Rational Redesign of Monoamine Oxidase A into a Dehydrogenase to Probe ROS in Cardiac Aging.

Cardiac senescence is a typical chronic frailty condition in the elderly population, and cellular aging is often associated with oxidative stress. The mitochondrial-membrane flavoenzyme monoamine oxidase A (MAO A) catalyzes the oxidative deamination of neurotransmitters, and its expression increases in aged hearts. We produced recombinant human MAO A variants at Lys305 that play a key role in O2 reactivity leading to H2O2 production. The K305Q variant is as active as the wild-type enzyme, whereas K305M and K305S have 200-fold and 100-fold lower kcat values and similar Km. Under anaerobic conditions, K305M MAO A was normally reduced by substrate, whereas reoxidation by O2 was much slower but could be accomplished by quinone electron acceptors. When overexpressed in cardiomyoblasts by adenoviral vectors, the K305M variant showed enzymatic turnover similar to that of the wild-type but displayed decreased ROS levels and senescence markers. These results might translate into pharmacological treatments as MAO inhibitors may attenuate cardiomyocytes aging.

Monoamine oxidase-A is a novel driver of stress-induced premature senescence through inhibition of parkin-mediated mitophagy.

Cellular senescence, the irreversible cell cycle arrest observed in somatic cells, is an important driver of age-associated diseases. Mitochondria have been implicated in the process of senescence, primarily because they are both sources and targets of reactive oxygen species (ROS). In the heart, oxidative stress contributes to pathological cardiac ageing, but the mechanisms underlying ROS production are still not completely understood. The mitochondrial enzyme monoamine oxidase-A (MAO-A) is a relevant source of ROS in the heart through the formation of H2 O2 derived from the degradation of its main substrates, norepinephrine (NE) and serotonin. However, the potential link between MAO-A and senescence has not been previously investigated. Using cardiomyoblasts and primary cardiomyocytes, we demonstrate that chronic MAO-A activation mediated by synthetic (tyramine) and physiological (NE) substrates induces ROS-dependent DNA damage response, activation of cyclin-dependent kinase inhibitors p21cip , p16ink4a , and p15ink4b and typical features of senescence such as cell flattening and SA-ß-gal activity. Moreover, we observe that ROS produced by MAO-A lead to the accumulation of p53 in the cytosol where it inhibits parkin, an important regulator of mitophagy, resulting in mitochondrial dysfunction. Additionally, we show that the mTOR kinase contributes to mitophagy dysfunction by enhancing p53 cytoplasmic accumulation. Importantly, restoration of mitophagy, either by overexpression of parkin or inhibition of mTOR, prevents mitochondrial dysfunction and induction of senescence. Altogether, our data demonstrate a novel link between MAO-A and senescence in cardiomyocytes and provides mechanistic insights into the potential role of MAO-dependent oxidative stress in age-related pathologies.

Oleuropein Aglycone Protects against MAO-A-Induced Autophagy Impairment and Cardiomyocyte Death through Activation of TFEB.

Age-associated diseases such as neurodegenerative and cardiovascular disorders are characterized by increased oxidative stress associated with autophagy dysfunction. Oleuropein aglycone (OA), the main polyphenol found in olive oil, was recently characterized as an autophagy inducer and a promising agent against neurodegeneration. It is presently unknown whether OA can have beneficial effects in a model of cardiac stress characterized by autophagy dysfunction. Here, we explored the effects of OA in cardiomyocytes with overexpression of monoamine oxidase-A (MAO-A). This enzyme, by degrading catecholamine and serotonin, produces hydrogen peroxide (H2O2), which causes oxidative stress, autophagic flux blockade, and cell necrosis. We observed that OA treatment counteracted the cytotoxic effects of MAO-A through autophagy activation, as displayed by the increase of autophagic vacuoles and autophagy-specific markers (Beclin1 and LC3-II). Moreover, the decrease in autophagosomes and the increase in autolysosomes, indicative of autophagosome-lysosome fusion, suggested a restoration of the defective autophagic flux. Most interestingly, we found that the ability of OA to confer cardioprotection through autophagy induction involved nuclear translocation and activation of the transcriptional factor EB (TFEB). Our data provide strong evidence of the beneficial effects of OA, suggesting its potential use as a nutraceutical agent against age-related pathologies involving autophagy dysfunction, including cardiovascular diseases.

