73 results on '"RUOCCO MR"'
Search Results
2. Reactive oxygen species, Ki-Ras, and mitochondrial superoxide dismutase cooperate in nerve growth factor-induced differentiation of PC12 cells
- Author
-
Cassano S, Agnese S, D'Amato V, Papale M, Garbi C, Castagnola P, Ruocco MR, Castellano I, De Vendittis E, Santillo M, Amente S, Porcellini A, and Avvedimento EV.
- Abstract
Nerve growth factor (NGF) induces terminal differentiation in PC12, a pheochromocytoma-derived cell line. NGF binds a specific receptor on the membrane and triggers the ERK1/2 cascade, which stimulates the transcription of neural genes. We report that NGF significantly affects mitochondrial metabolism by reducing mitochondrial-produced reactive oxygen species and stabilizing the electrochemical gradient. This is accomplished by stimulation of mitochondrial manganese superoxide dismutase (MnSOD) both transcriptionally and post-transcriptionally via Ki-Ras and ERK1/2. Activation of MnSOD is essential for completion of neuronal differentiation because 1) expression of MnSOD induces the transcription of a neuronal specific promoter and neurite outgrowth, 2) silencing of endogenous MnSOD by small interfering RNA significantly reduces transcription induced by NGF, and 3) a Ki-Ras mutant in the polylysine stretch at the COOH terminus, unable to stimulate MnSOD, fails to induce complete differentiation. Overexpression of MnSOD restores differentiation in cells expressing this mutant. ERK1/2 is also downstream of MnSOD, as a SOD mimetic drug stimulates ERK1/2 with the same kinetics of NGF and silencing of MnSOD reduces NGF-induced late ERK1/2. Long term activation of ERK1/2 by NGF requires SOD activation, low levels of hydrogen peroxide, and the integrity of the microtubular cytoskeleton. Confocal immunofluorescence shows that NGF stimulates the formation of a complex containing membrane-bound Ki-Ras, microtubules, and mitochondria. We propose that active NGF receptor induces association of mitochondria with plasma membrane. Local activation of ERK1/2 by Ki-Ras stimulates mitochondrial SOD, which reduces reactive oxygen species and produces H(2)O(2). Low and spatially restricted levels of H(2)O(2) induce and maintain long term ERK1/2 activity and ultimately differentiation of PC12 cells.
- Published
- 2010
3. KEY AMINO ACID POSITIONS INVOLVED IN THE ACTIVITY, HEAT STABILITY AND COVALENT MODIFICATION OF RAT MITOCHONDRIAL MANGANESE SUPEROXIDE DISMUTASE
- Author
-
DE VENDITTIS, E, Castellano, I, DE VENDITTIS, A, Cecere, F, Cotugno, R, Chambery, A, DI MARO, A, Masullo, Mariorosario, and Ruocco, Mr
- Published
- 2009
4. The dimeric structure of Sulfolobus solfataricus thioredoxin A2 and the basis of its thermostability
- Author
-
Ruggiero, A, Masullo, Mariorosario, Marasco, D, Ruocco, Mr, Grimaldi, P, Arcari, P, Zagari, A, Vitagliano, L., Ruggiero, A., Masullo, Mariorosario, Marasco, Daniela, Ruocco, MARIA ROSARIA, Grimaldi, P., Arcari, Paolo, Zagari, Adriana, and Vitagliano, Luigi
- Subjects
Hot Temperature ,Sequence Homology, Amino Acid ,protein structure-stability ,Protein Conformation ,Protein Stability ,Archaeal Proteins ,Circular Dichroism ,Molecular Sequence Data ,protein structure-function ,Crystallography, X-Ray ,Thioredoxins ,protein dimer interface ,redox state ,Sulfolobus solfataricus ,Thermodynamics ,disulfide bond ,Amino Acid Sequence ,Protein Structure, Quaternary ,Dimerization ,X-ray crystallography - Abstract
The crystallographic characterization of SsTrxA2 reveals that the redox active center is buried by the dimeric interface of the protein. This suggests that pro- ductive interactions of the protein with its biological partners require a preliminary dissociation of the dimer. Although further studies are needed to fully address the biological implications of this finding, it can be specu- lated that this peculiar feature of SsTrxA2 may be impor- tant for the regulation of the protein activity. A struc- tural comparison of SsTrxA2 with the closely-related StTrxA2 suggests that minimal aminoacids substitutions may have a strong impact on the oligomerization state of the protein.
