Publications by authors named "Antonella Ferrante"

33 Publications

Insight into the Role of the STriatal-Enriched Protein Tyrosine Phosphatase (STEP) in A Receptor-Mediated Effects in the Central Nervous System.

Front Pharmacol 2021 19;12:647742. Epub 2021 Apr 19.

National Centre for Drug Research and Evaluation, Istituto Superiore di Sanitá, Rome, Italy.

The STriatal-Enriched protein tyrosine phosphatase STEP is a brain-specific tyrosine phosphatase that plays a pivotal role in the mechanisms of learning and memory, and it has been demonstrated to be involved in several neuropsychiatric diseases. Recently, we found a functional interaction between STEP and adenosine A receptor (AR), a subtype of the adenosine receptor family widely expressed in the central nervous system, where it regulates motor behavior and cognition, and plays a role in cell survival and neurodegeneration. Specifically, we demonstrated the involvement of STEP in AR-mediated cocaine effects in the striatum and, more recently, we found that in the rat striatum and hippocampus, as well as in a neuroblastoma cell line, the overexpression of the AR, or its stimulation, results in an increase in STEP activity. In the present article we will discuss the functional implication of this interaction, trying to examine the possible mechanisms involved in this relation between STEP and ARs.
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http://dx.doi.org/10.3389/fphar.2021.647742DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8090931PMC
April 2021

Adenosine A receptor inhibition reduces synaptic and cognitive hippocampal alterations in Fmr1 KO mice.

Transl Psychiatry 2021 Feb 5;11(1):112. Epub 2021 Feb 5.

National Center for Drug Research and Evaluation, Istituto Superiore di Sanità, Rome, Italy.

In fragile X syndrome (FXS) the lack of the fragile X mental retardation protein (FMRP) leads to exacerbated signaling through the metabotropic glutamate receptors 5 (mGlu5Rs). The adenosine A receptors (ARs), modulators of neuronal damage, could play a role in FXS. A synaptic colocalization and a strong permissive interaction between A and mGlu5 receptors in the hippocampus have been previously reported, suggesting that blocking ARs might normalize the mGlu5R-mediated effects of FXS. To study the cross-talk between A and mGlu5 receptors in the absence of FMRP, we performed extracellular electrophysiology experiments in hippocampal slices of Fmr1 KO mouse. The depression of field excitatory postsynaptic potential (fEPSPs) slope induced by the mGlu5R agonist CHPG was completely blocked by the AR antagonist ZM241385 and strongly potentiated by the AR agonist CGS21680, suggesting that the functional synergistic coupling between the two receptors could be increased in FXS. To verify if chronic AR blockade could reverse the FXS phenotypes, we treated the Fmr1 KO mice with istradefylline, an AR antagonist. We found that hippocampal DHPG-induced long-term depression (LTD), which is abnormally increased in FXS mice, was restored to the WT level. Furthermore, istradefylline corrected aberrant dendritic spine density, specific behavioral alterations, and overactive mTOR, TrkB, and STEP signaling in Fmr1 KO mice. Finally, we identified AR mRNA as a target of FMRP. Our results show that the pharmacological blockade of ARs partially restores some of the phenotypes of Fmr1 KO mice, both by reducing mGlu5R functioning and by acting on other AR-related downstream targets.
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http://dx.doi.org/10.1038/s41398-021-01238-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7864914PMC
February 2021

Adenosine A receptor stimulation restores cell functions and differentiation in Niemann-Pick type C-like oligodendrocytes.

Sci Rep 2019 07 5;9(1):9782. Epub 2019 Jul 5.

Research Coordination and Support Service, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161, Rome, Italy.

Niemann Pick type C (NPC) disease is a rare neurovisceral disorder. Mutations in npc1 gene induce an intracellular accumulation of unesterified cholesterol in the endosomal/lysosomal system causing cell death. We recently showed that stimulation of adenosine A receptors (AR) restores cholesterol accumulation in late endosomes/lysosomes in human NPC fibroblasts and neural cell lines transiently transfected with NPC1 siRNA, suggesting that these receptors might be targeted to contrast the disease. Since NPC1 disease is characterized by dysmyelination and maturational arrest of oligodendrocyte progenitors (OPs), in this study, we investigated whether AR stimulation could promote oligodendrocyte differentiation and myelin formation, thus overcoming these important neurological abnormalities. We developed a NPC1 pharmacological model, in which primary cultures of OPs are exposed to a cholesterol transport inhibitor to induce a NPC1-like phenotype characterized by several typical features such as (i) cholesterol accumulation, (ii) altered mitochondrial morphology and membrane potential, (iii) defect of autophagy and (iv) maturation arrest. The AR agonist CGS21680 normalized all NPC1-like features. The ability of CGS21680 of rescuing OP from maturational arrest and promoting their differentiation to mature OL, suggests that AR stimulation might be exploited to correct dysmyelination in NPC1, further supporting their therapeutic potential in the disease.
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http://dx.doi.org/10.1038/s41598-019-46268-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6611770PMC
July 2019

Adenosine A receptor as potential therapeutic target in neuropsychiatric disorders.

Pharmacol Res 2019 09 2;147:104338. Epub 2019 Jul 2.

National Centre for Drug Research and Evaluation, Istituto Superiore di Sanità, Roma, Italy.

Adenosine A receptor (AR) is a G-protein coupled receptor that regulates several important functions in the central nervous system. Large amount of preclinical data suggests that the AR could represent a target for the development of new therapeutic strategies for different neuropsychiatric conditions. In this review we will recapitulate and discuss the most relevant studies on the role of ARs in neurodegenerative, neurodevelopmental and psychiatric diseases, which led to suggest a therapeutic use of AR agonists in certain diseases (Niemann-Pick disease, autism-spectrum disorders, schizophrenia) and AR antagonists in others (Alzheimer's disease, Parkinson's disease, attention-deficit hyperactivity disorder, fragile X syndrome, depression, anxiety). Moreover, we will try to analyze which are the main obstacles to the conduction of clinical trials with AR ligands for the treatment of neuropsychiatric disease.
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http://dx.doi.org/10.1016/j.phrs.2019.104338DOI Listing
September 2019

Neuroprotective potential of adenosine A receptor partial agonists in experimental models of cerebral ischemia.

J Neurochem 2019 04 11;149(2):211-230. Epub 2019 Feb 11.

National Center for Drug Research and Evaluation, Istituto Superiore di Sanità, Rome, Italy.

