Hormesis and Oxidative Distress: Pathophysiology of Reactive Oxygen Species and the Open Question of Antioxidant Modulation and Supplementation.

Alterations of redox homeostasis leads to a condition of resilience known as hormesis that is due to the activation of redox-sensitive pathways stimulating cell proliferation, growth, differentiation, and angiogenesis. Instead, supraphysiological production of reactive oxygen species (ROS) exceeds antioxidant defence and leads to oxidative distress. This condition induces damage to biomolecules and is responsible or co-responsible for the onset of several chronic pathologies. Thus, a dietary antioxidant supplementation has been proposed in order to prevent aging, cardiovascular and degenerative diseases as well as carcinogenesis. However, this approach has failed to demonstrate efficacy, often leading to harmful side effects, in particular in patients affected by cancer. In this latter case, an approach based on endogenous antioxidant depletion, leading to ROS overproduction, has shown an interesting potential for enhancing susceptibility of patients to anticancer therapies. Therefore, a deep investigation of molecular pathways involved in redox balance is crucial in order to identify new molecular targets useful for the development of more effective therapeutic approaches. The review herein provides an overview of the pathophysiological role of ROS and focuses the attention on positive and negative aspects of antioxidant modulation with the intent to find new insights for a successful clinical application.

PKCα Inhibition as a Strategy to Sensitize Neuroblastoma Stem Cells to Etoposide by Stimulating Ferroptosis.

Cancer stem cells (CSCs) are a limited cell population inside a tumor bulk characterized by high levels of glutathione (GSH), the most important antioxidant thiol of which cysteine is the limiting amino acid for GSH biosynthesis. In fact, CSCs over-express xCT, a cystine transporter stabilized on cell membrane through interaction with CD44, a stemness marker whose expression is modulated by protein kinase Cα (PKCα). Since many chemotherapeutic drugs, such as Etoposide, exert their cytotoxic action by increasing reactive oxygen species (ROS) production, the presence of high antioxidant defenses confers to CSCs a crucial role in chemoresistance. In this study, Etoposide-sensitive and -resistant neuroblastoma CSCs were chronically treated with Etoposide, given alone or in combination with Sulfasalazine (SSZ) or with an inhibitor of PKCα (C2-4), which target xCT directly or indirectly, respectively. Both combined approaches are able to sensitize CSCs to Etoposide by decreasing intracellular GSH levels, inducing a metabolic switch from OXPHOS to aerobic glycolysis, down-regulating glutathione-peroxidase-4 activity and stimulating lipid peroxidation, thus leading to ferroptosis. Our results suggest, for the first time, that PKCα inhibition inducing ferroptosis might be a useful strategy with which to fight CSC chemoresistance.

Heme Oxygenase 1 in the Nervous System: Does It Favor Neuronal Cell Survival or Induce Neurodegeneration?

Heme oxygenase 1 (HO-1) up-regulation is recognized as a pivotal mechanism of cell adaptation to stress. Under control of different transcription factors but with a prominent role played by Nrf2, HO-1 induction is crucial also in nervous system response to damage. However, several lines of evidence have highlighted that HO-1 expression is associated to neuronal damage and neurodegeneration especially in Alzheimer's and Parkinson's diseases. In this review, we summarize the current literature regarding the role of HO-1 in nervous system pointing out different molecular mechanisms possibly responsible for HO-1 up-regulation in nervous system homeostasis and neurodegeneration.

Differentiation impairs Bach1 dependent HO-1 activation and increases sensitivity to oxidative stress in SH-SY5Y neuroblastoma cells.

Neuronal adaptation to oxidative stress is crucially important in order to prevent degenerative diseases. The role played by the Nrf2/HO-1 system in favoring cell survival of neuroblastoma (NB) cells exposed to hydrogen peroxide (H2O2) has been investigated using undifferentiated or all-trans retinoic acid (ATRA) differentiated SH-SY5Y cells. While undifferentiated cells were basically resistant to the oxidative stimulus, ATRA treatment progressively decreased cell viability in response to H2O2. HO-1 silencing decreased undifferentiated cell viability when exposed to H2O2, proving the role of HO-1 in cell survival. Conversely, ATRA differentiated cells exposed to H2O2 showed a significantly lower induction of HO-1, and only the supplementation with low doses of bilirubin (0,5-1 µM) restored viability. Moreover, the nuclear level of Bach1, repressor of HO-1 transcription, strongly decreased in undifferentiated cells exposed to oxidative stress, while did not change in ATRA differentiated cells. Furthermore, Bach1 was displaced from HO-1 promoter in undifferentiated cells exposed to H2O2, enabling the binding of Nrf2. On the contrary, in ATRA differentiated cells treated with H2O2, Bach1 displacement was impaired, preventing Nrf2 binding and limiting HO-1 transcription. In conclusion, our findings highlight the central role of Bach1 in HO-1-dependent neuronal response to oxidative stress.