Tight-Binding Inhibition of Human Monoamine Oxidase B by Chromone Analogs: A Kinetic, Crystallographic, and Biological Analysis.

Monoamine oxidase B (MAO-B) is a validated drug target for Parkinson's disease. Chromone derivatives were identified as novel potent and reversible MAO-B inhibitors, and herewith we report on a crystallographic and biochemical analysis to investigate their inhibition mechanism. The crystal structures of human MAO-B in complex with three chromone analogs bearing different substituents on the exocyclic aromatic ring (determined at 1.6-1.8 Å resolution) showed that they all bind in the active site cavity of the protein with the chromone moiety located in front of the FAD cofactor. These inhibitors form two hydrogen bonds with Tyr435 and Cys172 and perfectly fit the hydrophobic flat active site of human MAO-B. This is reflected in their tight-binding mechanism of inhibition with Ki values of 55, 17, and 31 nM for N-(3',4'-dimethylphenyl)-4-oxo-4 H-chromene-3-carboxamide (1), N-(3'-chlorophenyl)-4-oxo-4 H-chromene-3-carboxamide (2), and N-(3'-fluorophenyl)-4-oxo-4 H-chromene-3-carboxamide (3), respectively. These compounds were also 1000-fold more effective than l-deprenyl in reducing the cellular levels of reactive oxygen species (ROS).

Mechanism underlying the effect of long-term exposure to low dose of pesticides on DNA integrity.

Pesticides, including herbicides, insecticides and fungicides, are widely used in intensive agriculture. Recently, the long-term effects of pesticide exposure were found to be associated with many diseases. In this study, we evaluated the long-term effect of low-level exposure to a mixture of pesticides on DNA damage response (DDR) in relation to individual detoxifying variability. A residential population chronically exposed to pesticides was enrolled, biological/environmental pesticide levels; paroxonase 1 (PON-1) activity and 192 Q/R polymorphism and DDR were evaluated at three different periods of pesticide exposure. OGG1-dependent DNA repair activity was decreased in relation to pesticide exposure. The increase of DNA lesions and pesticide levels in the intensive pesticide-spraying period was independent on PON-1 activity. Next, human bronchial epithelial and neuronal cells were used as a model for in vitro evaluation of the mechanistic effect of pesticides. Pesticides induced mitochondrial dysfunction leading to ROS formation. ROS from mitochondria induced DNA damage, which in turn induced OGG1-dependent DNA repair activity through 8-oxoguanine DNA glycosylase 1 (OGG1) expression and activation. Even though OGG1 was overexpressed, an inhibition of its activity, associated with DNA lesion accumulation, was found at prolonged pesticide-exposure. A post-translational regulation of OGG1 by pesticide may be postulated. Taken together, long-term exposure to low-levels of pesticides affects DDR resulting in accumulation of DNA lesions that eventually may lead to cancer or neurological disorders.

Monoamine Oxidases, Oxidative Stress, and Altered Mitochondrial Dynamics in Cardiac Ageing.

The advances in healthcare over the past several decades have resulted in populations now living longer. With this increase in longevity, a wider prevalence of cardiovascular diseases is more common and known to be a major factor in rising healthcare costs. A wealth of scientific evidence has implicated cell senescence as an important component in the etiology of these age-dependent pathologies. A number of studies indicate that an excess of reactive oxygen species (ROS) contributes to trigger and accelerate the cardiac senescence processes, and a new role of monoamine oxidases, MAO-A and MAO-B, is emerging in this context. These mitochondrial enzymes regulate the level of catecholamines and serotonin by catalyzing their oxidative deamination in the heart. MAOs' expression substantially increases with ageing (6-fold MAO-A in the heart and 4-fold MAO-B in neuronal tissue), and their involvement in cardiac diseases is supposedly related to the formation of ROS, via the hydrogen peroxide produced during the substrate degradation. Here, we will review the most recent advances in this field and describe why MAOs could be effective targets in order to prevent age-associated cardiovascular disease.

