Autophagy and Autophagic Cell Death: Uncovering New Mechanisms Whereby Dehydroepiandrosterone Promotes Beneficial Effects on Human Health.

Dehydroepiandrosterone (DHEA) is the most abundant steroid hormone in human serum and a precursor of sexual hormones. Its levels, which are maximum between the age of 20 and 30, dramatically decline with aging thus raising the question that many pathological conditions typical of the elderly might be associated with the decrement of circulating DHEA. Moreover, since its very early discovery, DHEA and its metabolites have been shown to be active in many pathophysiological contexts, including cardiovascular disease, brain disorders, and cancer. Indeed, treatment with DHEA has beneficial effects for the cure of these and many other pathologies in vitro, in vivo, and in patient studies. However, the molecular mechanisms underlying DHEA effects have been only partially elucidated. Autophagy is a self-digestive process, by which cell homeostasis is maintained, damaged organelles removed, and cell survival assured upon stress stimuli. However, high rate of autophagy is detrimental and leads to a form of programmed cell death known as autophagic cell death (ACD). In this chapter, we describe the process of autophagy and the morphological and biochemical features of ACD. Moreover, we analyze the beneficial effects of DHEA in several pathologies and the molecular mechanisms with particular emphasis on its regulation of cell death processes. Finally, we review data indicating DHEA and structurally related steroid hormones as modulators of both autophagy and ACD, a research field that opens new avenues in the therapeutic use of these compounds.

Mitochondrial Hormesis links nutrient restriction to improved metabolism in fat cell.

Fasting promotes longevity by reprogramming metabolic and stress resistance pathways. However, although the impact on adipose tissue physiology through hormonal inputs is well established, the direct role of fasting on adipose cells is poorly understood. Herein we show that white and beige adipocytes, as well as mouse epididymal and subcutaneous adipose depots, respond to nutrient scarcity by acquiring a brown-like phenotype. Indeed, they improve oxidative metabolism through modulating the expression of mitochondrial- and nuclear-encoded oxidative phosphorylation genes as well as mitochondrial stress defensive proteins (UCP1, SOD2). Such adaptation is placed in a canonical mitohormetic response that proceeds via mitochondrial reactive oxygen species ((mt)ROS) production and redistribution of FoxO1 transcription factor into nucleus. Nuclear FoxO1 ((n)FoxO1) mediates retrograde communication by inducing the expression of mitochondrial oxidative and stress defensive genes. Collectively, our findings describe an unusual white/beige fat cell response to nutrient availability highlighting another health-promoting mechanism of fasting.

Dietary fat overload reprograms brown fat mitochondria.

Chronic nutrient overload accelerates the onset of several aging-related diseases reducing life expectancy. Although the mechanisms by which overnutrition affects metabolic processes in many tissues are known, its role on BAT physiology is still unclear. Herein, we investigated the mitochondrial responses in BAT of female mice exposed to high fat diet (HFD) at different steps of life. Although adult mice showed an unchanged mitochondrial amount, both respiration and OxPHOS subunits were strongly affected. Differently, offspring pups exposed to HFD during pregnancy and lactation displayed reduced mitochondrial mass but high oxidative efficiency that, however, resulted in increased bioenergetics state of BAT rather than augmented uncoupling respiration. Interestingly, the metabolic responses triggered by HFD were accompanied by changes in mitochondrial dynamics characterized by decreased content of the fragmentation marker Drp1 both in mothers and offspring pups. HFD-induced inactivation of the FoxO1 transcription factor seemed to be the up-stream modulator of Drp1 levels in brown fat cells. Furthermore, HFD offspring pups weaned with normal diet only partially reverted the mitochondrial dysfunctions caused by HFD. Finally these mice failed in activating the thermogenic program upon cold exposure. Collectively our findings suggest that maternal dietary fat overload irreversibly commits BAT unresponsiveness to physiological stimuli such as cool temperature and this dysfunction in the early stage of life might negatively modulate health and lifespan.

Glutathione Decrement Drives Thermogenic Program In Adipose Cells.