- Published
- 2009
5. Involvement of mitochondrial Mn-SOD in the defence of diclofenac-induced apoptosis in the SH-SY5Y neuroblastoma cell line
- Author
-
DE VENDITTIS, E, Zappelli, C, Cecere, F, Iuliano, A, Castellano, I, Grimaldi, P, Masullo, Mariorosario, Ruocco, Mr, DE VENDITTIS, Emmanuele, Zappelli, C., Cecere, F., Iuliano, A., Castellano, I., Grimaldi, P., Masullo, M., Ruocco, MARIA ROSARIA, Zappelli, C, Cecere, Francesca, Iuliano, A, Castellano, Immacolata, Grimaldi, P, and Masullo, Mariorosario
- Subjects
Neuroblastoma ,Diclofenac ,Apoptosis ,Superoxide dismutase - Abstract
Manganese superoxide dismutase (Mn-SOD) is a mitochondrial enzyme that dismutates two superoxide radicals into hydrogen peroxide and molecular oxygen. This enzyme is crucial for the defence against cellular reactive oxygen species (ROS), functioning as an essential anti-oxidant enzyme protecting critical targets from superoxide modification. Diclofenac is a non-steroidal antiinflammatory drug (NSAID) frequently used as an analgesic and in the treatment of rheumatic diseases; more recently, a number of experimental and clinical studies suggested its possible usage as an anticancer agent. Many reports have shown that diclofenac, as well as other NSAIDs, induce apoptosis in a variety of cell lines such as hepatic, gastric and renal, thus influencing their cellular redox state. On the other hand, a few data are available regarding the effects of these drugs on neuronal cells.Here we investigate diclofenac-induced apoptosis in the neuroblastoma cell line SH-SY5Y and the possible involvement of Mn-SOD in this process. Flow cytometric analysis of SH-SY5Y cells treated with diclofenac revealed a time- and dose-dependent increase of apoptotic nuclei. Moreover, the treatment of SH-SY5Y with diclofenac induces an increase in cellular ROS levels, as measured by oxidation-sensitive fluorescence probes. To evaluate the involvement of Mn-SOD in the cytotoxic effect induced by diclofenac, both protein level and enzyme activity have been evaluated in protein extracts obtained from SH-SY5Y cells grown in the absence or in the presence of diclofenac. Western blotting analysis showed that diclofenac decreases the levels of Mn-SOD; concomitantly, its enzymatic activity is reduced, as measured by a colorimetric assay on non-denaturing polyacrylamide gels. However, diclofenac does not affect the mRNA levels of Mn-SOD, as determined by RT-PCR experiments. When SH-SY5Y cells were cultured in the presence of a recombinant thioredoxin from the hyperthermophilic archaeon Sulfolobus solfataricus, a marked attenuation of the diclofenac-induced apoptosis was observed, together with an increase of the Mn-SOD levels. Furthermore, diclofenac induces a reduction of the mitochondrial membrane potential and a release of cytocrome c from mitochondria. These data suggest that mitochondria are involved in the diclofenac-induced apoptosis in SH-SY5Y neuroblastoma cell line and point to a possible role of Mn-SOD in this process.
- Published
- 2009
6. The thioredoxin system in the archaeon Sulfolobus solfataricus
- Author
-
Grimaldi, P, Lanzotti, M, Ruocco, Mr, Ruggiero, A, Arcari, P, Zagari, A, Vitagliano, L, Masullo, Mariorosario, P., Grimaldi, M., Lanzotti, Ruocco, MARIA ROSARIA, A., Ruggiero, Arcari, Paolo, A., Zagari, L., Vitagliano, and M., Masullo
- Published
- 2007
7. The thioredoxin system in the psychrophilic eubacterium Pseudoalteromonas haloplanktis
- Author
-
Cotugno, R, Salomone, G, Falasca, P, Evangelista, G, Ruocco, Mr, Masullo, Mariorosario, Raimo, G, and DE VENDITTIS, E.