Redox Homeostasis and Cellular Antioxidant Systems: Crucial Players in Cancer Growth and Therapy.

Reactive oxygen species (ROS) and their products are components of cell signaling pathways and play important roles in cellular physiology and pathophysiology. Under physiological conditions, cells control ROS levels by the use of scavenging systems such as superoxide dismutases, peroxiredoxins, and glutathione that balance ROS generation and elimination. Under oxidative stress conditions, excessive ROS can damage cellular proteins, lipids, and DNA, leading to cell damage that may contribute to carcinogenesis. Several studies have shown that cancer cells display an adaptive response to oxidative stress by increasing expression of antioxidant enzymes and molecules. As a double-edged sword, ROS influence signaling pathways determining beneficial or detrimental outcomes in cancer therapy. In this review, we address the role of redox homeostasis in cancer growth and therapy and examine the current literature regarding the redox regulatory systems that become upregulated in cancer and their role in promoting tumor progression and resistance to chemotherapy.

A novel mechanism for cyclic adenosine monophosphate-mediated memory formation: Role of amyloid beta.

Cyclic adenosine monophosphate (cAMP) regulates long-term potentiation (LTP) and ameliorates memory in healthy and diseased brain. Increasing evidence shows that, under physiological conditions, low concentrations of amyloid ß (Aß) are necessary for LTP expression and memory formation. Here, we report that cAMP controls amyloid precursor protein (APP) translation and Aß levels, and that the modulatory effects of cAMP on LTP occur through the stimulation of APP synthesis and Aß production.

HO-1 up-regulation: a key point in high-risk neuroblastoma resistance to bortezomib.

High-risk neuroblastoma (NB) is characterized by the development of chemoresistance, and bortezomib (BTZ), a selective inhibitor of proteasome, has been proposed in order to overcome drug resistance. Considering the involvement of the nuclear factor-erythroid-derived 2-like 2 (Nrf2) and heme oxygenase-1 (HO-1) in the antioxidant and detoxifying ability of cancer cells, in this study we have investigated their role in differently aggressive NB cell lines treated with BTZ, focusing on the modulation of HO-1 to improve sensitivity to therapy. We have shown that MYCN amplified HTLA-230 cells were slightly sensitive to BTZ treatment, due to the activation of Nrf2 that led to an impressive up-regulation of HO-1. BTZ-treated HTLA-230 cells down-regulated p53 and up-regulated p21, favoring cell survival. The inhibition of HO-1 activity obtained by Zinc (II) protoprophyrin IX (ZnPPIX) was able to significantly increase the pro-apoptotic effect of BTZ in a p53- and p21-independent way. However, MYCN non-amplified SH-SY5Y cells showed a greater sensitivity to BTZ in relation to their inability to up-regulate HO-1. Therefore, we have shown that HO-1 inhibition improves the sensitivity of aggressive NB to proteasome inhibition-based therapy, suggesting that HO-1 up-regulation can be used as a marker of chemoresistance in NB. These results open up a new scenario in developing a combined therapy to overcome chemoresistance in high-risk neuroblastoma.

Role of glutathione in cancer progression and chemoresistance.

Glutathione (GSH) plays an important role in a multitude of cellular processes, including cell differentiation, proliferation, and apoptosis, and disturbances in GSH homeostasis are involved in the etiology and progression of many human diseases including cancer. While GSH deficiency, or a decrease in the GSH/glutathione disulphide (GSSG) ratio, leads to an increased susceptibility to oxidative stress implicated in the progression of cancer, elevated GSH levels increase the antioxidant capacity and the resistance to oxidative stress as observed in many cancer cells. The present review highlights the role of GSH and related cytoprotective effects in the susceptibility to carcinogenesis and in the sensitivity of tumors to the cytotoxic effects of anticancer agents.

Impaired synthesis contributes to diabetes-induced decrease in liver glutathione.

