Nrf2-Keap1 signaling in oxidative and reductive stress.

Nrf2 and its endogenous inhibitor, Keap1, function as a ubiquitous, evolutionarily conserved intracellular defense mechanism to counteract oxidative stress. Sequestered by cytoplasmic Keap1 and targeted to proteasomal degradation in basal conditions, in case of oxidative stress Nrf2 detaches from Keap1 and translocates to the nucleus, where it heterodimerizes with one of the small Maf proteins. The heterodimers recognize the AREs, that are enhancer sequences present in the regulatory regions of Nrf2 target genes, essential for the recruitment of key factors for transcription. In the present review we briefly introduce the Nrf2-Keap1 system and describe Nrf2 functions, illustrate the Nrf2-NF-κB cross-talk, and highlight the effects of the Nrf2-Keap1 system in the physiology and pathophysiology of striated muscle tissue taking into account its role(s) in oxidative stress and reductive stress.

Natriuretic Peptides: The Case of Prostate Cancer.

Cardiac natriuretic peptides have long been known to act as main players in the homeostatic control of blood pressure, salt and water balance. However, in the last few decades, new properties have been ascribed to these hormones. A systematic review of English articles using MEDLINE Search terms included prostate cancer, inflammation, cardiac hormones, atrial natriuretic peptide, and brain natriuretic peptide. Most recent publications were selected. Natriuretic peptides are strongly connected to the immune system, whose two branches, innate and adaptive, are finely tuned and organized to kill invaders and repair injured tissues. These peptides control the immune response and act as anti-inflammatory and immune-modulatory agents. In addition, in cancers, natriuretic peptides have anti-proliferative effects by molecular mechanisms based on the inhibition/regulation of several pathways promoting cell proliferation and survival. Nowadays, it is accepted that chronic inflammation is a crucial player in prostate cancer development and progression. In this review, we summarize the current knowledge on the link between prostate cancer and inflammation and the potential use of natriuretic peptides as anti-inflammatory and anticancer agents.

The Role of Cyclo(His-Pro) in Neurodegeneration.

Neurodegenerative diseases may have distinct genetic etiologies and pathological manifestations, yet share common cellular mechanisms underpinning neuronal damage and dysfunction. These cellular mechanisms include excitotoxicity, calcium dysregulation, oxidative damage, ER stress and neuroinflammation. Recent data have identified a dual role in these events for glial cells, such as microglia and astrocytes, which are able both to induce and to protect against damage induced by diverse stresses. Cyclo(His-Pro), a cyclic dipeptide derived from the hydrolytic removal of the amino-terminal pyroglutamic acid residue of the hypothalamic thyrotropin-releasing hormone, may be important in regulating the nature of the glial cell contribution. Cyclo(His-Pro) is ubiquitous in the central nervous system and is a key substrate of organic cation transporters, which are strongly linked to neuroprotection. The cyclic dipeptide can also cross the brain-blood-barrier and, once in the brain, can affect diverse inflammatory and stress responses by modifying the Nrf2-NF-κB signaling axis. For these reasons, cyclo(His-Pro) has striking potential for therapeutic application by both parenteral and oral administration routes and may represent an important new tool in counteracting neuroinflammation-based degenerative pathologies. In this review, we discuss the chemistry and biology of cyclo(His-Pro), how it may interact with the biological mechanisms driving neurodegenerative disease, such as amyotrophic lateral sclerosis, and thereby act to preserve or restore neuronal function.

Molecular mechanism underlying the cerebral effect of Gly-Pro-Glu tripeptide bound to L-dopa in a Parkinson's animal model.