Adipose tissue metabolically adapts to external stimuli. We demonstrate that the induction of the thermogenic program in white adipocytes, through cold exposure in mice or in vitro adrenergic stimulation, is accompanied by a decrease in the intracellular content of glutathione (GSH). Moreover, the treatment with a GSH depleting agent, buthionine sulfoximine (BSO), recapitulates the effect of cold exposure resulting in the induction of thermogenic program. In particular, BSO treatment leads to enhanced uncoupling respiration as demonstrated by increased expression of thermogenic genes (e.g. Ucp1, Ppargc1a), augmented oxygen consumption and decreased mitochondrial transmembrane potential. Buffering GSH decrement by pre-treatment with GSH ester prevents the up-regulation of typical markers of uncoupling respiration. We demonstrate that FoxO1 activation is responsible for the conversion of white adipocytes into a brown phenotype as the "browning" effects of BSO are completely abrogated in cells down-regulating FoxO1. In mice, the BSO-mediated up-regulation of uncoupling genes results in weight loss that is at least in part ascribed to adipose tissue mass reduction. The induction of thermogenic program has been largely proposed to counteract obesity-related diseases. Based on these findings, we propose GSH as a novel therapeutic target to increase energy expenditure in adipocytes.

Oxidative myocardial damage in human cocaine-related cardiomyopathy.

AIMS: The pathogenesis of cocaine-related cardiomyopathy (CCM) is still unclear. Oxidative damage from cocaine-generated reactive oxygen species (ROS) overcoming myocardial antioxidant reserve has been hypothesized by experimental studies. METHODS AND RESULTS: Ten (2.3%) of 430 consecutive cases with dilated cardiomyopathy (DCM) were attributed to CCM. Endomyocardial biopsies from CCM were retrospectively investigated with histology, electron microscopy, immunohistochemistry (graded 0-3), and Western blot analysis for inducible nitric oxide synthase (iNOS) and nitrotyrosine. Oxidative damage to DNA was investigated by immunostaining for 8-hydroxydeoxyguanosine (8-OHdG), while apoptosis and necrosis were evaluated by in situ ligation with hairpin probes. Myocardial anti-oxidant reserve was evaluated through assessment of superoxide dismutase (SOD1-2) and catalase (CT) activity in two frozen samples from each patient. Results were compared with idiopathic DCM and normal controls. Cardiomyocytes were bigger and myocardial fibrosis was more pronounced in CCM than in the DCM cohort. Contraction band necrosis was always detectable only in CCM with sparse lymphocytic infiltrates in three cases. Both iNOS and nitrotyrosine were significantly more expressed in CCM than in DCM. Immunostaining for 8-OHdG, cardiomyocyte apoptosis, and necrosis were significantly increased in CCM compared with controls and DCM. Myocardial SOD1 and CT activity was significantly decreased compared with DCM and controls, and correlated with cell death and severity of left ventricular dysfunction. CONCLUSION: Oxidative stress is a major mechanism of myocardial damage in human CCM. It concurs with calcium overload to myocyte dysfunction and death.

Glutathione: new roles in redox signaling for an old antioxidant.

The physiological roles played by the tripeptide glutathione have greatly advanced over the past decades superimposing the research on free radicals, oxidative stress and, more recently, redox signaling. In particular, GSH is involved in nutrient metabolism, antioxidant defense, and regulation of cellular metabolic functions ranging from gene expression, DNA and protein synthesis to signal transduction, cell proliferation and apoptosis. This review will be focused on the role of GSH in cell signaling by analysing the more recent advancements about its capability to modulate nitroxidative stress, autophagy, and viral infection.

FoxO1 at the nexus between fat catabolism and longevity pathways.

Adipose tissue should not be considered a simple fat sink but a specialized system that promptly and dynamically responds to variations of nutrients, to fulfil its major role in whole-body energy homeostasis. Perturbation of energy storage and utilization, as well as the expansion of adipose tissue during ageing, are hallmarks of several inflammation-related metabolic disorders. Studies using model organisms have provided significant insight into the genetic factors and environmental conditions that influence adipose tissue function and cause the failure of its homeostasis. It is now clear that reduced caloric intake has a major impact on adipose tissue function and can provide a path towards better health and the avoidance of age-related chronic diseases. An intricate and evolutionary conserved signalling network is necessary to manage adipocyte response to nutrients. The transcription factor FoxO1 plays a leading role in integrating dietary conditions, insulin signalling and the down-stream response of adipocytes to maintain metabolic balance. Here we review recent insights on the novel role of FoxO1 in regulating lipid catabolism through the induction of adipose triglyceride lipase (ATGL) and lysosomal lipase (Lipa) in adipocytes during nutrient restriction. In particular, we highlight the nutrient-sensing and hormone-independent feature of FoxO1 activity and illustrate how, by potentiating lipid breakdown, the FoxO1 signalling cascade could induce pro-longevity adaptive responses in adipose tissue.

Inhibition of age-related cytokines production by ATGL: a mechanism linked to the anti-inflammatory effect of resveratrol.