Organic honey supplementation reverses pesticide-induced genotoxicity by modulating DNA damage response.

SCOPE: Glyphosate (GLY) and organophosphorus insecticides such as chlorpyrifos (CPF) may cause DNA damage and cancer in exposed individuals through mitochondrial dysfunction. Polyphenols ubiquitously present in fruits and vegetables, have been viewed as antioxidant molecules, but also influence mitochondrial homeostasis. Here, honey containing polyphenol compounds was evaluated for its potential protective effect on pesticide-induced genotoxicity. METHODS AND RESULTS: Honey extracts from four floral organic sources were evaluated for their polyphenol content, antioxidant activity, and potential protective effects on pesticide-related mitochondrial destabilization, reactive oxygen and nitrogen species formation, and DNA damage response in human bronchial epithelial and neuronal cells. The protective effect of honey was, then evaluated in a residential population chronically exposed to pesticides. The four honey types showed a different polyphenol profile associated with a different antioxidant power. The pesticide-induced mitochondrial dysfunction parallels ROS formation from mitochondria (mtROS) and consequent DNA damage. Honey extracts efficiently inhibited pesticide-induced mtROS formation, and reduced DNA damage by upregulation of DNA repair through NFR2. Honey supplementation enhanced DNA repair activity in a residential population chronically exposed to pesticides, which resulted in a marked reduction of pesticide-induced DNA lesions. CONCLUSION: These results provide new insight regarding the effect of honey containing polyphenols on pesticide-induced DNA damage response.

MicroRNA-126 induces autophagy by altering cell metabolism in malignant mesothelioma.

Autophagy favors both cell survival and cancer suppression, and increasing evidence reveals that microRNAs (MIRs) regulate autophagy. Previously we reported that MIR126 is downregulated in malignant mesothelioma (MM). Therefore, we investigated the role of MIR126 in the regulation of cell metabolism and autophagy in MM models. We report that MIR126 induces autophagic flux in MM cells by downregulating insulin receptor substrate-1 (IRS1) and disrupting the IRS1 signaling pathway. This was specific to MM cells, and was not observed in non-malignant cells of mesothelial origin or in MM cells expressing MIR126-insensitive IRS1 transcript. The MIR126 effect on autophagy in MM cells was recapitulated by IRS1 silencing, and antagonized by IRS1 overexpression or antisense MIR126 treatment. The MIR126-induced loss of IRS1 suppressed glucose uptake, leading to energy deprivation and AMPK-dependent phosphorylation of ULK1. In addition, MIR126 stimulated lipid droplet accumulation in a hypoxia-inducible factor-1α (HIF1α)-dependent manner. MIR126 also reduced pyruvate dehydrogenase kinase (PDK) and acetyl-CoA-citrate lyase (ACL) expression, leading to the accumulation of cytosolic citrate and paradoxical inhibition of pyruvate dehydrogenase (PDH) activity. Simultaneous pharmacological and genetic intervention with PDK and ACL activity phenocopied the effects of MIR126. This suggests that in MM MIR126 initiates a metabolic program leading to high autophagic flux and HIF1α stabilization, incompatible with tumor progression of MM. Consistently, MIR126-expressing MM cells injected into immunocompromised mice failed to progress beyond the initial stage of tumor formation, showing that increased autophagy has a protective role in MM.

Peripheral Skin Temperature and Circadian Biological Clock in Shift Nurses after a Day off.