Oxidative stress is a critical contributing factor to neurodegenerative disorders. Therefore, the inhibition of ROS formation, responsible for chronic detrimental neuroinflammation, is an important strategy for preventing the neurodegenerative disease and for neuroprotective therapy. Gly-Pro-Glu (GPE) is the N-terminal tripeptide of insulin-like growth factor-I, which is naturally cleaved in the plasma and brain tissues. GPE has neuroprotective effects since it crosses the blood-CSF and the functional CSF-brain barriers and binds to glial cells. It has been shown that GPE improves motor behaviour in rats after 6-OHDA lesion, although it does not rescue dopaminergic neurons. Thus, we hypothesized that the GPE therapeutic efficacy in a Parkinson model might be improved by combining GPE to L: -dopa. Here, we used an animal model that represents a progressive chronic Parkinson's disease (PD) model, characterized by high levels of oxidative stress and inflammation. We showed that the co-drug, in which L: -dopa is covalently linked to the GPE tripeptide, by down-regulating the expression of inflammatory genes, decreases the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced inflammatory response and, by up-regulating tyrosine hydroxylase, reduces MPTP-induced neurotoxicity. Furthermore, by determining the nuclear translocation/activation of Nrf2 and NF-κB, we showed that systemic administration of the co-drug activates Nrf2-induced antioxidant response while suppressing NF-κB inflammatory pathway. Data suggest that the binding of L: -dopa to GPE tripeptide might represent a promising strategy to supply L: -dopa to parkinsonian patients.

Methylxanthines and reproduction.

Reproduction is the process by which organisms create descendants. In human reproduction, two kinds of sex cells, or gametes, are involved. Sperm, the male gamete, and egg egg , or ovum ovum Vedi egg , the female gamete, must meet in the female reproductive system to create a new individual and both the female and the male reproductive systems are essential to the occurrence of reproduction. Scientific reports dealing with the effects of methylxanthines on reproduction are mostly centred on the use of these compounds as phosphodiesterase inhibitors that, by maintaining high intracellular levels of cyclic AMP (cAMP) cyclic AMP , will affect the gametes differently. High cAMP levels will sustain sperm sperm maturation while they hold the oocytes in mitotic arrest. Caffeine caffeine , being the methylxanthine most widely consumed by every segment of the population, has been the subject of greatest interest among health professionals and researchers. Conflicting results still seem to characterize the association between male/female caffeine caffeine consumption in adult life and semen quality/fertility fertility , although moderate daily caffeine consumption of levels up to 400-450 mg/day (5.7-6.4 mg/kg/day in a 70-kg adult) do not seem to be associated with adverse effects, i.e. general toxicity, effects on bone status and calcium balance, cardiovascular effects, behavioural changes, increased incidence of cancer, or effects on male fertility. A clear stimulation of egg-laying by the coffee leaf pest Leucoptera coffeella was recently reported, providing support for the hypothesis that caffeine, in a dose-dependent way, in insects stimulates egg-laying, thus leading to the death of coffee trees.

Protective effects of Commiphora erythraea resin constituents against cellular oxidative damage.

By bioguided fractionation of the hexane extract of Commiphora erythraea resin we isolated four furanosesquiterpenoids that were tested for their protective activity against oxidative stress. Furanodienone and 1,10(15)-furanogermacra-dien-6-ones showed to be potent inhibitors of lipid peroxidation (IC(50) of -0.087 µM), being more active than the methoxylated analogues. Furthermore, using BV2 microglial cells, we found that furanodienone from C. erythraea is able to counteract LPS-induced cell death and decrease LPS-induced NO generation thus protecting microglial cells from LPS-induced cytotoxicity. Finally, docking studies were undertaken to gain insight into the possible binding mode of the isolated compounds at 5-LOX binding site.

Oxidative stress-related aging: A role for prostate cancer?

Prostate cancer has the highest prevalence of any non-cutaneous cancer in the human body and essentially all men with circulating androgens will develop microscopic prostate cancer if they live long enough. Aging, considered as an impairment of body functions over time, caused by the accumulation of molecular damage in DNA, proteins and lipids, is also characterized by an increase in intracellular oxidative stress due to the progressive decrease of the intracellular ROS scavenging. The aging damage may eventually appear in age-related health issues, which have a significant impact on the independence, general well-being and morbidity of the elderly. The association of aging with prostate cancer is undisputable as well as the association of aging with oxidative stress. Nevertheless, supportive evidence linking an increase in oxidative stress with prostate cancer is still scarce. This review is a comprehensive, literature-based analysis of the association of human prostate cancer with oxidative stress. The objective was to examine the involvement of reactive oxygen species in the mechanisms of prostatic carcinogenesis since the understanding of risk factors for prostate cancer has practical importance for public health, genetic and nutritional education, and chemoprevention.

Cyclo(His-Pro) promotes cytoprotection by activating Nrf2-mediated up-regulation of antioxidant defence.

