The Domains of Human Nutrition: The Importance of Nutrition Education in Academia and Medical Schools.

Human nutrition encompasses an extremely broad range of medical, social, commercial, and ethical domains and thus represents a wide, interdisciplinary scientific and cultural discipline. The high prevalence of both disease-related malnutrition and overweight/obesity represents an important risk factor for disease burden and mortality worldwide. It is the opinion of Federation of the Italian Nutrition Societies (FeSIN) that these two sides of the same coin, with their sociocultural background, are related to a low "nutritional culture" secondary, at least in part, to an insufficient academic training for health-care professionals (HCPs). Therefore, FeSIN created a study group, composed of delegates of all the federated societies and representing the different HCPs involved in human nutrition, with the aim of identifying and defining the domains of human nutrition in the attempt to more clearly define the cultural identity of human nutrition in an academically and professionally oriented perspective and to report the conclusions in a position paper. Three main domains of human nutrition, namely, basic nutrition, applied nutrition, and clinical nutrition, were identified. FeSIN has examined the areas of knowledge pertinent to human nutrition. Thirty-two items were identified, attributed to one or more of the three domains and ranked considering their diverse importance for academic training in the different domains of human nutrition. Finally, the study group proposed the attribution of the different areas of knowledge to the degree courses where training in human nutrition is deemed necessary (e.g., schools of medicine, biology, nursing, etc.). It is conceivable that, in the near future, a better integration of the professionals involved in the field of human nutrition will eventually occur based on the progressive consolidation of knowledge, competence, and skills in the different areas and domains of this discipline.

Psychosocial, behavioural, pedagogical, and nutritional proposals about how to encourage eating a healthy breakfast.

BACKGROUND: Even if more and more evidences have highlighted the importance of breakfast in the growth and development of children, from 10 to 30% of US and European children and adolescents regularly skip breakfast. Thus, there is still a lot to be done before breakfast becomes a daily habit. The aim of this paper is to try and understand how it is possible to overcome the real or imaginary difficulties associated with skipping breakfast by psychosocial, behavioural, pedagogical and nutritional proposals. DISCUSSION: Schools are the best context where perform healthy interventions because it is here that children learn about the importance of good health at an age when the school still plays a major role in their education. Some school interventions, based on solid theories as the Self Determination Theory and the Behaviour Analysis, have been implemented in the last years to promote health behaviour such as intake of fruit and vegetables and physical activities. Cognitive behaviour therapy is the most closely monitored type of treatment/cure for obesity in randomised controlled trials. Moreover some associations such as the National Association of Food Science Specialists have drawn an own method to encourage food education at school and promote the importance of prevention. These projects could be used as starting point to perform interventions focus on breakfast. SUMMARY: Increase the consumption of breakfast between children is very important. Efforts should be done to drawn new school projects based on scientific-evidences.

Breakfast: a multidisciplinary approach.

BACKGROUND: The role of breakfast as an essential part of an healthy diet has been only recently promoted even if breakfast practices were known since the Middle Age. The growing scientific evidences on this topic are extremely sector-based nevertheless breakfast could be regarded from different point of views and from different expertises. This approach, that take into account history, sociology, anthropology, medicine, psychology and pedagogy, is useful to better understand the value of this meal in our culture. The aim of this paper was to analyse breakfast-related issues based on a multidisciplinary approach with input by specialists from different fields of learning. DISCUSSION: Breakfast is now recommended as part of a diet because it is associated with healthier macro- and micronutrient intakes, body mass index and lifestyle. Moreover recent studies showed that breakfast improves cognitive function, intuitive perception and academic performance. Research demonstrates the importance of providing breakfast not only to children but in adults and elderly too. Although the important role breakfast plays in maintaining the health, epidemiological data from industrialised countries reveal that many individuals either eat a nutritionally unhealthy breakfast or skip it completely. SUMMARY: The historical, bio-psychological and educational value of breakfast in our culture is extremely important and should be recognized and stressed by the scientific community. Efforts should be done to promote this practice for the individual health and well-being.

p53 orchestrates the PGC-1α-mediated antioxidant response upon mild redox and metabolic imbalance.