The circadian biological clock is essentially based on the light/dark cycle. Some people working with shift schedules cannot adjust their sleep/wake cycle to the light/dark cycle, and this may result in alterations of the circadian biological clock. This study explored the circadian biological clock of shift and daytime nurses using non-invasive methods. Peripheral skin temperature, cortisol and melatonin levels in saliva, and Per2 expression in pubic hair follicle cells were investigated for 24 h after a day off. Significant differences were observed in peripheral skin temperature and cortisol levels between shift and daytime nurses. No differences in melatonin levels were obtained. Per2 maximum values were significantly different between the two groups. Shift nurses exhibited lower circadian variations compared to daytime nurses, and this may indicate an adjustment of the circadian biological clock to continuous shift schedules. Non-invasive procedures, such as peripheral skin temperature measurement, determination of cortisol and melatonin in saliva, and analysis of clock genes in hair follicle cells, may be effective approaches to extensively study the circadian clock in shift workers.

Combined circulating epigenetic markers to improve mesothelin performance in the diagnosis of malignant mesothelioma.

OBJECTIVES: Malignant mesothelioma (MM) is a highly aggressive tumor with poor prognosis. A major challenge is the development and application of early and highly reliable diagnostic marker(s). Serum biomarkers, such as 'soluble mesothelin-related proteins' (SMRPs), is the most studied and frequently used in MM. However, the low sensitivity of SMRPs for early MM limits its value; therefore, additional biomarkers are required. In this study, two epigenetically regulated markers in MM (microRNA-126, miR-126, and methylated thrombomodulin promoter, Met-TM) were combined with SMRPs and evaluated as a potential strategy to detect MM at an early stage. MATERIALS AND METHODS: A total of 188 subjects, including 45 MM patients, 99 asbestos-exposed subjects, and 44 healthy controls were prospectively enrolled, serum samples collected, and serum levels of SMRPs, miR-126 and Met-TM evaluated. Logistic regression analysis was performed to evaluate the diagnostic value of the three biomarkers. Using this approach, the performance of the '3-biomarker classifier' was tested by calculating the overall probability score of the MM and control samples, respectively, and the ROC curve was generated. RESULTS AND CONCLUSION: The combination of the three biomarkers was the best predictor to differentiate MM patients from asbestos-exposed subjects and healthy controls. The accuracy and cancer specificity was confirmed in a second validation cohort and lung cancer population. We propose that the combination of the two epigenetic biomarkers with SMRPs as a diagnosis for early MM overcomes the limitations of using SMRPs alone.

Circadian Modulation of 8-Oxoguanine DNA Damage Repair.

The DNA base excision repair pathway is the main system involved in the removal of oxidative damage to DNA such as 8-Oxoguanine (8-oxoG) primarily via the 8-Oxoguanine DNA glycosylase (OGG1). Our goal was to investigate whether the repair of 8-oxoG DNA damage follow a circadian rhythm. In a group of 15 healthy volunteers, we found a daily variation of Ogg1 expression and activity with higher levels in the morning compared to the evening hours. Consistent with this, we also found lower levels of 8-oxoG in morning hours compared to those in the evening hours. Lymphocytes exposed to oxidative damage to DNA at 8:00 AM display lower accumulation of 8-oxoG than lymphocytes exposed at 8:00 PM. Furthermore, altered levels of Ogg1 expression were also observed in a group of shift workers experiencing a deregulation of circadian clock genes compared to a control group. Moreover, BMAL1 knockdown fibroblasts with a deregulated molecular clock showed an abolishment of circadian variation of Ogg1 expression and an increase of OGG1 activity. Our results suggest that the circadian modulation of 8-oxoG DNA damage repair, according to a variation of Ogg1 expression, could render humans less susceptible to accumulate 8-oxoG DNA damage in the morning hours.

Circadian gene expression and extremely low-frequency magnetic fields: an in vitro study.

