Research Center San Raffaele Pisana

Rome, Italy

Research Center San Raffaele Pisana

Rome, Italy
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Filomeni G.,University of Rome Tor Vergata | Cardaci S.,University of Rome Tor Vergata | Da Costa Ferreira A.M.,University of Sao Paulo | Rotilio G.,University of Rome Tor Vergata | And 3 more authors.
Biochemical Journal | Year: 2011

We have demonstrated previously that the complex bis[(2-oxindol-3-ylimino)- 2-(2-aminoethyl)pyridine-N,N′]copper(II), named [Cu(isaepy) 2], induces AMPK (AMP-activated protein kinase)-dependent/p53-mediated apoptosis in tumour cells by targeting mitochondria. In the present study, we found that p38 MAPK (p38 mitogen-activated protein kinase) is the molecular link in the phosphorylation cascade connecting AMPK to p53. Transfection of SH-SY5Y cells with a dominant-negative mutant of AMPK resulted in a decrease in apoptosis and a significant reduction in phospho-active p38 MAPK and p53. Similarly, reverse genetics of p38 MAPK yielded a reduction in p53 and a decrease in the extent of apoptosis, confirming an exclusive hierarchy of activation that proceeds via AMPK/p38 MAPK/p53. Fuel supplies counteracted [Cu(isaepy) 2]-induced apoptosis and AMPK/p38 MAPK/p53 activation, with glucose being the most effective, suggesting a role for energetic imbalance in [Cu(isaepy) 2] toxicity. Co-administration of 3BrPA (3-bromopyruvate), a well-known inhibitor of glycolysis, and succinate dehydrogenase, enhanced apoptosis and AMPK/p38 MAPK/p53 signalling pathway activation. Under these conditions, no toxic effectwas observed in SOD(superoxide dismutase)-overexpressing SH-SY5Y cells or in PCNs (primary cortical neurons), which are, conversely, sensitized to the combined treatment with [Cu(isaepy) 2] and 3BrPA only if grown in low-glucose medium or incubated with the glucose-6-phosphate dehydrogenase inhibitor dehydroepiandrosterone. Overall, the results suggest that NADPH deriving from the pentose phosphate pathway contributes to PCN resistance to [Cu(isaepy) 2] toxicity and propose its employment in combination with 3BrPA as possible tool for cancer treatment. © The Authors Journal compilation © 2011 Biochemical Society.


Filomeni G.,University of Rome Tor Vergata | Desideri E.,University of Rome Tor Vergata | Desideri E.,Research Center San Raffaele Pisana | Cardaci S.,University of Rome Tor Vergata | And 6 more authors.
Autophagy | Year: 2010

Kaempferol, a dietary cancer chemopreventive polyphenol, has been reported to trigger apoptosis in several tumor histotypes, but the mechanism underlying this phenomenon is not fully understood. Here, we demonstrate that in HeLa cells, kaempferol induces energetic failure due to inhibition of both glucose uptake and complex I of the mitochondrial respiratory chain. As adaptive response, cells activate autophagy, the occurrence of which was established cytofluorometrically, upon acridine orange staining, and immunochemically, by following the increase of the autolysosome-associated form of the microtubule-associated protein light chain 3 (LC3-II). Autophagy is an early and reversible process occurring as survival mechanisms against apoptosis. Indeed, chemical inhibition of autophagy, by incubations with monensin, wortmannin, 3-methyladenine, or by silencing Atg5, significantly increases the extent of apoptosis, which takes place via the mitochondrial pathway, and shortens the time in which the apoptotic markers are detectable. We also demonstrate that autophagy depends on the early activation of the AMP-activated protein kinase (AMPK)/mTOR-mediated pathway. The overexpression of dominant negative AMPK results in a decrease of autophagic cells, a decrement of LC3-II levels, and a significant increase of apoptosis. Experiments performed with another carcinoma cell line yielded the same results, suggesting for kaempferol a unique mechanism of action. © 2010 Landes Bioscience.


Cardaci S.,University of Rome Tor Vergata | Filomeni G.,University of Rome Tor Vergata | Rotilio G.,University of Rome Tor Vergata | Rotilio G.,Research Center San Raffaele Pisana | And 2 more authors.
Biochemical Journal | Year: 2010

BH4 (tetrahydrobiopterin) induces neuronal demise via production of ROS (reactive oxygen species). In the present study we investigated the mechanisms of its toxicity and the redox signalling events responsible for the apoptotic commitment in SH-SY5Y neuroblastoma cells and inmouse primary cortical neurons. We identified in p38MAPK/p53 a BH4-responsive pro-apoptotic signalling axis, as demonstrated by the recovery of neuronal viability achieved by gene silencing or pharmacological inhibition of both p38MAPK and p53. BH4-induced oxidative stress was characterized by a decrease in the GSH/GSSG ratio, an increase in protein carbonylation and DNA damage. BH4 toxicity and the redox-activated apoptotic pathway were counteracted by the H2O2-scavengers catalase and N-acetylcysteine and enhanced by the GSH neo-synthesis inhibitor BSO (buthionine sulfoximine). We also demonstrated that BH4 impairs glucose uptake and utilization, which was prevented by catalase administration. This effect contributes to the neuronal demise, exacerbating BH4-induced nuclear damage and the activation of the pro-apoptotic p38MAPK/p53 axis. Inhibition of glucose uptake was also observed upon treatment with 6-hydroxydopamine, another redox-cycling molecule, suggesting a common mechanism of action for autooxidizable neurotoxins. © The Authors.