AIMS: The transcriptional coactivator peroxisome proliferator-activated receptor-γ coactivator-1 α (PPARGC1A or PGC-1α) is a powerful controller of cell metabolism and assures the balance between the production and the scavenging of pro-oxidant molecules by coordinating mitochondrial biogenesis and the expression of antioxidants. However, even though a huge amount of data referring to the role of PGC-1α is available, the molecular mechanisms of its regulation at the transcriptional level are not completely understood. In the present report, we aim at characterizing whether the decrease of antioxidant glutathione (GSH) modulates PGC-1α expression and its downstream metabolic pathways. RESULTS: We found that upon GSH shortage, induced either by its chemical depletion or by metabolic stress (i.e., fasting), p53 binds to the PPARGC1A promoter of both human and mouse genes, and this event is positively related to increased PGC-1α expression. This effect was abrogated by inhibiting nitric oxide (NO) synthase or guanylate cyclase, implicating NO/cGMP signaling in such a process. We show that p53-mediated PGC-1α upregulation is directed to potentiate the antioxidant defense through nuclear factor (erythroid-derived 2)-like2 (NFE2L2)-mediated expression of manganese superoxide dismutase (SOD2) and γ-glutamylcysteine ligase without modulating mitochondrial biogenesis. INNOVATION AND CONCLUSIONS: We outlined a new NO-dependent signaling axis responsible for survival antioxidant response upon mild metabolic stress (fasting) and/or oxidative imbalance (GSH depletion). Such signaling axis could become the cornerstone for new pharmacological or dietary approaches for improving antioxidant response during ageing and human pathologies associated with oxidative stress.

Low-Shear Modeled Microgravity Enhances Salmonella Enterica Resistance to Hydrogen Peroxide Through a Mechanism Involving KatG and KatN.

Studies carried out in recent years have established that growth under conditions of reduced gravity enhances Salmonella enterica serovar Typhimurium virulence. To analyze the possibility that this microgravity-induced increase in pathogenicity could involve alterations in the ability of Salmonella to withstand oxidative stress, we have compared the resistance to hydrogen peroxide of various Salmonella enterica strains grown under conditions of low shear modeled microgravity (LSMMG) or normal gravity (NG). We have found that growth in LSMMG significantly enhances hydrogen peroxide resistance of all the strains analyzed. This effect is abolished by deletion of the genes encoding for the catalases KatG and KatN, whose activity is markedly modulated by growth in LSMMG. In addition, we have observed that Salmonella enterica serovar Typhimurium strains lacking Hfq, RpoE, RpoS or OxyR are still more resistant to oxidative stress when grown in LSMMG than in NG conditions, indicating that these global gene regulators are not responsible for the microgravity-induced changes in KatG and KatN activity. As Salmonella likely encounters low shear conditions in the intestinal tract, our observations suggest that alterations in the relative activity of KatG and KatN could enhance Salmonella resistance to the reactive oxygen species produced also during natural infections.

Glutamine deprivation enhances antitumor activity of 3-bromopyruvate through the stabilization of monocarboxylate transporter-1.

Anticancer drug efficacy might be leveraged by strategies to target certain biochemical adaptations of tumors. Here we show how depriving cancer cells of glutamine can enhance the anticancer properties of 3-bromopyruvate, a halogenated analog of pyruvic acid. Glutamine deprival potentiated 3-bromopyruvate chemotherapy by increasing the stability of the monocarboxylate transporter-1, an effect that sensitized cells to metabolic oxidative stress and autophagic cell death. We further elucidated mechanisms through which resistance to chemopotentiation by glutamine deprival could be circumvented. Overall, our findings offer a preclinical proof-of-concept for how to employ 3-bromopyruvate or other monocarboxylic-based drugs to sensitize tumors to chemotherapy.

p38(MAPK) and ERK1/2 dictate cell death/survival response to different pro-oxidant stimuli via p53 and Nrf2 in neuroblastoma cells SH-SY5Y.