Ageing is characterized by the expansion and the decreased vascularization of visceral adipose tissue (vAT), disruption of metabolic activities, and decline of the function of the immune system, leading to chronic inflammatory states. We previously demonstrated that, in vAT of mice at early state of ageing, adipocytes mount a stress resistance response consisting in the upregulation of ATGL, which is functional in restraining the production of inflammatory cytokines. Here, we found that, in the late phase of ageing, such an adaptive response is impaired. In particular, 24-months-old mice and aged 3T3-L1 adipocytes display affected expression of ATGL and its downstream PPARα-mediated lipid signalling pathway, leading to upregulation of TNFα and IL-6 production. We show that the natural polyphenol compound resveratrol (RSV) efficiently suppresses the expression of TNFα and IL-6 in an ATGL/PPARα dependent manner. Actually, adipocytes downregulating ATGL do not show a restored PPARα expression and display elevated cytokines production. Overall the results obtained highlight a crucial function of ATGL in inhibiting age-related inflammation and reinforce the idea that RSV could represent a valid natural compound to limit the onset and/or the exacerbation of the age-related inflammatory states.

S-nitrosoglutathione reductase deficiency-induced S-nitrosylation results in neuromuscular dysfunction.

AIMS: Nitric oxide (NO) production is implicated in muscle contraction, growth and atrophy, and in the onset of neuropathy. However, many aspects of the mechanism of action of NO are not yet clarified, mainly regarding its role in muscle wasting. Notably, whether NO production-associated neuromuscular atrophy depends on tyrosine nitration or S-nitrosothiols (SNOs) formation is still a matter of debate. Here, we aim at assessing this issue by characterizing the neuromuscular phenotype of S-nitrosoglutathione reductase-null (GSNOR-KO) mice that maintain the capability to produce NO, but are unable to reduce SNOs. RESULTS: We demonstrate that, without any sign of protein nitration, young GSNOR-KO mice show neuromuscular atrophy due to loss of muscle mass, reduced fiber size, and neuropathic behavior. In particular, GSNOR-KO mice show a significant decrease in nerve axon number, with the myelin sheath appearing disorganized and reduced, leading to a dramatic development of a neuropathic phenotype. Mitochondria appear fragmented and depolarized in GSNOR-KO myofibers and myotubes, conditions that are reverted by N-acetylcysteine treatment. Nevertheless, although atrogene transcription is induced, and bulk autophagy activated, no removal of damaged mitochondria is observed. These events, alongside basal increase of apoptotic markers, contribute to persistence of a neuropathic and myopathic state. INNOVATION: Our study provides the first evidence that GSNOR deficiency, which affects exclusively SNOs reduction without altering nitrotyrosine levels, results in a clinically relevant neuromuscular phenotype. CONCLUSION: These findings provide novel insights into the involvement of GSNOR and S-nitrosylation in neuromuscular atrophy and neuropathic pain that are associated with pathological states; for example, diabetes and cancer.

Nuclear recruitment of neuronal nitric-oxide synthase by α-syntrophin is crucial for the induction of mitochondrial biogenesis.

Neuronal nitric-oxide synthase (nNOS) has various splicing variants and different subcellular localizations. nNOS can be found also in the nucleus; however, its exact role in this compartment is still not completely defined. In this report, we demonstrate that the PDZ domain allows the recruitment of nNOS to nuclei, thus favoring local NO production, nuclear protein S-nitrosylation, and induction of mitochondrial biogenesis. In particular, overexpression of PDZ-containing nNOS (nNOSα) increases S-nitrosylated CREB with consequent augmented binding on cAMP response element consensus sequence on peroxisome proliferator-activated receptor γ co-activator (PGC)-1α promoter. The resulting PGC-1α induction is accompanied by the expression of mitochondrial genes (e.g., TFAM, MtCO1) and increased mitochondrial mass. Importantly, full active nNOS lacking PDZ domain (nNOSß) does not localize in nuclei and fails in inducing the expression of PGC-1α. Moreover, we substantiate that the mitochondrial biogenesis normally accompanying myogenesis is associated with nuclear translocation of nNOS. We demonstrate that α-Syntrophin, which resides in nuclei of myocytes, functions as the upstream mediator of nuclear nNOS translocation and nNOS-dependent mitochondrial biogenesis. Overall, our results indicate that altered nNOS splicing and nuclear localization could be contributing factors in human muscular diseases associated with mitochondrial impairment.

Punctum on two different transcription factors regulated by PGC-1α: nuclear factor erythroid-derived 2-like 2 and nuclear respiratory factor 2.