Cerebral ischemia is the second most common cause of death and a major cause of disability worldwide. Available therapies are based only on anticoagulants or recombinant tissue plasminogen activator. Extracellular adenosine increases during ischemia and acts as a neuroprotective endogenous agent mainly by activating adenosine A receptors (A Rs) which control calcium influx, glutamate release, membrane potential, and metabolism. Accordingly, in many experimental paradigms it has been already demonstrated that the stimulation of A R with full agonists is able to reduce ischemia-related structural and functional brain damage; unfortunately, cardiovascular side effects and desensitization of A R induced by these compounds have strongly limited their exploitation in stroke therapy so far. Among the newly emerging compounds, A R partial agonists could be almost free of side effects and equally effective. Therefore, we decided to evaluate the neuroprotective potential of two A R partial agonists, namely 2'-dCCPA and 3'-dCCPA, in in vitro and ex vivo experimental models of cerebral ischemia. Within the experimental paradigm of oxygen-glucose deprivation in vitro in human neuroblastoma (SH-SY5Y) cells both A R partial agonists increased cell viability. Considering the high level of expression of A Rs in the hippocampus and the susceptibility of CA1 region to hypoxia, we performed electrophysiological experiments in this subfield. The application of 7 min of oxygen-glucose deprivation constantly produces an irreversible synaptic failure in all the C57Bl/6 mice hippocampal slices evaluated; both tested compounds allowed a significant recovery of synaptic transmission. These findings demonstrate that A R and its partial agonists are still of interest for cerebral ischemia therapy. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.
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http://dx.doi.org/10.1111/jnc.14660DOI Listing
April 2019

Neuronal adenosine A receptor overexpression is neuroprotective towards 3-nitropropionic acid-induced striatal toxicity: a rat model of Huntington's disease.

Purinergic Signal 2018 09 16;14(3):235-243. Epub 2018 May 16.

National Center for Drug Research and Evaluation, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161, Rome, Italy.

The A adenosine receptor (AR) is widely distributed on different cellular types in the brain, where it exerts a broad spectrum of pathophysiological functions, and for which a role in different neurodegenerative diseases has been hypothesized or demonstrated. To investigate the role of neuronal ARs in neurodegeneration, we evaluated in vitro and in vivo the effect of the neurotoxin 3-nitropropionic acid (3-NP) in a transgenic rat strain overexpressing ARs under the control of the neural-specific enolase promoter (NSEA rats). We recorded extracellular field potentials (FP) in corticostriatal slice and found that the synaptotoxic effect of 3-NP was significantly reduced in NSEA rats compared with wild-type animals (WT). In addition, after exposing corticostriatal slices to 3-NP 10 mM for 2 h, we found that striatal cell viability was significantly higher in NSEA rats compared to control rats. These in vitro results were confirmed by in vivo experiments: daily treatment of female rats with 3-NP 10 mg/kg for 8 days induced a selective bilateral lesion in the striatum, which was significantly reduced in NSEA compared to WT rats. These results demonstrate that the overexpression of the AR selectively at the neuronal level reduced 3-NP-induced neurodegeneration, and suggest an important function of the neuronal AR in the modulation of neurodegeneration.
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http://dx.doi.org/10.1007/s11302-018-9609-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6107463PMC
September 2018

The adenosine A receptor agonist T1-11 ameliorates neurovisceral symptoms and extends the lifespan of a mouse model of Niemann-Pick type C disease.

Neurobiol Dis 2018 02 25;110:1-11. Epub 2017 Oct 25.

National Center for Drug Research and Evaluation, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.

Niemann-Pick C is a fatal neurovisceral disorder caused, in 95% of cases, by mutation of NPC1 gene. Therapeutic options are extremely limited and new "druggable" targets are highly warranted. We previously demonstrated that the stimulation of the adenosine A receptor (AR) normalized the pathological phenotype of cellular models of NPC1. Since the validation of ARs as a therapeutic target for NPC1 can be obtained only conducting studies in in vivo models of the disease, in the present paper, the effects of two agonists of ARs were evaluated in the mouse model Balb/c Npc1, hereafter indicated as NPC1-/-. The agonists CGS21680 (2.5 and 5mg/kg/day by intraperitoneal injection) and T1-11 (50mg/kg/day in drinking water) were administered at a presymptomatic stage of the disease of NPC1-/- mice (PN28 and PN30, respectively); the experimental groups were the following: vehicle-treated WT mice (N=16 for both CGS and T1-11 treatments); vehicle-treated NPC1-/- mice (N=14 for CGS and 12 for T1-11 treatment); CGS-treated NPC1-/- mice (N=7) and T1-11-treated NPC1-/- mice (N=11). The efficacy of the treatments was evaluated by comparing vehicle-treated and CGS or T1-11-treated NPC1-/- mice for their motor deficits (analyzed by both rotarod and footprint tests), hippocampal cognitive impairment (by Novel Object Recognition (NOR) test), cerebellar neurodegeneration (Purkinje neurons counting), and cholesterol and sphingomyelin accumulation in spleen and liver. Finally, the effect of both agonists on survival was evaluated by applying a humane late endpoint (weight loss >30% of peak weight, punched posture and reduced activity in the cage). The results demonstrated that, while CGS21680 only slightly attenuated cognitive deficits, T1-11 ameliorated motor coordination, significantly improved cognitive impairments, increased the survival of Purkinje neurons and reduced sphingomyelin accumulation in the liver. More importantly, it significantly prolonged the lifespan of NPC1-/- mice. In vitro experiments conducted in a neuronal model of NPC1 demonstrated that the ability of T1-11 to normalize cell phenotype was mediated by the selective activation of ARs and modulation of intracellular calcium levels. In conclusion, our results fully confirm the validity of ARs as a new target for NPC1 treatment. As soon as new ligands with improved pharmacokinetic characteristics (i.e. orally active, with brain bioavailability and metabolic stability) will be obtained, AR agonists could represent a breakthrough in the treatment of NPC.
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http://dx.doi.org/10.1016/j.nbd.2017.10.013DOI Listing
February 2018

Striatal adenosine-cannabinoid receptor interactions in rats over-expressing adenosine A2A receptors.

J Neurochem 2016 Mar 24;136(5):907-17. Epub 2015 Nov 24.

Department Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanità, Rome, Italy.

