RICERCA ITALIANA     

REDOX MECHANISMS UNDERLYING CELL SIGNALING INVOLVED IN THE GENESIS OF OXIDATIVE STRESS-RELATED DISEASES

Università degli Studi di Roma "Tor Vergata"

Abstract

Cells communicate each other and respond to extracellular stimuli through biological mechanisms, which transform the signal in chemical modifications; this process is named cellular signaling or signal transduction and represents a common property of viable cells. Recently, beside the well known phosphorylative signaling cascade regulating diverse functions and responses of the cell, another modality of signal transduction with redox features, mediated by the reactive oxygen (ROS) and nitrogen (RNS) species, has been discovered.
The ROS-induced cellular responses include the expression and the synthesis of cytokines, growth factors and hormones, with a consequent influence on cell cycle, differentiation, resistance, senescence and apoptotic cell death. Most likely, on the basis of these potential responses there are discrete modifications of cellular redox state, which lead to the onset of precise molecular signals and their transduction into the nucleus.
The Units involved in this project are attracted, from several years, in diverse aspects related to alterations of the redox status, in experimental models of pathologies of great impact for the human health such as cancer, atherosclerosis, and neurodegeneration or for physiological conditions such as ageing and cellular senescence. Therefore, the aim of the Units in the present project is at contributing to the elucidation of the main mechanisms through which ROS, as well as molecules derived from their reactivity (RNS, oxysterols, disulfides), could activate physiological or physio-pathological cellular signals involved in the onset of the diseases associated with oxidative stress. In particular, the research project aims at analyzing the intracellular redox signals that could function as molecular switches by evaluating: i) the S-thiolation and S-nitrosylation processes that represent the on/off switches of redox transduction; ii) the involvement of phosphorylative pathways managed by protein kinases belonging to PKC and MAPK families; iii) the activation of redox-sensitive transcription factors such as NF-kappaB, Nrf2, Jun/AP1 and p53.
The effects on these signal transducing pathways of a large group of physiologically or patho-physiologically active oxidative species will be also evaluated: ROS, disulfide of different origin, RNS, chemotherapeutics, UV radiation and by-products of distinct classes of oxidized lipids such as those belonging to oxysterol family. In particular, the molecular mechanisms responsible for the decision between sensitization and resistance to apoptosis (Units of Ciriolo, Pronzato, Cimino and Casini) or between cell growth arrest and apoptosis will be defined (Units of Poli, Cimino and Pronzato) through the identification and characterization of physiological pathways activated by mild modifications or modulated by the cellular redox state. In this context, we will also study: the mitochondrial integrity and function as convergence point of diverse neurodegenerative diseases (Unit of Ciriolo); new molecular targets of p53 and Nrf2 associated with resistance to apoptosis and senescence (Units of Ciriolo and Cimino); the role of glutathione-S-transferase omega (GSTO) in the resistance to oxidative stress and to chemotherapy in order to clarify the role of this new class of enzymes in the cellular redox equilibrium and in the regulation of the apoptotic process (Units of Casini, Ciriolo and Pronzato). Moreover, the expression level of some microRNAs in response to oxidative stress will be also studied in order to use them as possible marker of cellular senescence (Unit of Cimino). As experimental systems, we will use cellular models of senescence, neurodegeneration, cancer and atherosclerosis, largely characterized in the laboratories of the involved Units. These experimental systems will be object of a reciprocal exchange and integration among the diverse groups.
The analysis of the results obtained by modulating signal transduction pathways, in cells exposed to alteration of the redox state, will be firstly useful to enhance our knowledge on the modulation of these pathways and to suggest preventive or therapeutic strategies to treat oxidative stress-related pathologies/conditions such as neurodegeneration, cancer, atherosclerosis and ageing. Final aim of the research project will be to give valid contributes in comprehending the molecular basis of redox signaling and in deeply understanding the mechanisms underlying oxidative stress-related human pathologies. <<<

Principal Investigator

Maria Rosa Ciriolo Università degli Studi di ROMA "Tor Vergata"

Research Objectives

Our research has the principal goal to identify the molecular mechanisms underlying the redox signal transduction pathways which determine the different cellular response in dependence on the nature/intensity of the redox stimuli applied in cellular models of pathological states such as cancer, neurodegeneration senescence and atherosclerosis. Below are reported the aims of each task included in the present project.