Redox changes are often reported as causative of neoplastic transformation and chemoresistance, but are also exploited as clinical tools to selectively kill tumor cells. We previously demonstrated that gastrointestinal-derived tumor histotypes are resistant to ROS-based treatments by means of the redox activation of Nrf2, but highly sensitive to disulfide stressors triggering apoptosis via the redox induction of Trx1/p38(MAPK)/p53 signaling pathway. Here, we provide evidence that neuroblastoma SH-SY5Y has a complete opposite behavior, being sensitive to H2O2, but resistant to the glutathione (GSH)-oxidizing molecule diamide. Consistent with these observations, the apoptotic pathway activated upon H2O2 treatment relies upon Trx1 oxidation, and is mediated by the p38(MAPK)/p53 signaling axis. Pre-treatment with different antioxidants, pharmacological inhibitor of p38(MAPK), or small interfering RNA against p53 rescue cell viability. On the contrary, cell survival to diamide relies upon redox activation of Nrf2, in a way independent on Keap1 oxidation, but responsive to ERK1/2 activation. Chemical inhibition of GSH neo-synthesis or ERK1/2 phosphorylation, as well as overexpression of the dominant-negative form of Nrf2 sensitizes cells to diamide toxicity. In the searching for the molecular determinant(s) unifying these phenomena, we found that SH-SY5Y cells show high GSH levels, but exhibit very low GPx activity. This feature allows to efficiently buffer disulfide stress, but leaves them being vulnerable to H2O2-mediated insult. The increase of GPx activity by means of selenium supplementation or GPx1 ectopic expression completely reverses death phenotype, indicating that the response of tumor cells to diverse oxidative stimuli deeply involves the entire GSH redox system.

Neuroprotection of kaempferol by autophagy in models of rotenone-mediated acute toxicity: possible implications for Parkinson's disease.

This study aims to elucidate the processes underlying neuroprotection of kaempferol in models of rotenone-induced acute toxicity. We demonstrate that kaempferol, but not quercetin, myricetin or resveratrol, protects SH-SY5Y cells and primary neurons from rotenone toxicity, as a reduction of caspases cleavage and apoptotic nuclei are observed. Reactive oxygen species (ROS) levels and mitochondrial carbonyls decrease significantly. Mitochondrial network, transmembrane potential and oxygen consumption are also deeply preserved. We demonstrate that the main event responsible for the kaempferol-mediated antiapoptotic and antioxidant effects is the enhancement of mitochondrial turnover by autophagy. Indeed, fluorescence and electron microscopy analyses show an increase of the mitochondrial fission rate and mitochondria-containing autophagosomes. Moreover, the autophagosome-bound microtubule-associated protein light chain-3 (LC3-II) increases during kaempferol treatment and chemical/genetic inhibitors of autophagy abolish kaempferol protective effects. Autophagy affords protection also toward other mitochondrial toxins (1-methyl-4-phenyilpiridinium, paraquat) used to reproduce the typical features of Parkinson's disease (PD), but is inefficient against apoptotic stimuli not directly affecting mitochondria (H(2)O(2), 6-hydroxydopamine, staurosporine). Striatal glutamatergic response of rat brain slices is also preserved by kaempferol, suggesting a more general protection of kaempferol in Parkinson's disease. Overall, the data provide further evidence for kaempferol to be identified as an autophagic enhancer with potential therapeutic capacity.

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.

Modulation of intracellular glutathione affects adipogenesis in 3T3-L1 cells.

Impairment of redox homeostasis has been extensively associated with obesity, as a consequence of the chronic inflammatory state present in overweight subjects. Deregulation of glutathione (GSH), the most important non-enzymatic intracellular anti-oxidant, induces insulin resistance in mature adipocytes, but data are lacking about its effects on adipogenesis. In this report we demonstrate that during adipogenesis of 3T3-L1 cells the GSH/GSSG ratio decreases, shifting redox status towards oxidizing conditions. Moreover, we demonstrate that inhibition of GSH synthesis, obtained by treatment with L-buthionine-sulfoximine (BSO), enhances C/EBPß LAP/LIP ratio and PPARγ expression during mitotic clonal expansion (MCE) stimulating adipogenesis. On the contrary, GSH ethyl ester (GSHest) supplementation completely abrogates this process also in the presence of BSO. GSH decrement during the first 24 h of adipogenesis is sufficient to induce higher triglyceride accumulation in differentiated adipocytes with respect to control, whereas GSHest treatment inhibits lipid droplets formation. We further demonstrate that Resveratrol (RV) could exert anti-adipogenic properties also by increasing GSH content through γ-glutamyl-cysteine ligase (GCL) induction. Overall data indicate that in pre-adipocytes the decrease of GSH accelerates adipogenesis, suggesting that the use of agents able to maintain GSH redox status in adipose tissue, such as RV, could be promising in stopping the lipogenic loop of obesity.

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.

Neuronal nitric oxide synthase interacts with Sp1 through the PDZ domain inhibiting Sp1-mediated copper-zinc superoxide dismutase expression.