It is well known that circadian clocks are mainly regulated by light targeting signaling pathways in the hypothalamic suprachiasmatic nucleus. However, an entrainment mediated by non-photic sensory stimuli was also suggested for peripheral clocks. Exposure to extremely low frequency (ELF) electromagnetic fields might affect circadian rhythmicity. The goal of this research was to investigate effects of ELF magnetic fields (ELF-MF) on circadian clock genes in a human fibroblast cell line. We found that an ELF-MF (0.1 mT, 50 Hz) exposure was capable of entraining expression of clock genes BMAL1, PER2, PER3, CRY1, and CRY2. Moreover, ELF-MF treatment induced an alteration in circadian clock gene expression previously entrained by serum shock stimulation. These results support the hypothesis that ELF-MF may be able to drive circadian physiologic processes by modulating peripheral clock gene expression.

Wood dust exposure induces cell transformation through EGFR-mediated OGG1 inhibition.

A high risk of neoplastic transformation of nasal and paranasal sinuses mucosa is related to the occupational exposure to wood dust. However, the role of occupational exposures in the aetiology of the airway cancers remains largely unknown. Here, an in vitro model was performed to investigate the carcinogenic effect of wood dusts. Human bronchial epithelial cells were incubated with hard and soft wood dusts and the DNA damage and response to DNA damage evaluated. Wood dust exposure induced accumulation of oxidised DNA bases, which was associated with a delay in DNA repair activity. By exposing cells to wood dust at a prolonged time, wood dust-initiated cells were obtained. Initiated-cells were able to form colonies in soft agar, and to induce blood vessel formation. These cells showed extensive autophagy, reduced DNA repair, which was associated with reduced OGG1 expression and oxidised DNA base accumulation. These events were found related to the activation of EGFR/AKT/mTOR pathway, through phosphorylation and subsequent inactivation of tuberin. The persistence in the tissue of wood dusts, their repetitious binding with EGFR may continually trigger the activation switch, leading to chronic down-regulation of genes involved in DNA repair, leading to cell transformation and proliferation.

Low perceived social support is associated with CD8+CD57+ lymphocyte expansion and increased TNF-α levels.

Social support has been supposed to have a positive impact on the function of the immune system. However, the relationship between perceived social support and immune function has not yet been fully investigated. In this cross-sectional study, we investigated the link between perceived social support and lymphocyte subpopulations and cytokines. 232 healthy subjects provided a blood sample and completed the Multidimensional Scale of Perceived Social Support (MSPSS) questionnaire. Lymphocyte immunophenotypes and cytokines were determined. Significantly increased CD8+CD57+ lymphocytes and TNF-α levels were found in group with low perceived social support. Multivariate linear regression corrected for possible confounders confirmed a significant role of perceived social support in predicting the number of CD8+CD57+ lymphocyte and TNF-α levels. This study supports the association between perceived social support and immune function. In particular, poor social support may be related to a state of chronic inflammation sustained by CD8+CD57+ lymphocyte expansion and increased TNF-α levels.

MicroRNA-126 suppresses mesothelioma malignancy by targeting IRS1 and interfering with the mitochondrial function.

AIMS: MiR126 was found to be frequently lost in many types of cancer, including malignant mesothelioma (MM), which represents one of the most challenging neoplastic diseases. In this study, we investigated the potential tumor suppressor function of MiR126 in MM cells. The effect of MiR126 was examined in response to oxidative stress, aberrant mitochondrial function induced by inhibition of complex I, mitochondrial DNA (mtDNA) depletion, and hypoxia. RESULTS: MiR126 was up-regulated by oxidative stress in nonmalignant mesothelial (Met5A) and MM (H28) cell lines. In Met5A cells, rotenone inhibited MiR126 expression, but mtDNA depletion and hypoxia up-regulated MiR126. However, these various stimuli suppressed the levels of MiR126 in H28 cells. MiR126 affected mitochondrial energy metabolism, reduced mitochondrial respiration, and promoted glycolysis in H28 cells. This metabolic shift, associated with insulin receptor substrate-1 (IRS1)-modulated ATP-citrate lyase deregulation, resulted in higher ATP and citrate production. These changes were linked to the down-regulation of IRS1 by ectopic MiR126, reducing Akt signaling and inhibiting cytosolic sequestration of Forkhead box O1 (FoxO1), which promoted the expression of genes involved in gluconeogenesis and oxidative stress defense. These metabolic changes induced hypoxia-inducible factor-1α (HIF1α) stabilization. Consequently, MiR126 suppressed the malignancy of MM cells in vitro, a notion corroborated by the failure of H28(MiR126) cells to form tumors in nude mice. INNOVATION AND CONCLUSION: MiR126 affects mitochondrial energy metabolism, resulting in MM tumor suppression. Since MM is a fatal neoplastic disease with a few therapeutic options, this finding is of potential translational importance.