Adenosine A2A receptors (A2 A Rs) and cannabinoid CB1 receptors (CB1 Rs) are highly expressed in the striatum, where they functionally interact and form A2A /CB1 heteroreceptor complexes. We investigated the effects of CB1 R stimulation in a transgenic rat strain over-expressing A2 A Rs under the control of the neural-specific enolase promoter (NSEA2A rats) and in age-matched wild-type (WT) animals. The effects of the CB1 R agonist WIN 55,212-2 (WIN) were significantly lower in NSEA2A rats than in WT animals, as demonstrated by i) electrophysiological recordings of synaptic transmission in corticostriatal slices; ii) the measurement of glutamate outflow from striatal synaptosomes and iii) in vivo experiments on locomotor activity. Moreover, while the effects of WIN were modulated by both A2 A R agonist (CGS 21680) and antagonists (ZM 241385, KW-6002 and SCH-442416) in WT animals, the A2 A R antagonists failed to influence WIN-mediated effects in NSEA2A rats. The present results demonstrate that in rats with genetic neuronal over-expression of A2 A Rs, the effects mediated by CB1 R activation in the striatum are significantly reduced, suggesting a change in the stoichiometry of A2A and CB1 receptors and providing a strategy to dissect the involvement of A2 A R forming or not forming heteromers in the modulation of striatal functions. These findings add additional evidence for the existence of an interaction between striatal A2 A Rs and CB1 Rs, playing a fundamental role in the regulation of striatal functions. We studied A2A -CB1 receptor interaction in transgenic rats over-expressing adenosine A2A receptors under the control of the neuron-specific enolase promoter (NSEA2A ). In these rats, we demonstrated a reduced effect of the CB1 receptor agonist WIN 55,212-2 in the modulation of corticostriatal synaptic transmission and locomotor activity, while CB1 receptor expression level did not change with respect to WT rats. A reduction in the expression of A2A -CB1 receptor heteromers is postulated.
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http://dx.doi.org/10.1111/jnc.13421DOI Listing
March 2016

Modulation of Rho GTPases rescues brain mitochondrial dysfunction, cognitive deficits and aberrant synaptic plasticity in female mice modeling Rett syndrome.

Eur Neuropsychopharmacol 2015 Jun 30;25(6):889-901. Epub 2015 Mar 30.

Department of Cell Biology and Neuroscience, Istituto Superiore di Sanità, Roma, Italy.

Rho GTPases are molecules critically involved in neuronal plasticity and cognition. We have previously reported that modulation of brain Rho GTPases by the bacterial toxin CNF1 rescues the neurobehavioral phenotype in MeCP2-308 male mice, a model of Rett syndrome (RTT). RTT is a rare X-linked neurodevelopmental disorder and a genetic cause of intellectual disability, for which no effective therapy is available. Mitochondrial dysfunction has been proposed to be involved in the mechanism of the disease pathogenesis. Here we demonstrate that modulation of Rho GTPases by CNF1 rescues the reduced mitochondrial ATP production via oxidative phosphorylation in the brain of MeCP2-308 heterozygous female mice, the condition which more closely recapitulates that of RTT patients. In RTT mouse brain, CNF1 also restores the alterations in the activity of the mitochondrial respiratory chain (MRC) complexes and of ATP synthase, the molecular machinery responsible for the majority of cell energy production. Such effects were achieved through the upregulation of the protein content of those MRC complexes subunits, which were defective in RTT mouse brain. Restored mitochondrial functionality was accompanied by the rescue of deficits in cognitive function (spatial reference memory in the Barnes maze), synaptic plasticity (long-term potentiation) and Tyr1472 phosphorylation of GluN2B, which was abnormally enhanced in the hippocampus of RTT mice. Present findings bring into light previously unknown functional mitochondrial alterations in the brain of female mice modeling RTT and provide the first evidence that RTT brain mitochondrial dysfunction can be rescued by modulation of Rho GTPases.
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http://dx.doi.org/10.1016/j.euroneuro.2015.03.012DOI Listing
June 2015

Expression, pharmacology and functional activity of adenosine A1 receptors in genetic models of Huntington's disease.

Neurobiol Dis 2014 Nov 15;71:193-204. Epub 2014 Aug 15.

Istituto Superiore di Sanità, Department of Therapeutic Research and Medicines Evaluation, Viale Regina Elena 299, 00161 Rome, Italy.

Adenosine A1 receptor (A1R) stimulation exerts beneficial effects in response to various insults to the brain and, although it was found neuroprotective in a lesional model of Huntington's disease (HD), the features of this receptor in genetic models of HD have never been explored. In the present study we characterized the expression, affinity and functional effects of A1Rs in R6/2 mice (the most widely used transgenic model of HD) and in a cellular model of HD. Binding studies revealed that the density of A1Rs was significantly reduced in the cortex and the striatum of R6/2 mice compared to age-matched wild-type (WT), while receptor affinity was unchanged. The selective A1R agonist cyclopentyladenosine (CPA, 300nM) was significantly more effective in reducing synaptic transmission in corticostriatal slices from symptomatic R6/2 than in age-matched WT mice. Such an effect was due to a stronger inhibition of glutamate release from the pre-synaptic terminal. The different functional activities of A1Rs in HD mice were associated also to a different intracellular signaling pathway involved in the synaptic effect of CPA. In fact, while the PKA pathway was involved in both genotypes, p38 MAPK inhibitor SB203580 partially prevented synaptic effects of CPA in R6/2, but not in WT, mice; moreover, CPA differently modulated the phosphorylation status of p38 in the two genotypes. In vitro studies confirmed a different behavior of A1Rs in HD: CPA (100 nM for 5h) modulated cell viability in STHdh(Q111/Q111) (mhttHD cells), without affecting the viability of STHdh(Q7/Q7) (wthtt cells). This effect was prevented by the application of SB203580. Our results demonstrate that in the presence of the HD mutation A1Rs undergo profound changes in terms of expression, pharmacology and functional activity. These changes have to be taken in due account when considering A1Rs as a potential therapeutic target for this disease.
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http://dx.doi.org/10.1016/j.nbd.2014.08.013DOI Listing
November 2014

The stimulation of adenosine A2A receptors ameliorates the pathological phenotype of fibroblasts from Niemann-Pick type C patients.

J Neurosci 2013 Sep;33(39):15388-93

Department of Cell Biology and Neuroscience, and Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanità, 00161 Rome, Italy.