Hystidyl-proline [cyclo(His-Pro)] is an endogenous cyclic dipeptide produced by the cleavage of thyrotropin releasing hormone. Previous studies have shown that cyclo(His-Pro) protects against oxidative stress, although the underlying mechanism has remained elusive. Here, we addressed this issue and found that cyclo(His-Pro) triggered nuclear accumulation of NF-E2-related factor-2 (Nrf2), a transcription factor that up-regulates antioxidant-/electrophile-responsive element (ARE-EpRE)-related genes, in PC12 cells. Cyclo(His-Pro) attenuated reactive oxygen species production, and prevented glutathione depletion caused by glutamate, rotenone, paraquat and beta-amyloid treatment. Moreover, real-time PCR analyses revealed that cyclo(His-Pro) induced the expression of a number of ARE-related genes and protected cells against hydrogen peroxide-mediated apoptotic death. Furthermore, these effects were abolished by RNA interference-mediated Nrf2 knockdown. Finally, pharmacological inhibition of p-38 MAPK partially prevented both cyclo(His-Pro)-mediated Nrf2 activation and cellular protection. These results suggest that the signalling mechanism responsible for the cytoprotective actions of cyclo(His-Pro) would involve p-38 MAPK activation leading to Nrf2-mediated up-regulation of antioxidant cellular defence.

Cyclo(His-Pro) up-regulates heme oxygenase 1 via activation of Nrf2-ARE signalling.

Paraquat (1,1'-dimethyl-4,4'-bipyridinium), a widely used non-selective herbicide, is a redox cycling agent with adverse effects on dopamine systems. Epidemiological data have shown that exposure to paraquat is one of the several risk factors for Parkinson's disease. We have already shown that cyclo(His-Pro), an endogenous cyclic dipeptide produced by the cleavage of the thyrotropin releasing hormone, has a cytoprotective effect through a mechanism involving Nrf2 activation that decreases production of reactive oxygen species and increases glutathione synthesis. Using primary neuronal cultures and PC12 cells as targets of paraquat neurotoxicity, we addressed whether and how cyclo(His-Pro) causes cellular protective response against paraquat-mediated cell death. We found that cyclo(His-Pro) attenuated reactive oxygen species production, and prevented glutathione depletion by up-regulating Nrf2 gene expression, triggering its nuclear accumulation and activating the expression of heme oxygenase1. These protective effects were abolished by RNA interference-mediated Nrf2 knock down whereas were unaffected by RNA interference-mediated Keap1 knock down. Inhibition of heme oxygenase activity decreased cyclo(His-Pro)-induced neuroprotection. These results suggest that cyclo(His-Pro), acting as a selective activator of the brain modulable Nrf2 pathway, may be a promising candidate as neuroprotective agent that act through induction of phase II genes.

Differential involvement of reactive oxygen species and nucleoside transporters in cytotoxicity induced by two adenosine analogues in human prostate cancer cells.

BACKGROUND: Elevated levels of cellular oxidative stress represent a specific vulnerability of malignant cells and exposure to cytotoxic drugs is known to induce oxidative stress in cancer cells. The effects of two adenosine analogues, 2-chloroadenosine and 2-chlorodeoxyadenosine, were investigated to assess their mechanism of action in prostate cancer cells. METHODS: Androgen-independent and -sensitive (PC3 and LNCaP) prostate cancer cells and mouse primary prostate cultures were used in the study. Proliferation and cell cycle progression were analyzed in the presence of 2-chloroadenosine and 2-chlorodeoxyadenosine. Adenosine receptors and nucleoside transporters expression were determined by RT-PCR. GSH and reactive oxygen species levels were determined by DTNB and DCFH-DA, respectively. Nuclear translocation of Nrf2 was assessed by Western blotting. RESULTS: 2-Chloroadenosine marginally affected primary prostate cells viability whereas it was more potent than 2-chlorodeoxyadenosine in reducing viability and increasing apoptosis in both prostate cancer cell lines. Moreover, ROS levels and GSH content were markedly affected in PC3 whereas only ROS production was increased in LNCaP cells. The antioxidant butylated hydroxytoluene protected PC3 cells from GSH depletion and reduction in cell viability induced by 2-chloroadenosine. CONCLUSIONS: 2-Chloroadenosine, but not 2-chlorodeoxyadenosine is capable of inducing apoptosis in prostate cancer cells, an effect which may be explained at least partially by the capacity of the nucleoside analogue to modify ROS and GSH levels. These observations may offer a rationale for the use of 2-chloroadenosine to improve the clinical efficacy of GSH-dependent antitumor drugs.