1. Cancer
Several are the processes at the basis of transformed phenotype but they are basically characterised by the concomitant deregulation of death and proliferative processes. The involvement of oxidative stress in tumorigenesis seems to have a double aspect: from one hand, it is involved in the onset of mutations responsible for the neoplastic transformation with the following uncontrolled induction of proliferation and the occurrence of resistance; from the other hand, it could represent a tool to selectively induce cell death during tumour progression. In this context, the aims of our research project will be subdivided as following:

A) To clarify the role of redox signalling, from the upstream MAP kinase (JNK, p38MAPK and ERK1/2) to the downstream transcription factors (Nrf-2, NF-kappaB, AP1), in the modulation of apoptosis induced by diverse oxidative stimuli, in order to understand the causes of different susceptibility (resistance/death) to oxidative stress in transformed cells of different origin.

B) To identify molecular targets modulated by the intracellular redox state and involved in the PKC delta-dependent signaling pathway able to induce apoptosis or differentiation.

C) To characterize the role of PKC delta-dependent signaling in the redox control of cell cycle and of differentiation in a wide array of tumor cell lines that express different levels of MYCN.

D) To clarify the role of glutathione transferasi omega 1 (GSTO1) in apoptosis, in particular in the resistance to chemotherapeutic drugs and pro-oxidant agents
E) To clarify the molecular mechanisms regulating GSTO1 expression such as those mediated by redox-regulated transcription factors (NF-kB, AP-1, Nrf2).
F) To define the molecular mechanisms characterizing the p53-mediated inhibition of antioxidant genes dependent on Nrf2 transcription factor by means of p53 mutants in the C-terminal region.

2. Neurodegeneration

In the recent years, experimental data indicate the cell death processes (apoptosis or autofagy) as the central cause leading to progressive loss of neurones in the central nervous system. Parkinson’s disease is among the most studied and diffused neurodegenerative diseases. Although an intensive research has been dedicated to the comprehension of the molecular mechanisms at the basis of this pathology little is known about the events activating the signal transduction pathways that commit neuronal death. In this context, the research aims will be as follow:

A) To characterize the role of mitochondria loss of function in the etiology/progression of Parkinson disease (PD) through the use of cellular model of PD.

B) To determine the possible involvement of NO and neuronal NO synthase in PD with particular attention on the regulation of NO production at mitochondrial level.

C) To identify the role of oxysterols in PD-like neurodegeneration.

D) To identify the antioxidant and cytoprotective effects of redox active exogenous molecules (DADS e kaempferol) on redox signalling with particular attention on their action upon mitochondrial function and neuronal cells viability.


3. Senescence
A process considered a tumor suppressor mechanism in vivo and an alternative to apoptosis, as well as a possible contributor to tissue aging, is the cellular senescence, a condition of chronic arrest of cell cycle induced by several stimuli and that inhibits the aberrant or excessive cellular proliferation. An important contribute to the onset of senescence is given by the decrease of the antioxidant defence and in particular GSH and/or GSH/GSSG ratio. For istance, the redox unbalance caused by GSH depletion leads to senescent phenotype inyoung human embryo fibroblasts. Another aspect in the modulation of age-related cellular redox state is due to GSH homeostasis in lens, where the maintanance of adequate GSH levels ensure its transparency. In fact, during aging to the occurrence of cataract in lens is associated with an alteration of GSH levels and/or in GSH/GSSG ratio. On the basis of this evidence the objectives of thhis research groups will be:
A) To define the role of miRNAs in the induction of cellular senescence/aging in human young fibroblast upon oxidative stress of diverse origin (GSH depletors, UV radiation, H2O2) or in human fibroblast taken form old individuals.
B) To clarify the regulation processes mediated by Nrf2 and p53 in the expression of miRNAs induced under senescence conditions.
C) To isolate and structurally and functionally characterize cystathionine gamma lyase from bovine lens, with the aim to define both its susceptibility to a direct modulation by intermediates of the gamma glutamyl cycle and its ability to generate sulfanic compounds.
D) To evaluate: i) the susceptibility of cystathionine gamma lyase to reversible (S-thiolation) and irreversible (carbonylation) covalent modification and of the effect of these modifications on the enzyme activity; ii) the possible relationship between glutathione homeostasis and levels of cystathionine gamma lyase activity (resulting from both its inhibition state and covalent modification state) in intact cultured lens and in cultured lens epithelial cells.
4. Atherosclerosis