Diabetes-induced glutathione (GSH) decrease is usually ascribed to GSH oxidation. Here we investigate, in streptozotocin-treated rats, if impairment of GSH synthesis contributes to GSH decrease in diabetic liver, and if antioxidant treatments can provide protection. Diabetic rats were divided into 3 groups: untreated diabetic rats (UD); N-acetyl-cysteine (NAC)-treated diabetic rats; taurine (TAU)-treated diabetic rats; a group of non-streptozotocin-treated rats was used as control (CTR). All rats were sacrificed at 40 weeks of age. Diabetes induced hepatic glutathione decrease, but oxidized glutathione (GSSG) did not increase significantly. Accumulations of cysteine and cysteinyl-glycine in UD suggest respectively decreased glutathione synthesis and increased loss through the plasma membrane with subsequent degradation. Decreased expression of γ-glutamyl-cysteine synthetase in UD is consistent with repressed GSH synthesis. Moreover, diabetes caused increase of GSSG/GSH ratio and induction of heme oxygenase-1, both signs of oxidative stress. Supplementation with NAC or TAU resulted in amelioration of glutathione levels, probably depending on antioxidant activity, more efficient glutathione synthesis and decreased GSH loss and degradation. In conclusion, impaired synthesis and increased loss and degradation of GSH appear to contribute to a decrease in GSH levels in diabetic liver. NAC and TAU are able to partially protect from oxidative stress and GSH decrease, while enhancing GSH synthesis and restricting GSH loss.

Resistance of neuroblastoma GI-ME-N cell line to glutathione depletion involves Nrf2 and heme oxygenase-1.

Cancer cell survival is known to be related to the ability to counteract oxidative stress, and glutathione (GSH) depletion has been proposed as a mechanism to sensitize cells to anticancer therapy. However, we observed that GI-ME-N cells, a neuroblastoma cell line without MYCN amplification, are able to survive even if GSH-depleted by l-buthionine-(S,R)-sulfoximine (BSO). Here, we show that in GI-ME-N cells, BSO activates Nrf2 and up-regulates heme oxygenase-1 (HO-1). Silencing of Nrf2 restrained HO-1 induction by BSO. Inhibition of HO-1 and silencing of Nrf2 or HO-1 sensitized GI-ME-N cells to BSO, leading to reactive oxygen/nitrogen species overproduction and decreasing viability. Moreover, targeting the Nrf2/HO-1 axis sensitized GI-ME-N cells to etoposide more than GSH depletion. Therefore, we have provided evidence that in GI-ME-N cells, the Nrf2/HO-1 axis plays a crucial role as a protective factor against cellular stress, and we suggest that the inhibition of Nfr2/HO-1 signaling should be considered as a central target in the clinical battle against neuroblastoma.

Human mesangial cells resist glycoxidative stress through an antioxidant response.

The generation of advanced glycation end-products (AGE), the interaction with their receptors, the generation of reactive oxygen species, and the modulation of intracellular redox equilibrium are believed to be the main factors causing alterations of mesangial cell physiology leading to diabetic nephropathy. Normal human primary mesangial cells were exposed to glycoxidative stress by culture in high glucose (HG) or treatment with AGE for up to 6 days. In both cases only a moderate generation of reactive oxygen species and production of HNE-protein adducts were induced while protein nitrotyrosination was not affected. Moreover, HG and AGE caused a significant antioxidant response, confirmed by the induction of heme oxygenase 1 and the consumption of vitamin E. Glutathione was decreased only by HG. Mesangial cell proliferation and viability were slightly affected by HG and AGE. Furthermore, both treatments failed to influence TGF-ß1 and MCP-1 secretion and to modulate RAGE and collagen IV expression. We believe that normal human mesangial cells can resist glycoxidative stress by the observed antioxidant response. These results support the concept that mesangial cells are only partly responsible for the onset and progression of diabetic nephropathy and that the role of other cell types, such as podocytes and endothelial cells, should be taken into consideration.

PKC delta and NADPH oxidase in retinoic acid-induced neuroblastoma cell differentiation.

The role of reactive oxygen species (ROS) in the regulation of signal transduction processes has been well established in many cell types and recently the fine tuning of redox signalling in neurons received increasing attention. With regard to this, the involvement of NADPH oxidase (NOX) in neuronal pathophysiology has been proposed but deserves more investigation. In the present study, we used SH-SY5Y neuroblastoma cells to analyse the role of NADPH oxidase in retinoic acid (RA)-induced differentiation, pointing out the involvement of protein kinase C (PKC) delta in the activation of NOX. Retinoic acid induces neuronal differentiation as revealed by the increased expression of MAP2, the decreased cell doubling rate, and the gain in neuronal morphological features and these events are accompanied by the increased expression level of PKC delta and p67(phox), one of the components of NADPH oxidase. Using DPI to inhibit NOX activity we show that retinoic acid acts through this enzyme to induce morphological changes linked to the differentiation. Moreover, using rottlerin to inhibit PKC delta or transfection experiments to overexpress it, we show that retinoic acid acts through this enzyme to induce MAP2 expression and to increase p67(phox) membrane translocation leading to NADPH oxidase activation. These findings identify the activation of PKC delta and NADPH oxidase as crucial steps in RA-induced neuroblastoma cell differentiation.