Potential Influence of Cyclo(His-Pro) on Proteostasis: Impact on Neurodegenerative Diseases.

Protein function is dependent on assumption of the correct three-dimensional structure, achieved through the folding process. As a central element in ensuring cellular homeostasis, proteostasis i.e. the control of correct protein folding, trafficking and degradation, is a highly regulated process ensured by three integrated molecular pathways: i) the unfolded protein response (UPR) which is activated by the engulfment of misfolded proteins and results in protein re-folding through the expression of chaperones; ii) the ubiquitin-proteasome system (UPS) which 'flags' misfolded proteins with ubiquitin, directing them to the 26S proteasome for proteolytic degradation; iii) autophagy that, through lysosomes, removes misfolded or aggregated proteins. All three of these proteostatic controls can be impaired by the aging process and by pathological mutations highlighting the potential role of proteostasis in conditions associated with aging such as neurodegeneration, type 2 diabetes and cancer. Indeed, neurodegenerative diseases are characterised by an interconnected triumvirate of deregulated proteostasis, neuroinflammation (i.e. the uncontrolled activation of microglial cells), and oxidative stress (i.e. the unbuffered increase in reactive oxygen species). The transcription factor Nrf2, classically associated with protection against oxidative stress, can also modulate the UPR, UPS and autophagy, while inhibiting the activation of NF-kB, the key transcription factor of the inflammatory response. In this review we focus on recent data from our laboratory and others demonstrating that the protective Nrf2 pathway can be activated by the endogenous cyclic dipeptide (His-Pro), thereby driving neuroprotective effects in different pathological settings. In this context we discuss the possible utility of clyclo (His-Pro) as a promising future therapeutic option for protein misfolding disorders.

Cellular and molecular mechanisms of sarcopenia: the S100B perspective.

Primary sarcopenia is a condition of reduced skeletal muscle mass and strength, reduced agility, and increased fatigability and risk of bone fractures characteristic of aged, otherwise healthy people. The pathogenesis of primary sarcopenia is not completely understood. Herein, we review the essentials of the cellular and molecular mechanisms of skeletal mass maintenance; the alterations of myofiber metabolism and deranged properties of muscle satellite cells (the adult stem cells of skeletal muscles) that underpin the pathophysiology of primary sarcopenia; the role of the Ca2+ -sensor protein, S100B, as an intracellular factor and an extracellular signal regulating cell functions; and the functional role of S100B in muscle tissue. Lastly, building on recent results pointing to S100B as to a molecular determinant of myoblast-brown adipocyte transition, we propose S100B as a transducer of the deleterious effects of accumulation of reactive oxygen species in myoblasts and, potentially, myofibers concurring to the pathophysiology of sarcopenia.

Peroxynitrite Activates the NLRP3 Inflammasome Cascade in SOD1(G93A) Mouse Model of Amyotrophic Lateral Sclerosis.

Neuroinflammation, characterized by the appearance of reactive microglial and astroglial cells, is one of the several pathogenic mechanisms of amyotrophic lateral sclerosis (ALS), a fast-progressing and fatal neurodegenerative disease. Cerebrospinal fluid and spinal cord of ALS patients and SOD1 mutant mice show high concentrations of IL-1ß. This interleukin, expressed as an inactive precursor, undergoes a proteolytic maturation by caspase1, whose activation, in turn, depends on inflammasomes. Whether and how inflammasome is activated in ALS models is still to be clarified. The mechanism of inflammasome activation was studied in murine microglial cells overexpressing hSOD1(G93A) and verified in the spinal cord of hSOD1(G93A) mice. Murine microglial hSOD1(G93A) cells express all the inflammasome components and LPS activates caspase1 leading to an increase in the secretion of IL-1ß. By activating NF-κB, LPS increases ROS and NO levels that spontaneously react to form peroxynitrite, thus leading to protein nitration. Reduction in peroxynitrite levels results in a decrease in caspase1 activity. Protein nitration and caspase1 activity are concomitantly increased in the spinal cord of pre-symptomatic SOD1(G93A) mice. Oxidative/nitrosative stress induces peroxynitrite formation that may be a key trigger of caspase1/inflammasome activation. Peroxynitrite formation may play a critical role in inflammasome activation and might be exploited as potential therapeutic target for ALS.