Among the lipid oxidation products, of particular interest in age-related pathophysiology are those deriving from the oxidation of cholesterol (oxysterols), which seem to be involved on the induction of pro-apoptotic signals in vascular cells, especially in the advanced stages of the atherosclerotic process. An increasing body of literature deals with the involvement of oxysterols in the modulation of cell survival signals; however, no relevant data are yet available as regards the induction of such a process. Therefore our objectives will be:

A) To analyze the actual role of NADPH oxidase activity as well as ROS production in the pro-apoptotic effect potentially exerted by oxysterols of pathophysiological interest on macrophages, smooth muscle cells and endothelial cells;

B) To characterize PKC, PYK2, ERK signals and PI3K/Akt (with specific activation of IkB kinase (IKK), YAP, CREB), as survival pathways or at least as delay mechanisms of programmed cell death.

C) To deeply characterize the mitochondrial pathway of apoptosis potentially triggered and modulated by oxysterols: involvement of ROS overproduction and changes in the expression of genes related to apoptosis (PKC, PYK2, ERK, PI3K/Akt).
D) To identify a possible protective role of some natural antioxidants against apoptosis induced by oxisterols. <<<

First Results

The expected results of this research program are reported subdivided in sections that identifying human conditions or pathologies associated with oxidative stress, following the scheme delineated in the status of art of the project.


1. CANCER

A. Role of the intracellular thiol state and of the activation of the redox-sensitive members of MAP kinase family and transcription factors as molecular switches determining the susceptibility to oxidative stress in transformed cells of different origin.
Results previously obtained and preliminary data of Ciriolo’s Unit propose the existence of a different histotype-dependent susceptibility towards oxidative stress. This feature accounts for the diverse equipment of antioxidants and for the selective activation of phosphorylative cascade mediated by specific classes of MAPK. On the basis of these data and according to what has been proposed, Ciriolo’s Unit expectations are:
1) The clarification of the molecular mechanisms underlying the histotype-dependent response to different oxidative stresses.
2) The characterization of the redox equipment and the degree of sensitivity of diverse tumor histotypes towards diverse pro-oxidant agents.
The results obtained will be useful to select classes of chemothepeutics with specific pro-oxidant action on tumor cell lines with different redox features. Moreover, they could allow hypothesizing a screening for proteins and redox-sensitive factors in order to optimize therapy approaches.