Rotating-shift nurses after a day off: peripheral clock gene expression, urinary melatonin, and serum 17-ß-estradiol levels.

OBJECTIVE: Impairment of clock gene expression and changes in melatonin and 17-ß-estradiol levels may constitute biological alterations underlying the increased risk of breast cancer among shift workers. The aim of this study was to compare levels of selected core clock gene expression, 6-sulfatoxymelatonin (aMT6s), and 17-ß-estradiol between rotational shift work (SW) and daytime (DT) workers after a day off. METHODS: The cross-sectional study comprised 60 nurses with ≥2 years of SW and 56 permanent DT nurses. Transcript levels of circadian genes BMAL1, CLOCK, NPAS2, CRY1, CRY2, PER1, PER2, PER3, and REVERBα were determined by quantitative real-time polymerase chain reaction (PCR) in lymphocytes. All participants were tested in the early follicular phase of the menstrual cycle. Samples were collected at the beginning of the morning-shift after a regular night's sleep on a day off. Chronotype and sociodemographic characteristics were also evaluated. RESULTS: We found a significantly higher expression of BMAL1, CLOCK, NPAS2, PER1, PER2, and REVERBα and a lower expression of PER3, CRY1 and CRY2 among SW compared to DT nurses. SW participants did not demonstrate a significant difference in aMT6s levels, but they did show significantly higher 17-ß-estradiol levels compared to DT nurses. Multiple linear regression analysis confirmed the role of SW on expression of BMAL1 (ß 0.21, P=0.040), CLOCK (ß 0.35, P=0.008), NPAS2 (ß 0.30, P=0.012), PER1 (ß 0.33, P=0.008), PER2 (ß 0.19, P=0.047), PER3 (ß -0.27, P=0.012), CRY1 (ß -0.33, P=0.002), CRY2 (ß -0.31, P=0.005), REVERBα (ß 0.19, P=0.045), and on 17-ß-estradiol levels (ß 0.32, P=0.003). The analysis also confirmed the role of chronotype as an independent factor for PER1 (ß 0.48, P=0.001) and PER2 (ß -0.22, P=0.022) expression, and 17-ß-estradiol levels (ß 0.26, P=0.011). CONCLUSIONS: Rotating SW nurses show alterations in peripheral clock gene expression and 17-ß-estradiol levels at the beginning of the morning shift after a day off.

Occupational styrene exposure induces stress-responsive genes involved in cytoprotective and cytotoxic activities.

OBJECTIVE: The aim of this study was to evaluate the expression of a panel of genes involved in toxicology in response to styrene exposure at levels below the occupational standard setting. METHODS: Workers in a fiber glass boat industry were evaluated for a panel of stress- and toxicity-related genes and associated with biochemical parameters related to hepatic injury. Urinary styrene metabolites (MA+PGA) of subjects and environmental sampling data collected for air at workplace were used to estimate styrene exposure. RESULTS: Expression array analysis revealed massive upregulation of genes encoding stress-responsive proteins (HSPA1L, EGR1, IL-6, IL-1ß, TNSF10 and TNFα) in the styrene-exposed group; the levels of cytokines released were further confirmed in serum. The exposed workers were then stratified by styrene exposure levels. EGR1 gene upregulation paralleled the expression and transcriptional protein levels of IL-6, TNSF10 and TNFα in styrene exposed workers, even at low level. The activation of the EGR1 pathway observed at low-styrene exposure was associated with a slight increase of hepatic markers found in highly exposed subjects, even though they were within normal range. The ALT and AST levels were not affected by alcohol consumption, and positively correlated with urinary styrene metabolites as evaluated by multiple regression analysis. CONCLUSION: The pro-inflammatory cytokines IL-6 and TNFα are the primary mediators of processes involved in the hepatic injury response and regeneration. Here, we show that styrene induced stress responsive genes involved in cytoprotection and cytotoxicity at low-exposure, that proceed to a mild subclinical hepatic toxicity at high-styrene exposure.