Niemann-Pick type C1 (NPC1) disease is a rare neurovisceral disorder characterized by intracellular accumulation of unesterified cholesterol, sphingolipids, and other lipids in the lysosomal compartment. A deregulation of lysosomal calcium has been identified as one of the earliest steps of the degenerative process. Since adenosine A2A receptors (A2ARs) control lysosome trafficking and pH, which closely regulates lysosomal calcium, we hypothesized a role for these receptors in NPC1. The aim of this study was to evaluate the effects of the A2AR agonist CGS21680 on human control and NPC1 fibroblasts. We show that CGS21680 raises lysosomal calcium levels and rescues mitochondrial functionality (mitochondrial inner membrane potential and expression of the complex IV of the mitochondrial respiratory chain), which is compromised in NPC1 cells. These effects are prevented by the selective blockade of A2ARs by the antagonist ZM241385. The effects of A2AR activation on lysosomal calcium are not mediated by the cAMP/PKA pathway but they appear to involve the phosphorylation of ERK1/2. Finally, CGS21680 reduces cholesterol accumulation (Filipin III staining), which is the main criterion currently used for identification of a compound or pathway that would be beneficial for NPC disease, and such an effect is prevented by the Ca(2+) chelator BAPTA-AM. Our findings strongly support the hypothesis that A2AR agonists may represent a therapeutic option for NPC1 and provide insights on their mechanisms of action.
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http://dx.doi.org/10.1523/JNEUROSCI.0558-13.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6618463PMC
September 2013

Cocaine-induced changes of synaptic transmission in the striatum are modulated by adenosine A2A receptors and involve the tyrosine phosphatase STEP.

Neuropsychopharmacology 2014 Feb 30;39(3):569-78. Epub 2013 Aug 30.

Department Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanità, Roma, Italy.

The striatum is a brain area implicated in the pharmacological action of drugs of abuse. Adenosine A2A receptors (A2ARs) are highly expressed in the striatum and mediate, at least in part, cocaine-induced psychomotor effects in vivo. Here we studied the synaptic mechanisms implicated in the pharmacological action of cocaine in the striatum and investigated the influence of A2ARs. We found that synaptic transmission was depressed in corticostriatal slices after perfusion with cocaine (10 μM). This effect was reduced by the A2AR antagonist ZM241385 and almost abolished in striatal A2AR-knockout mice (mice lacking A2ARs in striatal neurons, stA2ARKO). The effect of cocaine on synaptic transmission was also prevented by the protein tyrosine phosphatases (PTPs) inhibitor sodium orthovanadate (Na3VO4). In synaptosomes prepared from striatal slices, we found that the activity of striatal-enriched protein tyrosine phosphatase (STEP) was upregulated by cocaine, prevented by ZM241385, and absent in synaptosomes from stA2ARKO. The role played by STEP in cocaine modulation of synaptic transmission was investigated in whole-cell voltage clamp recordings from medium spiny neurons of the striatum. We found that TAT-STEP, a peptide that renders STEP enzymatically inactive, prevented cocaine-induced reduction in AMPA- and NMDA-mediated excitatory post-synaptic currents, whereas the control peptide, TAT-myc, had no effect. These results demonstrate that striatal A2ARs modulate cocaine-induced synaptic depression in the striatum and highlight the potential role of PTPs and specifically STEP in the effects of cocaine.
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http://dx.doi.org/10.1038/npp.2013.229DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3895235PMC
February 2014

BDNF prevents NMDA-induced toxicity in models of Huntington's disease: the effects are genotype specific and adenosine A2A receptor is involved.

J Neurochem 2013 Apr 27;125(2):225-35. Epub 2013 Feb 27.

Section of Central Nervous System Pharmacology, Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanità, Rome, Italy.

NMDA receptor-mediated excitotoxicity is thought to play a pivotal role in the pathogenesis of Huntington's disease (HD). The neurotrophin brain-derived neurotrophic factor (BDNF), which is also highly involved in HD and whose effects are modulated by adenosine A2 ARs, influences the activity and expression of striatal NMDA receptors. In electrophysiology experiments, we investigated the role of BDNF toward NMDA-induced effects in HD models, and the possible involvement of A2ARs. In corticostriatal slices from wild-type mice and age-matched symptomatic R6/2 mice (a model of HD), NMDA application (75 μM) induced a transient or a permanent (i.e., toxic) reduction of field potential amplitude, respectively. BDNF (10 ng/mL) potentiated NMDA effects in wild-type, while it protected from NMDA toxicity in R6/2 mice. Both effects of BDNF were prevented by A2 AR blockade. The protective effect of BDNF against NMDA-induced toxicity was reproduced in a cellular model of HD. These findings may have very important implications for the neuroprotective potential of BDNF and A2 AR ligands in HD.
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http://dx.doi.org/10.1111/jnc.12177DOI Listing
April 2013

Effects of chronic caffeine intake in a mouse model of amyotrophic lateral sclerosis.

J Neurosci Res 2013 Apr 30;91(4):585-92. Epub 2013 Jan 30.

Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanità, Rome, Italy.

Caffeine is a nonselective adenosine receptor antagonist; chronic consumption has proved protective toward neurodegenerative diseases such as Parkinson's and Alzheimer's diseases. The present study was designed to determine whether caffeine intake affected survival and/or motor performance in a transgenic model of amyotrophic lateral sclerosis (ALS). SOD1(G93A) mice received caffeine through drinking water from 70 days of age until death. Body weight, motor performance and survival were evaluated. Furthermore, the expression of adenosine A(2A) receptors (A(2A) Rs), glial glutamate transporter (GLT1), and glial fibrillar acidic protein (GFAP) were evaluated by Western blotting. The results showed that caffeine intake significantly shortened the survival of SOD1(G93A) mice (log rank test, P = 0.01) and induced a nonsignificant advancing of disease onset. The expression of A(2A) R, GLT1, and GFAP was altered in the spinal cords of ALS mice, but caffeine did not influence their expression in either wild-type or SOD1(G93) mice. These data indicate that adenosine receptors may play an important role in ALS.
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http://dx.doi.org/10.1002/jnr.23185DOI Listing
April 2013

Unbalance of CB1 receptors expressed in GABAergic and glutamatergic neurons in a transgenic mouse model of Huntington's disease.

Neurobiol Dis 2012 Mar 23;45(3):983-91. Epub 2011 Dec 23.

Department of Therapeutic Research and Medicine Evaluation, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.

