytotoxicity is enhanced by either epidermal growth factor (EGF) or NMDA. These findings support the idea that GSH might represent the most important buffer of NO toxicity in neuronal cells, and indicate that the disruption of cellular redox buffering controlled by GSH makes neuronal cells susceptible to endogenous physiological flux of NO.

Pyruvate transamination and NAD biosynthesis enable proliferation of succinate dehydrogenase-deficient cells by supporting aerobic glycolysis.

Succinate dehydrogenase (SDH) is the mitochondrial enzyme converting succinate to fumarate in the tricarboxylic acid (TCA) cycle. SDH acts as a tumor suppressor with germline loss-of-function mutations in its encoding genes predisposing to aggressive familial neuroendocrine and renal cancer syndromes. Lack of SDH activity disrupts the TCA cycle, imposes Warburg-like bioenergetic features, and commits cells to rely on pyruvate carboxylation for anabolic needs. However, the spectrum of metabolic adaptations enabling SDH-deficient tumors to cope with a dysfunctional TCA cycle remains largely unresolved. By using previously characterized Sdhb-deleted kidney mouse cells, here we found that SDH deficiency commits cells to rely on mitochondrial glutamate-pyruvate transaminase (GPT2) activity for proliferation. We showed that GPT2-dependent alanine biosynthesis is crucial to sustain reductive carboxylation of glutamine, thereby circumventing the TCA cycle truncation determined by SDH loss. By driving the reductive TCA cycle anaplerosis, GPT2 activity fuels a metabolic circuit maintaining a favorable intracellular NAD+ pool to enable glycolysis, thus meeting the energetic demands of SDH-deficient cells. As a metabolic syllogism, SDH deficiency confers sensitivity to NAD+ depletion achieved by pharmacological inhibition of nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme of the NAD+ salvage pathway. Beyond identifying an epistatic functional relationship between two metabolic genes in the control of SDH-deficient cell fitness, this study disclosed a metabolic strategy to increase the sensitivity of tumors to interventions limiting NAD availability.

Linoleic acid potentiates CD8+ T cell metabolic fitness and antitumor immunity.

The metabolic state represents a major hurdle for an effective adoptive T cell therapy (ACT). Indeed, specific lipids can harm CD8+ T cell (CTL) mitochondrial integrity, leading to defective antitumor responses. However, the extent to which lipids can affect the CTL functions and fate remains unexplored. Here, we show that linoleic acid (LA) is a major positive regulator of CTL activity by improving metabolic fitness, preventing exhaustion, and stimulating a memory-like phenotype with superior effector functions. We report that LA treatment enhances the formation of ER-mitochondria contacts (MERC), which in turn promotes calcium (Ca2+) signaling, mitochondrial energetics, and CTL effector functions. As a direct consequence, the antitumor potency of LA-instructed CD8 T cells is superior in vitro and in vivo. We thus propose LA treatment as an ACT potentiator in tumor therapy.

Targeting aerobic glycolysis: 3-bromopyruvate as a promising anticancer drug.

The Warburg effect refers to the phenomenon whereby cancer cells avidly take up glucose and produce lactic acid under aerobic conditions. Although the molecular mechanisms underlying tumor reliance on glycolysis remains not completely clear, its inhibition opens feasible therapeutic windows for cancer treatment. Indeed, several small molecules have emerged by combinatorial studies exhibiting promising anticancer activity both in vitro and in vivo, as a single agent or in combination with other therapeutic modalities. Therefore, besides reviewing the alterations of glycolysis that occur with malignant transformation, this manuscript aims at recapitulating the most effective pharmacological therapeutics of its targeting. In particular, we describe the principal mechanisms of action and the main targets of 3-bromopyruvate, an alkylating agent with impressive antitumor effects in several models of animal tumors. Moreover, we discuss the chemo-potentiating strategies that would make unparalleled the putative therapeutic efficacy of its use in clinical settings.