Desideri E.,University of Rome Tor Vergata | Filomeni G.,University of Rome Tor Vergata | Filomeni G.,Research Center San Raffaele Pisana | Ciriolo M.R.,University of Rome Tor Vergata | Ciriolo M.R.,Research Center San Raffaele Pisana
Autophagy | Year: 2012

Glutathione (γ-L-glutamyl-L-cysteinyl-glycine, GSH ) is the most abundant low molecular weight, thiol-containing compound within the cells and has a primary role in the antioxidant defense and intracellular signaling. Here we demonstrated that nutrient deprivation led to a significant decrease of intracellular GSH levels in three different carcinoma cell lines. This phenomenon was dependent on ABCC 1-mediated GSH extrusion, along with GCL inhibition and, to a minor extent, the formation of GSH -protein mixed disulfides that synergistically contributed to the modulation of autophagy by shifting the intracellular redox state toward more oxidizing conditions. Modulation of intracellular GSH by inhibiting its de novo synthesis through incubation with buthionine sulfoximine, or by maintaining its levels through GSH ethyl ester, affected the oxidation of protein thiols, such as PRDXs and consequently the kinetics of autophagy activation. We also demonstrated that thiol-oxidizing or -alkylating agents, such as diamide and diethyl maleate activated autophagy, corroborating the evidence that changes in thiol redox state contributed to the occurrence of autophagy. © 2012 Landes Bioscience.


Di Giacomo G.,Research Center San Raffaele Pisana | Rizza S.,University of Rome Tor Vergata | Montagna C.,Research Center San Raffaele Pisana | Filomeni G.,Research Center San Raffaele Pisana | Filomeni G.,University of Rome Tor Vergata
International Journal of Cell Biology | Year: 2012

S-nitrosylation is a posttranslational modification of cysteine residues that has been frequently indicated as potential molecular mechanism governing cell response upon redox unbalance downstream of nitric oxide (over)production. In the last years, increased levels of S-nitrosothiols (SNOs) have been tightly associated with the onset of nitroxidative stress-based pathologies (e.g., cancer and neurodegeneration), conditions in which alterations of mitochondrial homeostasis and activation of cellular processes dependent on it have been reported as well. In this paper we aim at summarizing the current knowledge of mitochondria-related proteins undergoing S-nitrosylation and how this redox modification might impact on mitochondrial functions, whose impairment has been correlated to tumorigenesis and neuronal cell death. In particular, emphasis will be given to the possible, but still neglected implication of denitrosylation reactions in the modulation of mitochondrial SNOs and how they can affect mitochondrion-related cellular process, such as oxidative phosphorylation, mitochondrial dynamics, and mitophagy. Copyright © 2012 Giuseppina Di Giacomo et al.


Filomeni G.,University of Rome Tor Vergata | Filomeni G.,Research Center San Raffaele Pisana | Piccirillo S.,Research Center San Raffaele Pisana | Rotilio G.,University of Rome Tor Vergata | And 3 more authors.
Biochemical Pharmacology | Year: 2012

Redox changes are often reported as causative of neoplastic transformation and chemoresistance, but are also exploited as clinical tools to selectively kill tumor cells. We previously demonstrated that gastrointestinal-derived tumor histotypes are resistant to ROS-based treatments by means of the redox activation of Nrf2, but highly sensitive to disulfide stressors triggering apoptosis via the redox induction of Trx1/p38 MAPK/p53 signaling pathway. Here, we provide evidence that neuroblastoma SH-SY5Y has a complete opposite behavior, being sensitive to H 2O 2, but resistant to the glutathione (GSH)-oxidizing molecule diamide. Consistent with these observations, the apoptotic pathway activated upon H 2O 2 treatment relies upon Trx1 oxidation, and is mediated by the p38 MAPK/p53 signaling axis. Pre-treatment with different antioxidants, pharmacological inhibitor of p38 MAPK, or small interfering RNA against p53 rescue cell viability. On the contrary, cell survival to diamide relies upon redox activation of Nrf2, in a way independent on Keap1 oxidation, but responsive to ERK1/2 activation. Chemical inhibition of GSH neo-synthesis or ERK1/2 phosphorylation, as well as overexpression of the dominant-negative form of Nrf2 sensitizes cells to diamide toxicity. In the searching for the molecular determinant(s) unifying these phenomena, we found that SH-SY5Y cells show high GSH levels, but exhibit very low GPx activity. This feature allows to efficiently buffer disulfide stress, but leaves them being vulnerable to H 2O 2-mediated insult. The increase of GPx activity by means of selenium supplementation or GPx1 ectopic expression completely reverses death phenotype, indicating that the response of tumor cells to diverse oxidative stimuli deeply involves the entire GSH redox system. © 2012 Elsevier Inc. All rights reserved.


PubMed | Research Center San Raffaele Pisana
Type: | Journal: International journal of cell biology | Year: 2012

S-nitrosylation is a posttranslational modification of cysteine residues that has been frequently indicated as potential molecular mechanism governing cell response upon redox unbalance downstream of nitric oxide (over)production. In the last years, increased levels of S-nitrosothiols (SNOs) have been tightly associated with the onset of nitroxidative stress-based pathologies (e.g., cancer and neurodegeneration), conditions in which alterations of mitochondrial homeostasis and activation of cellular processes dependent on it have been reported as well. In this paper we aim at summarizing the current knowledge of mitochondria-related proteins undergoing S-nitrosylation and how this redox modification might impact on mitochondrial functions, whose impairment has been correlated to tumorigenesis and neuronal cell death. In particular, emphasis will be given to the possible, but still neglected implication of denitrosylation reactions in the modulation of mitochondrial SNOs and how they can affect mitochondrion-related cellular process, such as oxidative phosphorylation, mitochondrial dynamics, and mitophagy.

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