Cannabinoid CB1 receptors (CB1Rs) are known to be downregulated in patients and in animal models of Huntington's disease (HD). However, the functional meaning of this reduction, if any, is still unclear. Here, the effects of the cannabinoid receptor agonist WIN 55,212-2 (WIN) were investigated on striatal synaptic transmission and on glutamate and GABA release in symptomatic R6/2 mice, a genetic model of HD. The expression levels of CB1Rs in glutamatergic and GABAergic synapses were also evaluated. We found that in R6/2 mice, WIN effects on synaptic transmission and glutamate release were significantly increased with respect to wild type mice. On the contrary, a decrease in WIN-induced reduction of GABA release was found in R6/2 versus WT mice. The expression of CB1Rs in GABAergic neurons was drastically reduced, while CB1Rs levels in glutamatergic neurons were unchanged. These results demonstrate that the expression and functionality of CB1Rs are differentially affected in GABAergic and glutamatergic neurons in R6/2 mice. As a result, the balance between CB1Rs expressed by the two neuronal populations and, thus, the net effect of CB1R stimulation, is profoundly altered in HD mice.
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http://dx.doi.org/10.1016/j.nbd.2011.12.017DOI Listing
March 2012

Complex behavioral and synaptic effects of dietary branched chain amino acids in a mouse model of amyotrophic lateral sclerosis.

Mol Nutr Food Res 2011 Apr 5;55(4):541-52. Epub 2011 Jan 5.

Section of Neurotoxicology and Neuroendocrinology, Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Rome, Italy.

Scope: We hypothesized that chronic supplementation with branched chain amino acids (BCAAs) affects neurobehavioral development in vulnerable gene backgrounds.

Methods And Results: A murine model of amyotrophic lateral sclerosis (ALS), G93A mice bearing the mutated human superoxide dismutase 1 (SOD1) gene, and control mice received from 4 to 16 wk of age dietary supplementation with BCAAs at doses comparable to human usage. Motor coordination, exploratory behaviors, pain threshold, synaptic activity and response to glutamatergic stimulation in primary motor cortex slices were evaluated between the 8th and 16th week. The glial glutamate transporter 1 (GLT-1) and metabotropic glutamate 5 receptor (mGlu5R) were analyzed by immunoblotting in cortex, hippocampus and striatum. BCAAs induced hyperactivity, decreased pain threshold in wild-type mice and exacerbated the motor deficits of G93A mice while counteracting their abnormal pain response. Electrophysiology on G93A brain slices showed impaired synaptic function, reduced toxicity of GLT-1 blocking and increased glutamate toxicity prevented by BCAAs. Immunoblotting indicated down-regulation of GLT-1 and mGlu5R in G93A, both effects counteracted by BCAAs.

Conclusion: These results, though not fully confirming a role of BCAAs in ALS-like etiology in the genetic model, clearly indicate that BCAAs' complex effects on central nervous system depend on gene background and raise alert over their spread use.
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http://dx.doi.org/10.1002/mnfr.201000296DOI Listing
April 2011

Role of adenosine A(2A) receptors in modulating synaptic functions and brain levels of BDNF: a possible key mechanism in the pathophysiology of Huntington's disease.

ScientificWorldJournal 2010 Sep 1;10:1768-82. Epub 2010 Sep 1.

Department of Therapeutic Research and Medicine Evaluation, Istituto Superiore di Sanità, Rome.

In the last few years, accumulating evidence has shown the existence of an important cross-talk between adenosine A(2A) receptors (A(2A)Rs) and brain-derived neurotrophic factor (BDNF). Not only are A(2A)Rs involved in the mechanism of transactivation of BDNF receptor TrkB, they also modulate the effect of BDNF on synaptic transmission, playing a facilitatory and permissive role. The cAMP-PKA pathway, the main transduction system operated by A(2A)Rs, is involved in such effects. Furthermore, a basal tonus of A(2A)Rs is required to allow the regulation of BDNF physiological levels in the brain, as demonstrated by the reduced protein levels measured in A(2A)Rs KO mice. The crucial role of adenosine A(2A)Rs in the maintenance of synaptic functions and BDNF levels will be reviewed here and discussed in the light of possible implications for Huntington's disease therapy, in which a joint impairment of BDNF and A(2A)Rs seems to play a pathogenetic role.
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http://dx.doi.org/10.1100/tsw.2010.164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5763899PMC
September 2010

Influence of CGS 21680, a selective adenosine A(2A) receptor agonist, on NMDA receptor function and expression in the brain of Huntington's disease mice.

Brain Res 2010 Apr 4;1323:184-91. Epub 2010 Feb 4.

Department of Therapeutic Research and Medicine Evaluation, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161, Rome, Italy.

The effect of chronic treatment with the selective adenosine A(2A) receptor agonist CGS 21680 on N-Methyl-d-Aspartate (NMDA) receptor function and expression has been studied in the striatum and cortex of R6/2 mice, a genetic mouse model of Huntington's disease (HD). Starting from 8weeks of age, R6/2 and wild type (WT) mice were treated daily with CGS 21680 (0.5mg/kg i.p.) for 3weeks and the expression levels of NMDA receptor subunits were then evaluated. In addition, to study CGS 21680-induced changes in NMDA receptor function, NMDA-induced toxicity in corticostriatal slices from both R6/2 and WT mice was investigated. We found that CGS 21680 increased NR2A subunit expression and the NR2A/NR2B ratio in the cortex of R6/2 mice, having no effect in WT mice. In the striatum, CGS 21680 reduced NR1 expression in both R6/2 and WT mice while the effect on NR2A and NR2/NR2B expression was genotype-dependent, reducing and increasing their expression in WT and R6/2 mice, respectively. On the contrary, NMDA-induced toxicity in corticostriatal slices was not modified by the treatment in WT or HD mice. These results demonstrate that in vivo activation of A(2A) receptors modulates the subunit composition of NMDA receptors in the brain of HD mice.
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http://dx.doi.org/10.1016/j.brainres.2010.01.080DOI Listing
April 2010

Remodeling of striatal NMDA receptors by chronic A(2A) receptor blockade in Huntington's disease mice.

Neurobiol Dis 2010 Jan 3;37(1):99-105. Epub 2009 Oct 3.

Department of Therapeutic Research and Medicine Evaluation, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161, Rome, Italy.