Metabolic oxidative stress elicited by the copper(II) complex [Cu(isaepy)2] triggers apoptosis in SH-SY5Y cells through the induction of the AMP-activated protein kinase/p38MAPK/p53 signalling axis: evidence for a combined use with 3-bromopyruvate in neuroblastoma treatment.

We have demonstrated previously that the complex bis[(2-oxindol-3-ylimino)-2-(2-aminoethyl)pyridine-N,N']copper(II), named [Cu(isaepy)(2)], induces AMPK (AMP-activated protein kinase)-dependent/p53-mediated apoptosis in tumour cells by targeting mitochondria. In the present study, we found that p38(MAPK) (p38 mitogen-activated protein kinase) is the molecular link in the phosphorylation cascade connecting AMPK to p53. Transfection of SH-SY5Y cells with a dominant-negative mutant of AMPK resulted in a decrease in apoptosis and a significant reduction in phospho-active p38(MAPK) and p53. Similarly, reverse genetics of p38(MAPK) yielded a reduction in p53 and a decrease in the extent of apoptosis, confirming an exclusive hierarchy of activation that proceeds via AMPK/p38(MAPK)/p53. Fuel supplies counteracted [Cu(isaepy)(2)]-induced apoptosis and AMPK/p38(MAPK)/p53 activation, with glucose being the most effective, suggesting a role for energetic imbalance in [Cu(isaepy)(2)] toxicity. Co-administration of 3BrPA (3-bromopyruvate), a well-known inhibitor of glycolysis, and succinate dehydrogenase, enhanced apoptosis and AMPK/p38(MAPK)/p53 signalling pathway activation. Under these conditions, no toxic effect was observed in SOD (superoxide dismutase)-overexpressing SH-SY5Y cells or in PCNs (primary cortical neurons), which are, conversely, sensitized to the combined treatment with [Cu(isaepy)(2)] and 3BrPA only if grown in low-glucose medium or incubated with the glucose-6-phosphate dehydrogenase inhibitor dehydroepiandrosterone. Overall, the results suggest that NADPH deriving from the pentose phosphate pathway contributes to PCN resistance to [Cu(isaepy)(2)] toxicity and propose its employment in combination with 3BrPA as possible tool for cancer treatment.

Disruption of the M80-Fe ligation stimulates the translocation of cytochrome c to the cytoplasm and nucleus in nonapoptotic cells.

Native cytochrome c (cyt c) has a compact tertiary structure with a hexacoordinated heme iron and functions in electron transport in mitochondria and apoptosis in the cytoplasm. However, the possibility that protein modifications confer additional functions to cyt c has not been explored. Disruption of methionine 80 (M80)-Fe ligation of cyt c under nitrative stress has been reported. To model this alteration and determine if it confers new properties to cyt c, a cyt c mutant (M80A) was constitutively expressed in cells. M80A-cyt c has increased peroxidase activity and is spontaneously released from mitochondria, translocating to the cytoplasm and nucleus in the absence of apoptosis. Moreover, M80A models endogenously nitrated cyt c because nitration of WT-cyt c is associated with its translocation to the cytoplasm and nucleus. Further, M80A cyt c may up-regulate protective responses to nitrative stress. Our findings raise the possibility that endogenous protein modifications that disrupt the M80-Fe ligation (such as tyrosine nitration) stimulate nuclear translocation and confer new functions to cyt c in nonapoptotic cells.

p38(MAPK)/p53 signalling axis mediates neuronal apoptosis in response to tetrahydrobiopterin-induced oxidative stress and glucose uptake inhibition: implication for neurodegeneration.