GSH loss per se does not affect neuroblastoma survival and is not genotoxic.

Depletion of glutathione (GSH) by buthionine sulfoximine (BSO) has been reported to be toxic against some cancer cells and to sensitize many tumours including neuroblastoma (NB) to anticancer drugs. The balance between the production rate of reactive oxygen species (ROS) and the function of GSH affects the intracellular reduction-oxidation status, which is crucial for the regulation of several cellular physiological functions. To assess the role of glutathione in neuroblastoma therapy, the effect of sublethal concentrations of BSO was studied in a panel of neuroblastoma cell lines characterized by different MYCN status. We found that GSH depletion per se not accompanied by ROS overproduction, does not affect cell survival, and is not genotoxic but induces HO-1 expression in GI-ME-N cell line, a representative example of MYCN non-amplified NB cells, having the highest basal levels of GSH among the tested NB lines. These observations might open a novel therapeutic window based on the possibility of modulating the cellular 'activity' of GSH.

Oxidative stress and antioxidant defence in a healthy nonagenarian population.

Results on oxidative markers during ageing are not consistent throughout the scientific literature; however, successful ageing may depend on better ability to cope with oxidative stress. A previous study of ours showed that successful ageing could actually be related to enhanced response to oxidatively modified proteins. In this study, a healthy nonagenarian population (OVER-90) was examined for various blood oxidative biomarkers and compared with a healthy population of blood donors (age range, 23-66 years). Blood glutathione, both total (tGSH) and oxidised (GSSG), and total plasmatic antioxidant status were maintained in the OVER-90 at a level similar to the control population. Sulphydryl (sulfhydryl) groups and glutathione peroxidase (GPx) were instead decreased. The results are discussed in a possible unifying view: the OVER-90 population could possess a globally preserved antioxidant ability, though some signs of oxidative damage are present and some structures could be 'sacrificed' in order to keep the redox equilibrium.

Heme oxygenase 1 expression in rat liver during ageing and ethanol intoxication.

Heme oxygenase 1 (HO-1) expression is recognized as a marker of cellular response to oxidative stress; since ageing is believed to be related to oxidative "wear and tear", HO-1 may represent a candidate biomarker of ageing. In our study, the hepatic expression of HO-1 mRNA, evaluated by RT-PCR in 2.5-24 month-old rats, was higher at 6 months than at 2.5 months of age, but thereafter increased no further: on the contrary, a declining trend was observed. However, while 2.5 month-old rats responded to acute ethanol intoxication by displaying increased expression of liver HO-1 mRNA, and 6 month-old rats exhibited a mild response, 18 month-old rats did not show any response; this phenomenon suggests that during development and ageing the transcriptional response to oxidative stress decreases. In our view, the finding that HO-1 expression did not increase progressively during ageing may be explained by a decreased transcriptional ability to respond to stress in older animals, rather than by a reduction in oxidative stress.

Effects of Agelas oroides and Petrosia ficiformis crude extracts on human neuroblastoma cell survival.

Among marine sessile organisms, sponges (Porifera) are the major producers of bioactive secondary metabolites that defend them against predators and competitors and are used to interfere with the pathogenesis of many human diseases. Some of these biological active metabolites are able to influence cell survival and death, modifying the activity of several enzymes involved in these cellular processes. These natural compounds show a potential anticancer activity but the mechanism of this action is largely unknown. In this study, we investigated the effects of two Mediterranean sponges, Agelas oroides and Petrosia ficiformis on the viability of human neuroblastoma cells. Upon treatment with the methanolic extract of Petrosia ficiformis, a marked cytotoxic effect was observed at any concentration or time of exposure. In contrast, a time- and dose-dependent effect was monitored for Agelas oroides that induced the development of apoptotic features and ROS production in LAN5 cells. These events were suppressed by calpeptin or zVAD and by vitamin C suggesting that the cell death caused by Agelas oroides was calpain- and caspase-dependent and of oxidative nature. Comet assay showed that this methanolic extract was not able to produce a genotoxic effect. Future studies will be applied to investigate the effect of isolated bioactive compounds from crude extract of this sponge which are potentially useful for cancer therapeutics.