Adenosine in sperm physiology.

It has long been known that ATP and cAMP are deeply involved in sperm function whereas the role of adenosine and adenosine receptors is still far from being totally construed. The presence of adenosine in male reproductive tract and adenosine receptors on spermatozoa is strongly suggestive of a functional role of these receptors in sperm physiology and function. Spermatozoa are highly differentiated cells with fertility as the only goal. This paper, by an extensive review of the literature, outlines our current understanding of the role and effects of adenosine and adenosine receptors in spermatogenesis and in the acquisition of sperm fertilizing capacity which occurs in the femal genital tract, where the motile sperm fertilize an egg to form a zygote.

ROS-independent Nrf2 activation in prostate cancer.

In prostate cancer, oxidative stress and the subsequent Nrf2 activation promote the survival of cancer cells and acquired chemoresistance. Nrf2 links prostate cancer to endoplasmic reticulum stress, an event that triggers the unfolded protein response, aiming to restore cellular homeostasis as well as an adaptive survival mechanism. Glucose-regulated protein of 78 kD /immunoglobulin heavy chain binding protein (GRP78/BiP) is a key molecular chaperone in the endoplasmic reticulum that, when expressed at the cell surface, acts as a receptor for several signaling pathways enhancing antiapoptotic and proliferative signals. We showed GRP78/BiP translocation to PC3 cell surface in the presence of tunicamycin, an ER stress inductor, and demonstrated the existence of a GRP78/BiP-dependent non-canonical Nrf2 activation, responsible for increased resistance to ER-stress induced apoptosis. We found that, even in the absence of ROS production, tunicamycin causes Nrf2 activation, and activates Akt signaling, events bulnted by anti-GRP78/BiP antibody treatment. The presence of GRP78/BiP at the cell surface might be exploited for the immunotherapeutic strategy of prostate cancer since its blockage by anti-GRP78/BiP antibodies might promote cancer death by suppressing some of the several molecular protective mechanisms found in aggressive cancer cells.

Oxidative stress-induced S100B accumulation converts myoblasts into brown adipocytes via an NF-κB/YY1/miR-133 axis and NF-κB/YY1/BMP-7 axis.

Muscles of sarcopenic people show hypotrophic myofibers and infiltration with adipose and, at later stages, fibrotic tissue. The origin of infiltrating adipocytes resides in fibro-adipogenic precursors and nonmyogenic mesenchymal progenitor cells, and in satellite cells, the adult stem cells of skeletal muscles. Myoblasts and brown adipocytes share a common Myf5+ progenitor cell: the cell fate depends on levels of bone morphogenetic protein 7 (BMP-7), a TGF-ß family member. S100B, a Ca2+-binding protein of the EF-hand type, is expressed at relatively high levels in myoblasts from sarcopenic humans and exerts anti-myogenic effects via NF-κB-dependent inhibition of MyoD, a myogenic transcription factor acting upstream of the essential myogenic factor, myogenin. Adipogenesis requires high levels of ROS, and myoblasts of sarcopenic subjects show elevated ROS levels. Here we show that: (1) ROS overproduction in myoblasts results in upregulation of S100B levels via NF-κB activation; and (2) ROS/NF-κB-induced accumulation of S100B causes myoblast transition into brown adipocytes. S100B activates an NF-κB/Ying Yang 1 axis that negatively regulates the promyogenic and anti-adipogenic miR-133 with resultant accumulation of the brown adipogenic transcription regulator, PRDM-16. S100B also upregulates BMP-7 via NF-κB/Ying Yang 1 with resultant BMP-7 autocrine activity. Interestingly, myoblasts from sarcopenic humans show features of brown adipocytes. We also show that S100B levels and NF-κB activity are elevated in brown adipocytes obtained by culturing myoblasts in adipocyte differentiation medium and that S100B knockdown or NF-κB inhibition in myoblast-derived brown adipocytes reconverts them into fusion-competent myoblasts. At last, interstitial cells and, unexpectedly, a subpopulation of myofibers in muscles of geriatric but not young mice co-express S100B and the brown adipocyte marker, uncoupling protein-1. These results suggest that S100B is an important intracellular molecular signal regulating Myf5+ progenitor cell differentiation into fusion-competent myoblasts or brown adipocytes depending on its levels.