BH4 (tetrahydrobiopterin) induces neuronal demise via production of ROS (reactive oxygen species). In the present study we investigated the mechanisms of its toxicity and the redox signalling events responsible for the apoptotic commitment in SH-SY5Y neuroblastoma cells and in mouse primary cortical neurons. We identified in p38(MAPK)/p53 a BH4-responsive pro-apoptotic signalling axis, as demonstrated by the recovery of neuronal viability achieved by gene silencing or pharmacological inhibition of both p38(MAPK) and p53. BH4-induced oxidative stress was characterized by a decrease in the GSH/GSSG ratio, an increase in protein carbonylation and DNA damage. BH4 toxicity and the redox-activated apoptotic pathway were counteracted by the H2O2-scavengers catalase and N-acetylcysteine and enhanced by the GSH neo-synthesis inhibitor BSO (buthionine sulfoximine). We also demonstrated that BH4 impairs glucose uptake and utilization, which was prevented by catalase administration. This effect contributes to the neuronal demise, exacerbating BH4-induced nuclear damage and the activation of the pro-apoptotic p38(MAPK)/p53 axis. Inhibition of glucose uptake was also observed upon treatment with 6-hydroxydopamine, another redox-cycling molecule, suggesting a common mechanism of action for auto-oxidizable neurotoxins.

The heme synthesis-export system regulates the tricarboxylic acid cycle flux and oxidative phosphorylation.

Heme is an iron-containing porphyrin of vital importance for cell energetic metabolism. High rates of heme synthesis are commonly observed in proliferating cells. Moreover, the cell-surface heme exporter feline leukemia virus subgroup C receptor 1a (FLVCR1a) is overexpressed in several tumor types. However, the reasons why heme synthesis and export are enhanced in highly proliferating cells remain unknown. Here, we illustrate a functional axis between heme synthesis and heme export: heme efflux through the plasma membrane sustains heme synthesis, and implementation of the two processes down-modulates the tricarboxylic acid (TCA) cycle flux and oxidative phosphorylation. Conversely, inhibition of heme export reduces heme synthesis and promotes the TCA cycle fueling and flux as well as oxidative phosphorylation. These data indicate that the heme synthesis-export system modulates the TCA cycle and oxidative metabolism and provide a mechanistic basis for the observation that both processes are enhanced in cells with high-energy demand.

The Tricarboxylic Acid Cycle at the Crossroad Between Cancer and Immunity.

Significance: The tricarboxylic acid (TCA) cycle is a housekeeping metabolic pathway essential for generation of energy and biosynthetic intermediates. Alterations of the TCA cycle play a pivotal role in oncogenesis and inflammation. As such, some metabolic vulnerabilities, imposed by TCA cycle dysfunction in cancer, have been identified. Similarly, the TCA cycle appeared as an actionable pathway in immunopathologies. Recent Advances: Metabolic changes accompanying cell transformation have been usually considered as adaptive mechanisms to malignant transformation. The identification of oncogenic mutations in some TCA cycle enzymes changed this view, indicating altered mitochondrial metabolism as an instrumental mechanism for cancer initiation. Similarly, the observation that TCA cycle-derived metabolites have multiple signaling roles in immune cells supports the idea of this pathway as a metabolic rheostat of immune responses. Critical Issues: This review summarizes the crucial role of the TCA cycle in pathophysiology describing the post-translational and epigenetic impact of oncometabolites accumulation in cancer and immune cells. Future Directions: Additional studies will be necessary to further explore the role of oncometabolites in paracrine signaling and to identify genuine metabolic and nutritional liabilities imposed by TCA cycle dysfunction in cancer, hardly to be escaped by resistance mechanisms.

D-mannose suppresses macrophage IL-1ß production.

D-mannose is a monosaccharide approximately a hundred times less abundant than glucose in human blood. Previous studies demonstrated that supraphysiological levels of D-mannose inhibit tumour growth and stimulate regulatory T cell differentiation. It is not known whether D-mannose metabolism affects the function of non-proliferative cells, such as inflammatory macrophages. Here, we show that D-mannose suppresses LPS-induced macrophage activation by impairing IL-1ß production. In vivo, mannose administration improves survival in a mouse model of LPS-induced endotoxemia as well as decreases progression in a mouse model of DSS-induced colitis. Phosphomannose isomerase controls response of LPS-activated macrophages to D-mannose, which impairs glucose metabolism by raising intracellular mannose-6-phosphate levels. Such alterations result in the suppression of succinate-mediated HIF-1α activation, imposing a consequent reduction of LPS-induced Il1b expression. Disclosing an unrecognized metabolic hijack of macrophage activation, our study points towards safe D-mannose utilization as an effective intervention against inflammatory conditions.