In this report we demonstrate that neuronal nitric oxide synthase (nNOS) is able to interact with Sp1 both in vivo and in vitro. In particular, we show that such interaction is mediated by the N-terminal PDZ domain of full length nNOS (fl-nNOS). In fact nNOS mutant lacking the PDZ domain (ΔnNOS) displays an impaired ability to bind to Sp1, as demonstrated by co-immunoprecipitation experiments. The overexpression of fl-nNOS in SH-SY5Y cells leads to the formation of nNOS/Sp1 heterocomplex and inhibits the binding of Sp1 to DNA. Among the Sp1 target genes we looked at the possible alteration of binding to copper-zinc superoxide dismutase gene (sod1) promoter. We find that the interaction of nNOS with Sp1 leads to a significant decrease of SOD1 mRNA, protein level and activity. The overexpression of ΔnNOS results in an inability to sequester Sp1 and unaffected Sp1 DNA binding capacity, allowing sod1 to be expressed. The data reported give effort to the possible involvement of nNOS in regulating gene transcription in NO-independent manner giving an additional significance to the expression of specific nNOS splicing variants.

Under the ROS thiol network is the principal suspect for autophagy commitment.

Low molecular weight and protein sulphydryls undergo reactive oxygen species (ROS)-mediated oxidation. However, in contrast to the irreversible damages that oxidative conditions yield on biomolecules, the oxidation of reactive cysteines frequently results in reversible modifications, which represent the prototype of the molecular mechanisms underlying redox signaling. Many proteins involved in a wide range of cellular processes have been classified as "redoxsensitive," thereby modulating their function/activity dependent on the redox state of their critical thiols. Growing evidence from the past few years supports the idea that ROS production also correlates with the occurrence of autophagy. Nonetheless, the cysteine protease Atg4 remains the sole example of a protein whose redox regulation has been completely characterized and demonstrated to be necessary for the progression of autophagy. The principal aim of this commentary is to draw attention to the remarkable number of proteins that can fit the double role of: (i) being involved in autophagy, especially in autophagosome formation and (ii) sensing alterations of the cellular redox state by means of reactive cysteine residues. We will also attempt to provide a hypothetical model to explain the possible functional role of thiols in the occurrence of autophagy and outline a network of redox reactions likely concurring to allow the correct initiation and completion of autophagosomes.

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.

Peroxisome proliferator-activated receptor gamma co-activator 1alpha (PGC-1alpha) and sirtuin 1 (SIRT1) reside in mitochondria: possible direct function in mitochondrial biogenesis.

The transcriptional co-activator PGC-1alpha and the NAD(+)-dependent deacetylase SIRT1 are considered important inducers of mitochondrial biogenesis because in the nucleus they regulate transcription of nucleus-encoded mitochondrial genes. We demonstrate that PGC-1alpha and SIRT1 are also present inside mitochondria and are in close proximity to mtDNA. They interact with mitochondrial transcription factor A (TFAM) as assessed by confocal microscopy analysis and by blue native-PAGE. Nucleoid purification allowed us to identify SIRT1 and PGC-1alpha as proteins associated with native and cross-linked nucleoids, respectively. After mtDNA immunoprecipitation analysis, carried out on mitochondrial extracts, we found that PGC-1alpha is present on the same D-loop region recognized by TFAM. Finally, by oligonucleotide pulldown assay, we found PGC-1alpha and SIRT1 associated with the TFAM consensus sequence (human mitochondrial transcription factor-binding site H). The results obtained suggest that in mitochondria PGC-1alpha and SIRT1 may function as their nuclear counterparts and represent the genuine factors mediating the cross-talk between nuclear and mitochondrial genome. Finally, this work adds new knowledge on the function of SIRT1 and PGC-1alpha and highlights the direct involvement of such proteins in regulation of mitochondrial biogenesis.

Nutritional factors in human dispersals.

Nutrition can be defined as the biochemical network by which diet affects expression of genes giving rise to phenotypes that are able to successfully respond to environmental challenges, such as those resulting from dispersal to new habitats. A virtuous circle is generated between genes and diet via optimal nutrition, which provides metabolic support for the development of functions that in turn allow better exploitation of food resources in the new habitat. The present contribution will test this hypothesis by nutritional analysis of three sequential dispersals of Pleistocene hominins, which were accompanied and made successful by a dramatic expansion in brain size and function. Such anatomical/functional changes are likely to be related to specific mutations, but can be maintained across generations by the essential contribution of dietary factors since they are very expensive of both the energy and quality content of the diet. The importance of access to 'nutritionally dense' food, essentially meat, marks the forest to savannah transition, while that to a 'brain-specific diet', essentially a maritime pattern of food chain, seems distinctive of the inland to coast dispersal and of more recent out of Africa long-distance dispersals.