Excitotoxicity plays a major role in the pathogenesis of Huntington disease (HD), a fatal neurodegenerative disorder. Adenosine A(2A) receptors (A(2A)Rs) modulate excitotoxicity and have been suggested to play a pathogenetic role in HD. The main aim of this study was to evaluate the effect of A(2A)R blockade on the expression and functions of NMDA receptors in the striatum of HD mice (R6/2). We found that 3 weeks' treatment with SCH 58261 (0.01 mg/kg/day i.p. from the 8th week of age) modified NR1 and NR2A/NR2B expression in the striatum of R6/2 (Western blotting) while had no effect on NMDA-induced toxicity in corticostriatal slices (electrophysiological experiments). In conclusion, in vivo A(2A)R blockade induced a remodeling of NMDA receptors in the striatum of HD mice. Even though the functional relevance of the above effect remains to be fully elucidated, these results add further evidence to the modulatory role of A(2A)Rs in HD.
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http://dx.doi.org/10.1016/j.nbd.2009.09.012DOI Listing
January 2010

Adenosine A2A receptors enable the synaptic effects of cannabinoid CB1 receptors in the rodent striatum.

J Neurochem 2009 Sep 17;110(6):1921-30. Epub 2009 Jul 17.

Section of Central Nervous System Pharmacology, Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanità, Rome, Italy.

Adenosine A(2A), cannabinoid CB(1) and metabotropic glutamate 5 (mGlu(5)) receptors are all highly expressed in the striatum. The aim of the present work was to investigate whether, and by which mechanisms, the above receptors interact in the regulation of striatal synaptic transmission. By extracellular field potentials (FPs) recordings in corticostriatal slices, we demonstrated that the ability of the selective type 1 cannabinoid receptor (CB(1)R) agonist WIN55,212-2 to depress synaptic transmission was prevented by the pharmacological blockade or the genetic inactivation of A(2A)Rs. Such a permissive effect of A(2A)Rs towards CB(1)Rs does not seem to occur pre-synaptically as the ability of WIN55,212-2 to increase the R2/R1 ratio under a protocol of paired-pulse stimulation was not modified by ZM241385. Furthermore, the effects of WIN55,212-2 were reduced in slices from mice lacking post-synaptic striatal A(2A)Rs. The selective mGlu(5)R agonist (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) potentiated the synaptic effects of WIN55,212-2, and such a potentiation was abolished by A(2A)R blockade. Unlike the synaptic effects, the ability of WIN55,212-2 to prevent NMDA-induced toxicity was not influenced by ZM241385. Altogether, these results show that the state of activation of A(2A)Rs regulates the synaptic effects of CB(1)Rs and that A(2A)Rs may control CB(1) effects also indirectly, namely through mGlu(5)Rs.
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http://dx.doi.org/10.1111/j.1471-4159.2009.06282.xDOI Listing
September 2009

Region-specific neuroprotective effect of ZM 241385 towards glutamate uptake inhibition in cultured neurons.

Eur J Pharmacol 2009 Sep 18;617(1-3):28-32. Epub 2009 Jul 18.

Department of Therapeutic Research and Medicines Evaluation, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161, Rome, Italy.

Active uptake by neurons and glial cells is the main mechanism for maintaining extracellular glutamate at low, non-toxic concentrations. Adenosine A(2A) receptors regulate extracellular glutamate levels by acting on both the release and the uptake of glutamate. The aim of this study was to evaluate whether the inhibition of the effects of glutamate uptake blockers by adenosine A(2A) receptor antagonists resulted in neuroprotection. In cortical and striatal neuronal cultures, the application of l-trans-pyrrolidine-2,4-dicarboxylic acid (PDC, a transportable competitive inhibitor of glutamate uptake), induced a dose-dependent increase in lactate dehydrogenase (LDH) levels, an index of cytotoxicity. Such an effect of PDC was significantly reduced by pre-treatment with the adenosine A(2A) receptor antagonist ZM 241385 (50 nM) in striatal, but not cortical, cultures. The protective effects of ZM 241385 were specifically due to a counteraction of PDC effects, since ZM 241385 was totally ineffective in preventing the cytotoxicity induced by direct application of glutamate to cultures. These results indicate that adenosine A(2A) receptor antagonists prevent the toxic effects induced by a transportable competitive inhibitor of glutamate uptake, that such an effect specifically occurs in the striatum and that it does not depend on a direct blockade of glutamate-induced toxicity.
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http://dx.doi.org/10.1016/j.ejphar.2009.07.016DOI Listing
September 2009

Metabolomics using 1H-NMR of apoptosis and Necrosis in HL60 leukemia cells: differences between the two types of cell death and independence from the stimulus of apoptosis used.

Radiat Res 2008 Feb;169(2):170-80

Dipartimento di Ematologia, Oncologia e Medicina Molecolare, Instituto Superiore di Sanità, 00161 Rome, Italy.

High-resolution proton nuclear magnetic resonance ((1)H-NMR) spectroscopy was used to examine and compare the metabolic variations that occur in cells of the HL60 promyelocytic leukemia cell line after induction of apoptosis by ionizing radiation and the antineoplastic drug doxorubicin as well as after induction of necrosis by heating. Apoptosis and necrosis were confirmed by fluorescence microscopy using the chromatin stain Hoechst 33258, agarose gel electrophoresis of DNA, and determination of caspase 3 enzymatic activity. The 1H-NMR experiments revealed that the spectra of both samples containing apoptotic cells were characterized by the same trend of several important metabolites. Specifically, an increase in CH2 and CH3 mobile lipids, principally of CH2, decreases in glutamine and glutamate, choline-containing metabolites, taurine and reduced glutathione were observed. By contrast, the sample containing necrotic cells presented a completely different profile of 1H-NMR metabolites since it was characterized by a significant increase in all the metabolites examined, with the exception of CH2 mobile lipids, which remain unchanged, and reduced glutathione, which decreased. The results suggest that variations in 1H-NMR metabolites are specific to apoptosis independent of the physical or chemical nature of the stimulus used to induce this mode of cell death, while cells dying from necrosis are characterized by a completely different behavior of the same metabolites.
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http://dx.doi.org/10.1667/RR0958.1DOI Listing
February 2008

Temporal dynamics of 1H-NMR-visible metabolites during radiation-induced apoptosis in MG-63 human osteosarcoma spheroids.

Radiat Res 2006 Nov;166(5):734-45

Dipartimento di Ematologia, Oncologia e Medicina Molecolare, Istituto Superiore di Sanità, 00161, Rome, Italy.