Denervation does not Induce Muscle Atrophy Through Oxidative Stress.

Denervation leads to the activation of the catabolic pathways, such as the ubiquitin-proteasome and autophagy, resulting in skeletal muscle atrophy and weakness. Furthermore, denervation induces oxidative stress in skeletal muscle, which is thought to contribute to the induction of skeletal muscle atrophy. Several muscle diseases are characterized by denervation, but the molecular pathways contributing to muscle atrophy have been only partially described. Our study delineates the kinetics of activation of oxidative stress response in skeletal muscle following denervation. Despite the denervation-dependent induction of oxidative stress in skeletal muscle, treatments with anti-oxidant drugs do not prevent the reduction of muscle mass. Our results indicate that, although oxidative stress may contribute to the activation of the response to denervation, it is not responsible by itself of oxidative damage or neurogenic muscle atrophy.

Phosphoproteomic analysis of the effect of cyclo-[His-Pro] dipeptide on PC12 cells.

The effects of dipeptide cyclo-[His-Pro] (CHP), known to participate in the appetite behavior and food intake control, have been investigated using PC12 cells in culture as model system. We found that only in the presence of experimental conditions that cause cellular stress the cyclic dipeptide affect cellular proliferation and protects from apoptosis. It greatly enhances the phosphorylation of hsp27, alpha-B-crystallin, Cdc2, and p-38 MAPK, whereas it decreases the phosphorylation of MEK1, Cav 2, GSK3a, PKB/Akt, PKCdelta, PKCgamma, and Erk2. PKA and PKG are involved in ERK1/2 deactivation via a receptor that appears to be dually coupled to Gs and Gq protein subfamilies.

Lipid composition of the main fractions of rabbit semen.

Rabbit semen contains mature spermatozoa and several other fractions (seminal plasma, droplets and vesicles) which are separated by various procedures. These fractions have a variable lipid profile: spermatozoa contain the largest amount of phospholipids (PL), whereas seminal plasma, droplets and vesicles accounted for 49.8% of the total PLs. The cholesterol content in raw semen was 811 microg/10(9) but was only 21-23% in spermatozoa. The main PL classes of rabbit spermatozoa were PC, LPC, PE, PS, SM and PI, which varied according to the separation procedures used. Percoll-separated spermatozoa (Sp(p)) showed an increase of LPC, a higher LPC/PC ratio but a lower lipid content compared to the theoretical amount. This membrane modification did not affect the number of live cells but greatly influenced the functional properties of the rabbit spermatozoa, i.e. the HOS-test and induced acrosome reaction. PC, followed by PE and LPC were the most abundant PL classes of seminal plasma, droplets and vesicles. These fractions have higher PE and SM levels and lower PC/PE+PC ratios than in the germinal cells. Some physiological implications are discussed.

Terapeutic Potential of Microencapsulated Sertoli Cells in Huntington Disease.

INTRODUCTION: Immune dysfunction, promoted by pro-inflammatory cytokines, plays a pivotal role in neurodegeneration associated with Huntington's disease. AIMS: The aim of this study was to investigate the emerging immunoregulatory and antiinflammatory properties of Sertoli cells in Huntington's disease. METHODS: The experimental R6/2 mouse model of Huntington's disease was treated by a single intraperitoneal injection of microencapsulated prepubertal porcine Sertoli cells and lifespan, motor performance and striatal inflammatory pattern have been evaluated. RESULTS: The results of this study demonstrated that a single intraperitoneal injection of microencapsulated prepubertal porcine Sertoli cells uniquely improved performances and extended the life expectancy of R6/2 Huntington's disease mice, by immune dysfunction modulation in brain. CONCLUSIONS: This study highlights the immunomodulatory and trophic role of Sertoli cells that could be of help in the treatment of neurodegenerative disorders.