Styrene altered clock gene expression in serum-shocked cultured human fibroblasts.

The circadian clock can regulate the metabolic process of xenobiotics, but little is known as to circadian rhythms can be perturbed by xenobiotics. Styrene is a organic chemical widely used in occupational settings. The effects of styrene on the circadian genes of HuDE cells were evaluated after serum-shocking synchronization. A subtoxic dose of 100 µM of styrene altered the expression of clock genes BMAL1, PER2, PER3, CRY1, CRY2, and REV-ERB-α.

Alpha-tocopheryl succinate inhibits autophagic survival of prostate cancer cells induced by vitamin K3 and ascorbate to trigger cell death.

BACKGROUND: The redox-silent vitamin E analog α-tocopheryl succinate (α-TOS) was found to synergistically cooperate with vitamin K3 (VK3) plus ascorbic acid (AA) in the induction of cancer cell-selective apoptosis via a caspase-independent pathway. Here we investigated the molecular mechanism(s) underlying cell death induced in prostate cancer cells by α-TOS, VK3 and AA, and the potential use of targeted drug combination in the treatment of prostate cancer. METHODOLOGY/PRINCIPAL FINDINGS: The generation of ROS, cellular response to oxidative stress, and autophagy were investigated in PC3 prostate cancer cells by using drugs at sub-toxic doses. We evaluated whether PARP1-mediated apoptosis-inducing factor (AIF) release plays a role in apoptosis induced by the combination of the agents. Next, the effect of the combination of α-TOS, VK3 and AA on tumor growth was examined in nude mice. VK3 plus AA induced early ROS formation associated with induction of autophagy in response to oxidative stress, which was reduced by α-TOS, preventing the formation of autophagosomes. α-TOS induced mitochondrial destabilization leading to the release of AIF. Translocation of AIF from mitochondria to the nucleus, a result of the combinatorial treatment, was mediated by PARP1 activation. The inhibition of AIF as well as of PARP1 efficiently attenuated apoptosis triggered by the drug combination. Using a mouse model of prostate cancer, the combination of α-TOS, VK3 and AA was more efficient in tumor suppression than when the drugs were given separately, without deleterious side effects. CONCLUSIONS/SIGNIFICANCE: α-TOS, a mitochondria-targeting apoptotic agent, switches at sub-apoptotic doses from autophagy-dependent survival of cancer cells to their demise by promoting the induction of apoptosis. Given the grim prognosis for cancer patients, this finding is of potential clinical relevance.

Lipid peroxidation and paraoxonase-1 activity in celiac disease.

Paraoxonase-1 (PON1) plays an antioxidant and anti-inflammatory role. Aim of the study was to investigate the alteration of paraoxonase-1 activity in celiac disease (CD), an intestinal disorder characterized by toxic injury exerted by gluten peptides. Activities of PON1, levels of biochemical markers of lipid peroxidation and total antioxidant capacity were evaluated in serum obtained from 27 celiac patients (11 at diagnosis, 16 treated with gluten free diet) and 25 healthy subjects. Moreover, the serum susceptibility of Cu(2+)-induced lipid peroxidation was investigated in controls and patients. The results showed a lower PON1 activity in serum of both groups of celiac patients with respect to control subjects. PON1 activity in CD was related with markers of disease severity and was negatively correlated with the levels of lipid hydroperoxide and with the susceptibility of serum to lipid peroxidation induced in vitro by metal ions. The alteration of PON1 activity and markers of lipid peroxidation realized at lower extent in patients who were on a gluten-free diet.