The isatin-Schiff base copper(II) complex Cu(isaepy)2 acts as delocalized lipophilic cation, yields widespread mitochondrial oxidative damage and induces AMP-activated protein kinase-dependent apoptosis.

We previously demonstrated that Bis[(2-oxindol-3-ylimino)-2-(2-aminoethyl)pyridine-N,N']copper(II) [Cu(isaepy)(2)] was an efficient inducer of the apoptotic mitochondrial pathway. Here, we deeply dissect the mechanisms underlying the ability of Cu(isaepy)(2) to cause mitochondriotoxicity. In particular, we demonstrate that Cu(isaepy)(2) increases NADH-dependent oxygen consumption of isolated mitochondria and that this phenomenon is associated with oxy-radical production and insensitive to adenosine diphosphate. These data indicate that Cu(isaepy)(2) behaves as an uncoupler and this property is also confirmed in cell systems. Particularly, SH-SY5Y cells show: (i) an early loss of mitochondrial transmembrane potential; (ii) a decrease in the expression levels of respiratory complex components and (iii) a significant adenosine triphosphate (ATP) decrement. The causative energetic impairment mediated by Cu(isaepy)(2) in apoptosis is confirmed by experiments carried out with rho(0) cells, or by glucose supplementation, where cell death is significantly inhibited. Moreover, gastric and cervix carcinoma AGS and HeLa cells, which rely most of their ATP production on oxidative phosphorylation, show a marked sensitivity toward Cu(isaepy)(2). Adenosine monophosphate-activated protein kinase (AMPK), which is activated by events increasing the adenosine monophosphate:ATP ratio, is deeply involved in the apoptotic process because the overexpression of its dominant/negative form completely abolishes cell death. Upon glucose supplementation, AMPK is not activated, confirming its role as fuel-sensing enzyme that positively responds to Cu(isaepy)(2)-mediated energetic impairment by committing cells to apoptosis. Overall, data obtained indicate that Cu(isaepy)(2) behaves as delocalized lipophilic cation and induces mitochondrial-sited reactive oxygen species production. This event results in mitochondrial dysfunction and ATP decrease, which in turn triggers AMPK-dependent apoptosis.

Reactive oxygen species mediate p53 activation and apoptosis induced by sodium nitroprusside in SH-SY5Y cells.

Sodium nitroprusside (SNP) is a water-soluble iron nitrosyl complex clinically used as a powerful vasodilator for treatment of hypertension; and, in basic research, it has been used to mainly investigate the cytotoxic effects of nitrosative stress. Although NO is considered a pharmacologically active molecule, not all of the biological effects of SNP are dependent on its NO moiety. To elucidate the molecular executioner(s) responsible for SNP cytotoxicity, this study determines the involvement of oxidative stress in p53 activation and apoptotic induction elicited by SNP in SH-SY5Y neuroblastoma cells. We demonstrate that proapoptotic activity of SNP is independent of NO production, because SNP and its 2-day light-exhausted compound SNP(ex) trigger apoptosis to the same extent. We provide evidence for the occurrence of oxidative stress and oxidative damage during both SNP and SNP(ex) exposure and demonstrate that iron-derived reactive oxygen species (ROS) are the genuine mediators of their cytotoxicity. We show that p53 is equally activated upon both SNP and SNP(ex) treatments. Moreover, as demonstrated by small interfering RNA experiments, we indicate its primary role in the induction of apoptosis, suggesting the ineffectiveness of NO in its engagement. The attenuation of p53 levels, obtained by oxy-radical scavengers, is consistent with the recovery of cell viability and ROS decrease, demonstrate that SNP-mediated p53 activation is an event triggered by ROS and/or ROS-mediated damages. Together, our results suggest that investigations of the physiopathological effects of SNP should consider the role of ROS, other than NO, particularly in some conditions such as apoptotic induction and p53 activation.