The metabolic changes that occur as a function of time in MG-63 osteosarcoma three-dimensional tumor spheroids undergoing radiation-induced apoptosis were studied using high-resolution proton nuclear magnetic resonance ((1)H-NMR) spectroscopy. Specifically, the (1)H-NMR spectra of MG-63 spheroids collected at 24, 48 and 72 h after exposure to 5 Gy of ionizing radiation were compared to the spectra of their respective controls. Small spheroids (about 50-80 microm in diameter) with no hypoxic center were used. Apoptosis was verified by both staining of spheroid DNA with the Hoechst 33258 dye and determination of caspase 3 enzyme activity at the three times examined. The results demonstrate that, as the percentage of apoptosis rises with time after exposure to ionizing radiation, the metabolic changes that take place in MG-63 spheroids follow very precise temporal dynamics. In particular, significant time-related increases in both CH(2) and CH(3) mobile lipids, considered by many authors as markers of apoptosis, were observed. In addition, temporal variations were also observed in choline-containing metabolites, reduced glutathione (GSH), glutamine/glutamate, taurine, alanine, creatine/phosphocreatine and lactate. These data show that in addition to CH(2) and CH(3) lipids, other metabolites can also be extremely useful in a deeper understanding of the temporal dynamics of radiation-induced apoptosis. This comprehension is particularly important in spheroids, a cell model of great complexity that resembles in vivo tumors much more closely than monolayer cultures. Ultimately, it is hoped that such studies can help to evaluate the outcome of radiotherapy protocols more accurately.
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http://dx.doi.org/10.1667/RR0635.1DOI Listing
November 2006

Hypoxia and ionizing radiation: changes in adhesive properties and cell adhesion molecule expression in MG-63 three-dimensional tumor spheroids.

Cell Commun Adhes 2006 May-Jun;13(3):185-98

Dipartimento di Ematologia, Oncologia e Medicina Molecolare, Istituto Superiore di Sanità, Rome, Italy.

The effects of chemically induced hypoxia and ionizing radiation on the adhesive properties of MG-63 human osteosarcoma three-dimensional spheroids were investigated. Hypoxia was induced by addition of CoCl2 to small, nonhypoxic spheroids and verified by HIF-1alpha expression. In addition, the possible role of important cell adhesion molecules involved in tumor dissemination in inducing adhesive changes were also studied. In particular, two key integrins (i.e., the alpha chain of the fibronectin receptor, alpha5, and the alpha chain of the collagen receptor, alpha2), an important member of the immunoglobulin superfamily (CD54 or ICAM-1) and the strategic molecule CD44 (H-CAM, the principal receptor for hyaluronan) were examined. Because of the important role of fibronectin in adhesive processes, variations in this extracellular matrix component were also examined. The results seem to indicate that CoCl2-induced hypoxia greatly increases adhesion of MG-63 spheroids to both tissue culture plates and plates coated with fibronectin or collagen when compared to controls, while ionizing radiation induces a great decrease in this attachment. Furthermore, chemically induced hypoxia also partially inhibits the effects of ionizing radiation. The data also show that these adhesive changes are accompanied by concomitant variations in the expression of alpha5 and alpha2 integrins, CD44, and CD54 and fibronectin.
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http://dx.doi.org/10.1080/15419060600734153DOI Listing
September 2006

1H-NMR evidence for a different response to the same dose (2 Gy) of ionizing radiation of MG-63 human osteosarcoma cells and three-dimensional spheroids.

Anticancer Res 2006 Jan-Feb;26(1A):267-81

Dipartimento di Ematologia, Oncologia e Medicina Molecolare, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161, Rome, Italy.

High resolution proton nuclear magnetic resonance (1H-NMR) spectroscopy was used to examine the response of the MG-63 osteosarcoma cell line grown in monolayer and as 3-dimensional tumor spheroids to the same low dose (2 Gy) of ionizing radiation. The MG-63 cells and spheroids were irradiated at 24 h of growth and the 1H-NMR spectra of whole control and irradiated monolayer cells and of whole control and irradiated multicellular spheroids collected after another 24 h were compared. The 1H-NMR spectra of the perchloric acid extracts as well as the 2-dimensional 1H-NMR spectra of both pairs of cell systems were also obtained. Possible radiation-induced cell damage was determined by lactate dehydrogenase (LDH) release and variations in cell growth, while cell death was evaluated by chromatin dye Hoechst staining and DNA fragmentation assays. The results demonstrated that no cell damage took place, but that significant variations in numerous metabolites occured in both the monolayer cells and the spheroids after irradiation. Most of the changes observed were very similar in nature. In fact, significant increases in lactate, alanine, creatine and phosphocreatine and choline-containing metabolites and a significant decrease in glutathione (GSH) were observed in both cells and spheroids. However, while significant increases in CH2 and CH3 mobile lipids, glutamine/glutamate, taurine and inositol were seen in the spheroids, no variations in CH2 or CH3 lipids, glutamine/glutamate or taurine were recorded in the MG-63 cells grown in monolayer after irradiation. In addition, a significant decrease rather than a significant increase in inositol was also noted in the monolayer cells. The data presented seem to suggest that, although neither monolayer cells nor spheroids show apparent signs of damage after exposure to the same dose of ionizing radiation, very different cell death responses as well as very diverse antioxidant/osmoregulatory reactions were triggered by this stressing agent.
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March 2006

Increases in 1H-NMR mobile lipids are not always associated with overt apoptosis: evidence from MG-63 human osteosarcoma three-dimensional spheroids exposed to a low dose (2 Gy) of ionizing radiation.

Radiat Res 2006 Feb;165(2):131-41

Dipartimento di Ematologia, Oncologia e Medicina Molecolare, Istituto Superiore di Sanità, 00161, Rome, Italy.