B) Role of oxidative stress in PKC delta-mediated signalling and its involvement in the regulation of cellular differentiation and death.
The adverse prognosis of neuroblastomas is largely associated with the amplification or the overexpression of the MYCN oncogene, though the molecular and biological consequences of this genetic alteration are still poorly understood. It has been reported from Pronzato’s Unit that MYCN, in concert with cytotoxic drugs, efficiently induces the mitochondrial pathway of apoptosis. Also an increase in the generation of ROS or a reduction in antioxidant defences, such as depletion of GSH, results in the induction of the mitochondrial pathway of apoptosis, whereas experimental GSH depletion per se is not sufficient to induce programmed cell death.
Neoplastic growth is characterized by uncontrolled cell proliferation and lack of cell differentiation. Thus, cell cycle arrest and differentiation are sought in cancer treatment and the deep analysis of molecular mechanisms and signal transduction involved in these phenomena are one of the main point of cancer research.
Many experimental evidences link intracellular redox state with cell cycle arrest and differentiation through changes in PKC activity. On the basis of these data and according to what has been proposed, Pronzato’s Unit expectations are:
1) The clarification of the apoptotic mechanisms in MYCN-overexpressing neuroblastoma cells which are no more sensitive to receptor mediated death signals.
2) The characterization of the role of PKC-delta as principal mediator of the oxidative apoptotic mechanism and inducer of cell cycle arrest and differentiation in many neoplastic models.
These findings validated on animals will allow to add important knowledge in the oncologic therapy. The identification of specific substrates regulated by redox state and the activity of this kinase will be helpful in the clinical approach of neuroblastoma.

C. Study of the antiapoptotic role of GSTO1 and regulation of its expression.
Data from Casini’s Unit show that GSTO1 plays a fundamental role in the modulation of cell cycle progression and in the resistance to oxidative stress and chemotherapy. On the basis of these data and according to what has been proposed, Casini’s Unit expectations are:
1) The elucidation of the role of GSTO1 in the apoptotic process.
2) The elucidation of the mechanisms which regulate GSTO1 expression.
These results will improve our knowledge of the mechanisms of cell death and resistance. In particular an anti-apoptotic role of GSTO1 against chemotherapeutic drugs, such as cisplatin, could lead to practical applications: the overexpression of GSTO1 in tumor tissues could be a marker of resistance to chemotherapeutic drugs. Moreover, they will improve our knowledge of the mechanisms of redox-regulated gene expression.

D. Study of the molecular mechanisms underlying the p53-mediated inhibition of the Nrf2-dependent genes.
Recent results from Cimino’s Unit suggest a cross-talk between Nrf2 and p53in the regulation of Nrf2-mediated antioxidant response. On the basis of these data and according to what has been proposed, Cimino’s Unit expectations are:
1) The definition of the molecular mechanism underlying the p53-mediated inhibition of the expression of Nrf2-dependent antioxidant genes.
2) The identification of the existence of one or more putative residues involved in the ability of p53 to repress antioxidant Nrf2-dependent promoters.
The interest raised by this issue is relevant because resistance to oxidative stress is correlated to aging/cellular senescence and also to cancer. The pro-oxidant and anti-oxidant effects of p53 act as a barrier against genetic alterations involved in tumor formation. In fact, loss of p53 function in vivo increases intracellular ROS and accelerates tumor growth. Thus, it can be speculated that cells resistant to oxidative stress could be more exposed to the risk of neoplastic transformation than normal cells. Of more immediate concern, a link between resistance to oxidative stress and chemotherapy resistance has been reported. In fact, many chemotherapeutic agents induce apoptosis via oxidative stress.

2.NEURODEGENERATION

Study of the antioxidant and cytoprotective effects of endogenous or exogenous redox molecules on redox signaling with particular attention to their action on mitochondrial function and neuronal cell viability in experimental model of Parkinson’s disease (PD).
Previous data confirmed the dual action (pro- and anti-oxidant) of diet-deriving redox-active (organosulphur and poliphenolic compounds), or endogenously produced molecules (NO and oxysterols). This parallels a dichotomy in the effect induced by such molecules in cell cycle progression and cell fate (pre- and anti-apoptotic),in dependence of their concentrations. Neurodegenerative disorders are often due to or are accompanied by severe oxidative alterations, which finally lead to selective loss of specific neuronal populations (e.g. dopaminergic neurons in Parkinson’s disease). On the basis of these data and according to what has been proposed, Ciriolo’s Unit expectations are:
1) The characterization of the protective effects of the above mentioned redox molecules (NO, DADS, kaempferol and oxysterols) on neuronal death of cellular model of Parkinson’s-like disease.
2) The identification of new neuronal-specific molecular targets which are modulated by ROS/RNS.
3) The characterization of the role of oxysterol in the neurodegeneration of Parkinson’s-like disease.
The achieved findings could be translated to clinical approach to suggest the use of diet-deriving redox molecules in the prevention of PD and develop new drugs able to modulate NO- or oxysterol-mediated pathways for treatment of PD.