Reactive oxygen species: biological stimuli of neuroblastoma cell response.

Reactive oxygen species play a critical role in differentiation, proliferation and apoptosis acting as 'second messengers' able to regulate sulphydryl groups in signaling molecules as protein kinase C, a family of isoenzymes involved in many cellular responses and implicated in cell transformation. Neuroblastoma is characterised by the production of oxygen intermediates and L-buthionine-S,R-sulfoximine, a glutathione-depleting agent that has been tested in the clinics, exploits this biological peculiarity to induce cell death. The latter process is mediated by the oxidative activation of PKC delta which might be involved also in the production of reactive oxygen species, thus amplifying the apoptotic cascade.

Anti malondialdehyde-adduct immunological response as a possible marker of successful aging.

Contrasting results have been obtained by various researchers about oxidative markers of aging. In this study, a healthy over-90-year-old population was examined for various plasma oxidative biomarkers and compared with a healthy population of blood donors (age range 23-66). Plasma malondialdehyde (MDA), evaluated by means of the thiobarbituric acid test, was significantly higher in the over-90-year-old population, confirming the presence of increased lipoperoxidation in old age. The antibody titre against MDA-protein adducts, considered a marker of lipoperoxidative protein damage in vivo, was evaluated in an ELISA test, completely home made and calibrated versus a concentrated pool of human plasma; this antibody titre was significantly higher in the over-90-year-old population. Plasma vitamin E, evaluated in RP-HPLC, was not significantly different between the two groups. Plasma protein-bound carbonyls, a marker of oxidative protein damage, were measured with the 2,4-dinitrophenylhydrazine assay; their level in the over-90-year-old population was lower than in the blood donors. The higher antibody titre against MDA-adducts may result in protection against accumulation of oxidatively damaged proteins by enhancing their removal, and, together with the preserved plasma vitamin E level, it may endow over-90-year-olds with an especially efficient antioxidant profile. The low level of protein carbonyl might reflect the more efficient removal of damaged proteins.

Role of PKC-delta activity in glutathione-depleted neuroblastoma cells.

Protein kinases C (PKCs) are a family of isoenzymes sensitive to oxidative modifications and involved in the transduction signal pathways that regulate cell growth. As such, they can act as cellular sensors able to intercept intracellular redox changes and promote the primary adaptive cell response. In this study, we have demonstrated that PKC isoforms are specifically influenced by the amount of intracellular glutathione (GSH). The greatest GSH depletion is associated with a maximal reactive oxygen species (ROS) production and accompanied by an increase in the activity of the delta isoform and a concomitant inactivation of alpha. ROS generation induced early morphological changes in GSH-depleted neuroblastoma cells characterized, at the intracellular level, by the modulation of PKC-delta activity that was involved in the pathway leading to apoptosis. When cells were pretreated with rottlerin, their survival was improved by the ability of this compound to inhibit the activity of PKC-delta and to counteract ROS production. These results define a novel role of PKC-delta in the cell signaling pathway triggered by GSH loss normally associated with many neurodegenerative diseases and clinically employed in the treatment of neuroblastoma.

Role of PKC-dependent pathways in HNE-induced cell protein transport and secretion.

The beta isoforms of protein Kinase C (PKC) are closely involved in the regulation of cell protein transport and secretion. We have shown in different cellular types that treatment with HNE in a concentration range detectable in many pathophysiological conditions is able to induce selective activation of betaPKCs through direct interaction between the aldehyde and these isoenzymes. In isolated rat hepatocytes this specific isoenzyme activation plays a key role in the transport of procathepsin D from the trans-Golgi network to the endosomal-lysosomal compartment and in the exocytosis of mature cathepsin D. In NT2 neurons, HNE-mediated betaPKC activation induces an increase in intracellular amyloid beta production, without affecting full-length amyloid precursor protein expression. In a mouse macrophage-like cell line, the same beta isoform activation increases the release of the MCP-1 chemokine. Thus, pathophysiological HNE concentrations (0.1-1 microM) derived from a slight imbalance of the redox state are able to alter protein trafficking through beta PKC activation. These results suggest that mild oxidative stress and the PKC signal transduction pathway are closely involved in the pathophysiology of many diseases caused by changes in protein trafficking and release.