The metabolic changes that occur in MG-63 osteosarcoma three-dimensional tumor spheroids exposed to 2 Gy of ionizing radiation, a dose that is comparable to radiation therapy, were studied using high-resolution proton nuclear magnetic resonance ((1)H-NMR) spectroscopy. Specifically, the (1)H-NMR spectra of control and exposed MG-63 spheroids were compared. Small spheroids (about 50-80 microm in diameter) with no hypoxic center were used. The spectra of whole MG-63 spheroids as well as the perchloric acid extracts of these systems were evaluated. Cell damage was also examined by lactate dehydrogenase release and changes in cell growth. No cell damage was observed, but numerous metabolic changes took place in spheroids after exposure to ionizing radiation. In particular, significant increases in both CH(2) and CH(3) mobile lipids, considered by many authors as markers of apoptosis and also present in MG-63 spheroids undergoing overt apoptosis, were observed in spheroids irradiated with 2 Gy. However, the chromatin dye Hoechst 33258 and DNA fragmentation assays showed no overt apoptosis up to 7 days after irradiation with this low dose. Thus it is evident that increases in mobile lipids do not always indicate actual cell death. A detailed analysis of the other metabolic changes observed appears to suggest that the cell death program was initiated but not completed. In fact, the completely different behavior of two important cellular defense mechanisms, reduced glutathione and taurine, in spheroids irradiated with 2 Gy and in those undergoing overt apoptosis seems to indicate that these systems are protecting spheroids from actual cell death. In addition, these data also suggest that (1)H-NMR can be used to examine the effects of low doses of ionizing radiation in spheroids, a cell model of great complexity that closely resembles tumors in vivo. The importance of this possibility in relation to reaching the ultimate goal of a better evaluation of the outcome of radiotherapy protocols should not be ignored.
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http://dx.doi.org/10.1667/rr3500.1DOI Listing
February 2006

Increased cell compaction can augment the resistance of HT-29 human colon adenocarcinoma spheroids to ionizing radiation.

Int J Oncol 2006 Jan;28(1):111-8

Dipartimento di Ematologia, Oncologia e Medicina Molecolare, Istituto Superiore di Sanità, I-00161 Rome, Italy.

One of the most important questions in tumor biology is the understanding of the mechanisms responsible for the resistance of cancer cells to radiotherapy. In the present study, the possible role played by cell-cell interactions in determining the response of tumor cells to ionizing radiation was investigated. HT-29 colon adenocarcinoma spheroids were irradiated with a dose of 15 Gy in two different stages of growth characterized by diverse degrees of compaction: loosely organized spheroids (early spheroids) and compacted spheroids (late spheroids). Morphological analyses demonstrated that late spheroids were less damaged than early spheroids. Moreover, analyses of the cell cycle and cell death showed that ionizing radiation induced necrosis in early spheroids and apoptosis in late ones. From these results it can be concluded that late, compacted spheroids are more resistant to ionizing radiation than early, loose spheroids. In order to understand the mechanisms regulating this compaction-induced resistance of late spheroids, E-cadherin/beta-catenin complex expression and distribution were analyzed. In late spheroids, E-cadherin/beta-catenin complexes were shown to be tethered to the cytoskeleton, and since this organization has been demonstrated to strengthen cell-cell adhesion in other systems, it can be postulated that the same is true in HT-29 spheroids. In conclusion, it can be hypothesized that compaction of HT-29 spheroids is mediated by the reorganization of E-cadherin/beta-catenin complexes on the plasma membrane and that this compaction may be responsible for the increase in resistance of HT-29 spheroids to ionizing radiation.
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January 2006

A 50 Hz sinusoidal magnetic field does not damage MG-63 three-dimensional tumor spheroids but induces changes in their invasive properties.

Bioelectromagnetics 2006 Feb;27(2):132-41

Dipartimento di Ematologia, Oncologia e Medicina Molecolare, Istituto Superiore di Sanità, Rome, Italy.

The possibility that a sinusoidal 50 Hz magnetic field with a magnetic flux density of 1 mT can damage MG-63 osteosarcoma spheroids and induce variations in the invasive properties of these three-dimensional model systems after 2 days of exposure was investigated. Specifically, possible damage induced by these fields was examined by determining changes in spheroid surface morphology (light microscopy), growth (spheroid diameter and protein content determination), lactate dehydrogenase release, and reduced glutathione amount. Possible changes in the invasive properties were studied by invasion chambers. The results show no induction of cell damage by ELF fields while invasion chamber assays demonstrate a significant increase in the invasive potential of exposed spheroids. In order to determine if the fibronectin or hyaluronan receptors are involved, Western blot analysis was conducted on these two proteins. No significant variations were observed in either receptor in MG-63 multicellular tumor spheroids.
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http://dx.doi.org/10.1002/bem.20184DOI Listing
February 2006

Differential control of cholesterol and fatty acid biosynthesis in sensitive and multidrug-resistant LoVo tumor cells.

Anticancer Res 2003 Nov-Dec;23(6C):4737-46

Laboratorio di Ultrastrutture, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161, Rome, Italy.

Multidrug resistance (MDR) describes the decrease in sensitivity of tumor cells to a wide variety of cytotoxic compounds. Although a central role has been ascribed to the P-glycoprotein (Pgp) pump in MDR, lipids also appear to be extremely important. However, their precise role in MDR is not yet fully understood. It was the aim of the present paper to gain a deeper understanding of intracellular lipid equilibrium in both sensitive and MDR tumor cells. In particular, intracellular cholesterol biosynthesis and cholesterol esterification were examined in LoVo-sensitive and Pgp-overexpressing resistant cells. The data presented seem to suggest that the higher synthesis of cholesteryl ester and triglyceride observed in resistant with respect to wild-type cells is due to a greater production of fatty acids in these cells. The results are discussed in view of the possible roles of sterol regulatory element-binding proteins and Pgp in these phenomena.
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April 2004

MG-63 human osteosarcoma cells grown in monolayer and as three-dimensional tumor spheroids present a different metabolic profile: a (1)H NMR study.

FEBS Lett 2004 Jan;557(1-3):148-54

Dipartimento di Tecnologie e Salute, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy.

High resolution proton nuclear magnetic resonance ((1)H NMR) spectroscopy was used to determine if the same cell line (MG-63 human osteosarcoma cells) grown in monolayer or as small (about 50-80 microm in diameter), three-dimensional tumor spheroids with no hypoxic center has different metabolic characteristics. Consequently, the (1)H NMR spectra were obtained from both types of cultures and then compared. The results indicate that the type of cellular spatial array determines specific changes in MG-63 cells. In particular, small but significant differences in lactate and alanine indicating a perturbation in energy metabolism were observed in the two cell models. In addition, although variations in CH(2) and CH(3) groups were also seen, it is not possible at this time to establish if lipid metabolism is truly different in cells and spheroids.
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http://dx.doi.org/10.1016/s0014-5793(03)01466-2DOI Listing
January 2004