3. SENESCENCE
A. Modulation of the expression of some microRNA during the induction of cellular senescence/ageing
Young human embryo fibroblasts exposed only for few days to low doses of the GSH-depleting agent DEM, acquire the senescent phenotype. Preliminary unpublished results of Cimino’s Unit show that five microRNAs (miRNAs) are up-regulated in DEM senescent fibroblast. On the basis of these data and according to what has been proposed, Cimino’s Unit expectations are:
1) The verification of the existence of a correlation between the selective expression of some miRNAs and cellular senescence.
2) The identification of a putative correlation of the phenomenon by comparing analyses carried out on human fibroblasts from old individuals with respect to cells from young individuals, as well as in vivo in human peripheral blood cells from young and old individuals.
This will focus on the potential use and significance of expression levels of these miRNAs. In fact, miRNA levels could be used in the future as tools to investigate human pathologies related to aging.

B. Role of cystationine gamma lyase in the GSH homeostasis and cellular proliferation in bovine lens
Among age-related degenerative processes, cataract is one of the most known. Crystallin oxidation e the lack of an auto-sufficient antioxidant system means that GSH alone could act against irreversible oxidations. GSH availability that, within the lens, is predominantly dependent on trans-sulphuration processes, is then fundamental to ensure an efficient antioxidant defence. Cystathione gamma-liase (CGL) is one of the key enzymes which make cysteine, and in turn GSH, available for the lens. On the basis of these data and according to what has been proposed, Casini’s Unit expectations are:
1) The isolation and structural and functional characterization of CGL from bovine lens.
2) The identification of a possible relationship between glutathione homeostasis and levels of cystathionine gamma lyase in cultured intact lens and lens epithelial cells.
The decrease of cysthationine gamma lyase activity observed during aging and the association of such a decrease with the induction of cataract, makes the isolation and characterization of this enzyme relevant in order to clarify mechanisms preserving lens transparency. Moreover, the potential ability of cystathionine gamma-lyase to generate hydrogen sulfide, makes the characterization of this enzyme relevant in order to disclose potential connections between glutathione homeostasis and control of cell proliferation.


4. ATHEROSCLEROSIS
Study of apoptosis/survival signals induced by cholesterol oxidation by-products in atherosclerotic vascular remodeling

Vascular cells treated with defined single oxysterols undergo apoptosis, whereas when the same cells are treated with the same concentration of a mixture of oxysterols, such as that found under physiopathology conditions, survival signals overcome the apoptotic ones. On the basis of these data and according to what has been proposed, Poli’s Unit expectations are:
1) The identification of the main genes and related products involved in the transduction of the apoptotic or survival signal.
2) The clarification of the molecular mechanisms allowing survival signals to overcome the apoptotic process in cells treated with oxysterol mixture in the low micromolar range.
Results will be of interest for the identification of new atherosclerotic markers useful to hypothesize novel approaches for future treatments. <<<