BACKGROUND: The transcription factor nuclear factor-erythroid-derived 2-like 2 (official symbol: NFE2L2, alias: Nrf2) is a master regulator of antioxidant defense system, which makes it an attractive target for manipulations that aim to increase cellular resistance to oxidative stress. Nuclear respiratory factor 2 or GA binding protein transcription factor alpha (official symbol: GABPA, alias: NRF2) functions as a transcription factor that activates the expression of some key metabolic genes regulating cellular growth and nuclear genes required for mitochondrial respiration as well as mitochondrial DNA transcription and replication. SCOPE OF REVIEW: Despite the evident structural and functional differences, confusion has occurred in bibliographic databases due to the shared symbol NRF2 for these transcription factors. Such confusion has worsened after the discovery that the transcriptional co-activator peroxisome proliferator activated receptor gamma co-activator 1 alpha (PGC-1α) could control the signaling pathway of both NFE2L2 and GABPA through distinct molecular mechanisms. This review will summarize the implications of NFE2L2 and GABPA in various human patho-physiological conditions and how PGC-1α can regulate their different signaling axis. MAJOR CONCLUSIONS: This review underlines the overlapping functions between PGC-1α, NFE2L2 and GABPA, which alteration could induce the development of human pathological states. GENERAL SIGNIFICANCE: The comprehension of molecular mechanisms that modulate the intersection between these proteins will be important to identify new signaling axis involved in lifespan extension as well as novel targets for therapeutic interventions.

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.

Garlic-derived diallyl disulfide modulates peroxisome proliferator activated receptor gamma co-activator 1 alpha in neuroblastoma cells.

The peroxisome proliferator activated receptor gamma co-activator 1 alpha (PGC1α) is an inducible transcriptional co-activator with direct function in the induction of mitochondrial biogenesis. In the present report we show that, in SH-SY5Y neuroblastoma cells, garlic-derived diallyl disulfide (DADS) is able to increase PGC1α expression in a ROS-dependent manner and to induce mitochondrial biogenesis at early stage of treatment that precede cell cycle arrest and apoptosis outcome. In particular, we demonstrate that DADS elicits: i) the increase of PGC1α within nuclear compartment; ii) the decrease of PGC1α non-active acetylated form; iii) the induction of nuclear-encoded mitochondrial genes such as TFAM and TFBM1. We also show an accumulation of PGC1α within mitochondria along with an increased association with the regulatory D-Loop region of mtDNA and a concomitant augmented expression of mitochondrial RNA. Such events are related to a prompt elevation of mitochondrial mass, as assessed by evaluating the content of mtDNA. We show that the induction of mitochondrial biogenesis is directed to dampen the cytotoxic effect of DADS. Indeed, PGC1α overexpression or down-regulation prevents or exacerbates mtDNA loss and apoptosis. Overall the data highlight an anti-apoptotic role of PGC1α-mediated mitochondrial biogenesis in neuroblatoma cells and suggest PGC1α as a potential target for enhancing the effectiveness of therapy in aggressive neuroblastoma with high drug-resistance.

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.

Redox regulation of the influenza hemagglutinin maturation process: a new cell-mediated strategy for anti-influenza therapy.

AIM: The aim of this study was to determine whether GSH-C4, a hydrophobic glutathione derivative, affects in vitro and in vivo influenza virus infection by interfering with redox-sensitive intracellular pathways involved in the maturation of viral hemagglutinin (HA). RESULTS: GSH-C4 strongly inhibited influenza A virus replication in cultured cells and in lethally infected mice, where it also reduced lung damage and mortality. In cell-culture studies, GSH-C4 arrested viral HA folding; the disulfide-rich glycoprotein remained in the endoplasmic reticulum as a reduced monomer instead of undergoing oligomerization and cell plasma-membrane insertion. HA maturation depends on the host-cell oxidoreductase, protein disulfide isomerase (PDI), whose activity in infected cells is probably facilitated by virus-induced glutathione depletion. By correcting this deficit, GSH-C4 increased levels of reduced PDI and inhibited essential disulfide bond formation in HA. Host-cell glycoprotein expression in uninfected cells was unaffected by glutathione, which thus appears to act exclusively on glutathione-depleted cells. INNOVATION: All currently approved anti-influenza drugs target essential viral structures, and their efficacy is limited by toxicity and by the almost inevitable selection of drug-resistant viral mutants. GSH-C4 inhibits influenza virus replication by modulating redox-sensitive pathways in infected cells, without producing toxicity in uninfected cells or animals. Novel anti-influenza drugs that target intracellular pathways essential for viral replication ("cell-based approach") offer two important potential advantages: they are more difficult for the virus to adapt to and their efficacy should not be dependent on virus type, strain, or antigenic properties. CONCLUSION: Redox-sensitive host-cell pathways exploited for viral replication are promising targets for effective anti-influenza strategies.

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.

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.

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.