Carcinoma cells activate AMP-activated protein kinase-dependent autophagy as survival response to kaempferol-mediated energetic impairment.

Kaempferol, a dietary cancer chemopreventive polyphenol, has been reported to trigger apoptosis in several tumor histotypes, but the mechanism underlying this phenomenon is not fully understood. Here, we demonstrate that in HeLa cells, kaempferol induces energetic failure due to inhibition of both glucose uptake and Complex I of the mitochondrial respiratory chain. As adaptive response, cells activate autophagy, the occurrence of which was established cytofluorometrically, upon acridine orange staining, and immunochemically, by following the increase of the autolysosome-associated form of the microtubule-associated protein light chain 3 (LC3-II). Autophagy is an early and reversible process occurring as survival mechanisms against apoptosis. Indeed, chemical inhibition of autophagy, by incubations with monensin, wortmannin, 3-methyladenine, or by silencing Atg5, significantly increases the extent of apoptosis, which takes place via the mitochondrial pathway, and shortens the time in which the apoptotic markers are detectable. We also demonstrate that autophagy depends on the early activation of the AMP-activated protein kinase (AMPK)/mTOR-mediated pathway. The overexpression of dominant negative AMPK results in a decrease of autophagic cells, a decrement of LC3-II levels, and a significant increase of apoptosis. Experiments performed with another carcinoma cell line yielded the same results, suggesting for kaempferol a unique mechanism of action.

Tau dephosphorylation and microfilaments disruption are upstream events of the anti-proliferative effects of DADS in SH-SY5Y cells.

Garlic organosulphur compounds have been successfully used as redox anti-proliferative agents. In this work, we dissect the effects of diallyl disulphide (DADS) focusing on the events upstream of cell cycle arrest and apoptosis induced in neuroblastoma SH-SY5Y cells. We demonstrate that DADS is able to cause early morphological changes, cytoskeleton oxidation, microfilaments reduction and depolymerization of microtubules. These events are attenuated in cells stably overexpressing the antioxidant enzyme SOD1, suggesting that superoxide plays a crucial role in destabilizing cytoskeleton. Moreover, we evidence that the main microtubules-associated protein Tau undergoes PP1-mediated dephosphorylation as demonstrated by treatment with okadaic acid as well as by immunoreaction with anti-Tau-1 antibody, which specifically recognizes its dephosphorylated forms. Tau dephosphorylation is inhibited by the two-electron reductants NAC and GSH ester but not by SOD1. The inability of DADS to induce apoptosis in neuroblastoma-differentiated cells gives emphasis to the anti-proliferative activity of DADS, which can be regarded as a promising potent anti-neuroblastoma drug by virtue of its widespread cytoskeleton disrupting action on proliferating cells.

Redox mechanisms involved in the selective activation of Nrf2-mediated resistance versus p53-dependent apoptosis in adenocarcinoma cells.

We have investigated the role of reactive oxygen species and thiol-oxidizing agents in the induction of cell death and have shown that adenocarcinoma gastric (AGS) cells respond differently to the oxidative challenge according to the signaling pathways activated. In particular, apoptosis in AGS cells is induced via the mitochondrial pathway upon treatment with thiol-oxidizing agents, such as diamide. Apoptosis is associated with persistent oxidative damage, as evidenced by the increase in carbonylated proteins and the expression/activation of DNA damage-sensitive proteins histone H2A.X and DNA-dependent protein kinase. Resistance to hydrogen peroxide is instead associated with Keap1 oxidation and rapid translocation of Nrf2 into the nucleus. Sensitivity to diamide and resistance to hydrogen peroxide are correlated with GSH redox changes, with diamide severely increasing GSSG, and hydrogen peroxide transiently inducing protein-GSH mixed disulfides. We show that p53 is activated in response to diamide treatment by the oxidative induction of the Trx1/p38(MAPK) signaling pathway. Similar results were obtained with another carcinoma cell line, CaCo2, indicating that these findings are not limited to AGS cells. Our data suggest that thiol-oxidizing agents could be exploited as inducers of apoptosis in tumor histotypes resistant to ROS-producing chemotherapeutics.

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.