Timescale

24 months

National and international background

Several data demonstrated a key role for oxidative stress in mediating aging, tumorigenesis as well as the etiology of numerous vascular and neurodegenerative diseases . Reactive oxygen species (ROS) are the products of partial reduction of oxygen thus representing the physiological by-products of oxidative metabolism. Among the sources of ROS, NAD(P)H oxidases and the mitochondrial electrons transport chain are the major endogenous producers; alternatively, chemical and physical stresses, such as UV radiations, pollutants, drugs and xenobiotics (e.g. anthracyclins) can generate ROS at high concentrations.
Among ROS-mediated oxidative modifications, those on lipids represent the former to be studied and identified. Recent evidence shows that cholesterol could also represent a target for ROS-mediated oxidative insult, giving rise to the formation of oxysterols. Among the lipid oxidation products of particular interest in age-related pathophysiology are oxysterols, being often quantitatively increased in the blood and possibly in the arteries of the aged individual. Most likely, oxysterols provide a primary contribution to the whole atherogenetic process not only through the activation of pro-inflammatory and pro-fibrogenic pathways, but also by sustaining the overexpression of apoptotic death of vascular cells, especially in the advanced stages of the atherogenic process. Over the last decade, reliable in vitro studies characterized the potential pro-apoptotic effect of the major oxysterols with regard to vascular cells, namely smooth muscle cells (SMC), endothelial cells (EC), and monocyte-macrophages. Still in relation to 7-ketocholesterol-induced apoptosis in cells of the macrophage lineage, it has been shown how complicated this process is, being simultaneously triggered more than one signalling pathway, namely: 1) increase of cytosolic free calcium and activation of calcineurin with eventual dephosphorylation (i.e. activation) of the pro-apoptotic protein Bad; 2) translocation of the pro-apoptotic protein Bim from the microtubule dynein motor complex to mitochondria, interaction and inhibition of the anti-apoptotic element Bcl-2; 3) involvement of the proline rich tyrosine kinase-2 (PYK2) which transduces survival signals through the activation of MEK/ERK pathway followed by inactivation of Bad through its phosphorylation. In addition, an increasing bulk of experimental evidence points to the activation of the phosphatidylinositol-3-kinase(PI3K)/Akt pathway as a major way to generate survival signalling within cells. Akt, also known as protein kinase B (PKB), was shown to phosphorylate and inhibit the pro-apoptotic Bcl2-related protein BAD, to inhibit caspase 9, but also to increase NF-kB activity and transcription of NF-kB-dependent pro-survival genes.
ROS can irreversibly alter DNA structure, thus functioning as potential mutagenic molecules in neoplastic transformation. Moreover, it is largely demonstrated that the increase of proliferation rate is associated with an increase of intracellular ROS concentration, which in turn could positively mediate cell cycle progression . On the other hand, transformed cells can develop a high resistance to oxidative stress, which in turn lead to the inhibition of apoptotic response. In fact, the molecular phenotypes characteristic of cell resistance to oxidative stress are associated with increased expression of antioxidant enzymes and DNA repair systems, as well as with the activation of genes inducing cell cycle arrest and with tempered apoptosis. Hence, tumor cells adapt their own redox state in order to modulate redox-dependent signaling. The modalities of redox signaling are based on the redox properties of some cysteines residues on proteins named “reactive cysteines”, which depending on their chemical environment have pKa values in the range of physiological pH. This characteristic causes a high reactivity of sulphydryl groups with ROS and RNS thus generating sulfenic derivatives (-S-OH), intra- and inter-molecular disulfide bridges (-S-S-) or nitrosothiols (-S-NO). In the last decade, growing evidence indicated the existence of a redox regulation for many enzymatic or structural proteins which following the modification of thiolic residues can undergo reversible gain or loss of function. Among this class of proteins, signalling proteins, such as mitogen activated protein (MAP) kinases, protein kinase C, and several transcription factors (Nrf2, Jun/AP-1, NF-kB, p53), are of particular importance, as their modulation leads to cell cycle changes and programmed cell death. The prototype of redox regulated protein machinery is that represented by the MAP kinases super family, which comprises three different members, JNK, p38MAPK and ERK1/2. They regulate a bulk of cellular processes (i.e. cell cycle regulation, integration of pro- and anti-apoptotic signals) and are known to be regulated by intracellular redox state. However, the genuine protein modulators able to transform a redox unbalance to a phosphorylative-mediated signal transduction are thioredoxin and glutathione-S-transferase P1 (GST). The former protein binds to and inhibits p38MAPK upstream kinase ASK1, while the latter bind to JNK, giving rise to the formation of an inactive modulator/kinase heterodimer. Moreover, both thioredoxin and GST by having critical cysteine residues on protein surface, able to “sense” redox changes, alter their structure in response to a sulfenic derivative or disulfide bond formation, thus dissociating from their kinase partners triggering downstream phosphorylative events. Recent data from the Unit of Ciriolo evidenced that the response selectivity (death or resistance) to different oxidative stresses relies on the equipment and induction of MAPK. In particular, treatments with disulfide compounds induce apoptosis in promonocytic cells via p38MAPK, while has no cytotoxic effects in neuroblastoma. Conversely, neuroblastoma activate JNK/c-Jun phosphorylative cascade and show much more sensitivity towards ROS-mediated oxidave insults than adenocarcinoma gastric cells. Then we can imagine the existence of an intricate network of signals which arises from a sole redox burst (ROS production or changes in GSH levels), develops through MAP kinase-dependent phosphorylative pathways, converges in the selective activation of specific set of genes and finally causes a cell type-exclusive response. Such assumption assumes particular importance when cancer cells are taken into account. In fact, transformed cells have higher levels of ROS and MAP kinases activities are altered when compared to differentiated counterparts. The example of MAPK, however, is not isolated, since other protein kinases controlling cell fate are redox-regulated. Infact, the sensitivity of PKC family to oxidative imbalance and the existence of pro- and anti-apoptotic isoforms suggest that PKC can act as a molecular redox sensor able to promote cell survival or death. PKC family is subjected to a complex redox regulation: this enzyme is activated by phorbol 12-myristate 13 acetate (PMA) or diacylglycerol (DAG) and is regulated by phosphorylation, lipids and calcium. Selective oxidative modification at the amino-terminal regulatory domain generated activated PKC while alteration at the carboxy-terminal catalytic domain resulted in complete inactivation of the kinase. PKC delta in addition to be a target for redox modulation, is also involved in ROS generation and its pivotal role in stimulating NADPH oxidase activity has been reported.Thus, PKC delta has a well known anti-growth effect: it has been shown to have apoptotic properties and it is involved in the induction of cell cycle arrest and differentiation. In colon carcinoma cells, the increased expression of PKC delta causes cell cycle arrest in G1 phase through cyclin E and D1 down regulation, and PKC delta enzymatic inhibition increases cell growth through increase of cyclin E and D1 expression and p21 inhibition. PMA treatment is able to induce differentiation in promonocytic cell lines by the activation of different ROS generating systems as NADPH oxidase or COX2, via MAPK and p21 activation. More recently, the Unit of Pronzato has demonstrated that retinoic acid-induced neuronal differentiation via PKC delta activation increases cell susceptibility to glycoxidative stress. To identify new redox sensitive molecular targets is very important for employing them in antineoplastic strategies.
Beside a direct regulatory action upon target molecules, ROS/RNS can indirectly act on redox-sensitive proteins that represent the regulatory counterparts of the effector proteins. An example is that represented by Nrf2, a transcription factor that under resting conditions is usually engaged to actin filaments by means of Keap1. Under oxidative stress Nrf2 is released from Keap1, which is able to respond to oxidative/nitrosative stimuli by activating the antioxidant response. An increase in ROS and RNS levels induces redox modifications of Keap1. The occurring Keap1 structural changes let Nrf-2 to detach from Keap1, escape from cytoskeleton, migrate into the nucleus and bind to ARE sequences, codifying for antioxidant enzymes. Recently, the Unit of Cimino demonstrated a negative functional correlation between Nrf2 and p53, with the Nrf2-dependent gen transcription being repressed by p53, which is able to associate to Nrf2 promoter also in the absence of stress. Among Nrf2-dependent genes , those for the enzymes responsible for GSH neo-synthesis play a pivotal role. GSH is the most abundant low molecular weight thiol within cell compartments (1-10 mM). GSH homeostasis has a key role in the cellular defense against oxidative stress both for GSH capability to exist under different oxidation states (reduced or oxidized to intramolecular disulfide or to mixed disulfide with protein thiols), and for the several enzymatic systems mediating its reduction. Alterations of GSH/GSSG ratio normally reflect the redox alterations of the entire compartment or whole cell. In fact GSH directly scavenge ROS and RNS, and play a fundamental role in the maintenance of thiols from reactive cysteines. Recently, a new family of enzymes has emerged, which could play a critical role in the regulation of cellular redox equilibrium: the omega glutathione transferases (GSTO1). This is a new class of GST with special properties: they show only a minimal conjugating activity, which is typical of the other GSTs, but are able to perform reductase activities, such as the dehydroascorbate (DHA) reductase and the thioltransferase activities. Moreover, preliminary evidences from the Unit of Casini support the hypothesis that GSTO1 could play a role in cell cycle modulation, as GSTO1 overexpression is an event associated with conditions known to drive cells in G0/G1, suggesting a role of GSTO1 in cell cycle arrest and resistance to anti-cancer treatments. Such features are in fact associated with high GSTO1 levels in cells resistant towards chemotherapeutics and TNF-mediated death stimuli, probably caused by NF-kB. If from one hand resistance to oxidative stress induces tumor progression, from the other it can represent a tool to counteract aging and aging-related diseases, such as neurodegeneration. In the last decade many important discoveries contributed to the understanding of the regulation of life span in lower organisms, which indicate a strong correlation between cell resistance to oxidative stress and extension of life span. Cellular senescence is a process considered a tumor suppressor mechanism in vivo and an alternative pathway to apoptosis, as well as a possible contributor to tissue aging. Cellular senescence is a complex program with modifications of gene expression pattern. The Cimino’s Unit previously reported that young human embryo fibroblasts exposed to low doses of the GSH-depleting agent diethylmaleate (DEM) acquire the senescent phenotype. The Cimino’s Unit already performed a microarray analysis for 380 known human microRNAs (miRNAs), by using as probes RNAs from normal and DEM-treated young fibroblasts. miRNAs are a recently identified class of short, non-coding RNAs that act as “regulators” of protein synthesis via imperfect base pairing to the 3’ UTR region of target mRNAs. Preliminary results show that five miRNAs are up-regulated under DEM exposure. Alterations of both the levels of GSH and of the ratio GSH/GSSG have been reported to occur during aging. In the lens these alterations have been linked to cataract onset, both in animal models and in humans. The relevance of the transulfuration pathway in supplying cysteine for glutathione synthesis, already reported for several organs, has been recently suggested also for the lens. In particular, in this organ district, alterations of the levels of cystathionine gamma lyase (GCL), which catalyzes the rate limiting step of transulfuration pathway, have been reported to be associated both to aging and cataract onset. These evidences offer a possible explanation for the above mentioned decrease of GSH levels. Recent experimental evidences obtained by participants to this project suggest a potential regulatory action of Cys-Gly on glutathione synthesis through a modulation of CGL activity. Moreover, the ability of CGL to generate hydrogen sulfide through the generation of sulfanic compounds makes relevant to characterize CGL in order to define in the lens possible connections among GSH homeostasis, cellular redox state and mechanisms controlling cell proliferation and death. Neurodegeneration-related neuronal death is often associated with oxidative/nitrosative stress. The comprehension of the molecular mechanisms underlying neurodegeneration is of great importance to suggest new antioxidant-based therapies able to induce ROS scavenging. In fact, ROS are the principal mediators of the loss of mitochondrial integrity observed in such diseases, mainly in Parkinson’s disease (PD). Particularly, nigro-striatal dopaminergic cells show an increase of NO production due to an increase in the expression levels of the inducible (iNOS) and neuronal (nNOS) isoform of NO synthase. This phenomenon seems to account for mitochondrial dysfunction and the increase of Lewy bodies-contained nitrated proteins. As confirm, experimental evidence of the Unit of Ciriolo propose the anti-oxidant and mitochondria-protective action of some diet-deriving redox active molecules and the involvement of nNOS in the exacerbation of cytotoxicity in cellular models of PD. <<<