- Published
- 2007
8. A highly reactive cysteine is involved in the antioxidant function of superoxide dismutase from the psychrophilic eubacterium Pseudoalteromonas haloplanktis
- Author
-
Castellano, I, Ruocco, Mr, DI MARO, A, Chambery, A, Parente, A, Masullo, Mariorosario, DE VENDITTIS, E., Castellano, I, Ruocco, MARIA ROSARIA, DI MARO, A, Chambery, A, Parente, A, Masullo, M, and DE VENDITTIS, Emmanuele
- Subjects
Pseudoalteromonas haloplanktis ,psychrophile ,cysteine reactivity ,peroxynitrite resistance ,Superoxide dismutase - Abstract
Superoxide dismutase (SOD) is a metal enzyme playing a key role in the cell defence mechanism against the reactive oxygen species (ROS). High levels of ROS are involved in several pathologic states such as senescence, cell death and cancer. SODs are widely studied as potential therapeutic agents in pathologies correlated with oxidative stress. SODs are usually classified in two main structurally unrelated families on the basis of their metal content in the active site. The family of Fe- and Mn-SODs is found in eubacteria, archaea and mitochondria and this ubiquitous distribution probably reflects the most crucial antioxidant function of this enzyme. The modulation of SOD activity is essential during oxidative stress; data have been reported on the regulation of Fe- and Mn-SODs by covalent modifications. SODs isolated from extremophilic sources represent models to study the structure-function relationships of proteins adapted to extreme conditions, and the role of modifications in the enyme function. This report describes the biochemical and functional characterization of superoxide dismutase from the psychrophilic eubacterium Pseudoalteromonas haloplanktis (PhSOD). The enzyme, an iron-containing homodimer, is endowed with a high specific activity even at low temperatures, and displays a thermal resistance well above the growth temperature of P. haloplanktis. PhSOD is very sensitive to peroxynitrite, a physiological inactivator of mitochondrial Mn-SOD, acting on Tyr34. PhSOD has a unique and highly reactive cysteine residue (Cys57), located in a variable region of the protein. The three-dimensional model of PhSOD indicates that the structural organization of this region discriminates between dimeric and tetrameric SODs. Cys57 forms a disulfide bond with beta-mercaptoethanol in native conditions, whereas in specific denaturing conditions, two subunits of PhSOD form a covalent dimer. The modification by beta-mercaptoethanol has little effect on the enzyme activity, but significantly affects the tyrosine nitration, protecting the enzyme from inactivation. Covalent modification by cellular thiols, such as glutathione, on proteins containing highly reactive –SH groups plays an essential role in the regulation of the cellular redox state. Indeed, we have found that PhSOD is covalently modified by glutathione in vitro. Further investigation concerns the possible modification of PhSOD by glutathione in P. haloplanktis cells, upon induction of oxidative stress.
- Published
- 2006
9. 3D STRUCTURAL CHARACTERIZATION OF A THERMOSTABLE THIOREDOXIN REDUCTASE ISOLATED FROM SULFOLOBUS SOLFATARICUS
- Author
-
Ruggiero, A, Ruocco, Mr, Grimaldi, P, Masullo, Mariorosario, and Zagari, A.
- Published
- 2006
10. THE THIOREDOXIN SYSTEM IN SULFOLOBUS SOLFATARICUS
- Author
-
Ruggiero, A, Ruocco, Mr, Grimaldi, P, Masullo, Mariorosario, Zagari, A, and Vitagliano, L.
- Published
- 2006
11. Primary structure of superoxide dismutase from Pseudoalteromonas haloplanktis by a combination of automatic Edman degradation and ESI/Q-TOF mass spectrometry
- Author
-
DI MARO, A, Chambery, A, Castellano, I, Ruocco, Mr, Masullo, Mariorosario, DE VENDITTIS, E, Parente, A., DI MARO, A, Chambery, A, Castellano, Immacolata, Ruocco, MARIA ROSARIA, Masullo, M, DE VENDITTIS, Emmanuele, and Parente, A.
- Subjects
psychrophile ,Pseudoalteromonas haloplankti ,covalent modification ,Superoxide dismutase ,amino acid sequence - Abstract
Superoxide dismutase (SOD) is a metalloenzyme that has a protective effect against toxic superoxide radicals in both aerobic and anaerobic organisms. SODs have been classified into two families on the basis of their different structural folding and metal content (Cu/Zn in one family and Fe or Mn in the other one). SODs isolated from extremophilic organisms are suitable models to study the structure-function relationships and the molecular and evolutive mechanisms for the adaptation of proteins to extreme environments. We have previously isolated a SOD from the psychrophilic eubacterium Pseudoalteromonas haloplanktis (PhSOD), isolated from Antarctic marine sediments and adapted to grow at low temperatures. This enzyme has a specific activity of 6500 U/mg and according to preliminary characterization, PhSOD could be classified as a Fe-SOD. In this communication the rapid characterization of primary structure of PhSOD was determined using a combined approach based on automatic Edman degradatiion and electrospray ionization mass spectrometry (ESI-MS/MS). The information gathered by this approach combined with automated recording and interpretatiion of data enabled full primary structure determination of SOD with minimal time and material consumption (200 ug/10 nmoles). The primary structure of PhSOD was obtained using the following experimental steps: i) verification of the protein purity and identity by SDS-PAGE and ESI-MS; ii) enzymatic cleavage by endoproteinase Asp-N; iii) sequence determination of Asp-N peptides by Edmann degradation and iv) overlapping with tryptic peptides analysed by Q-Tof mass spectrometry and by homology with reference proteins. ESI-MS analysis of native PhSOD, obtained from RP-HPLC as the last purification step, showed that its molecular mass was 21328.50 +/- 0.40. Automated Edman degradation of peptides obtained from endoproteinase Asp-N and separated by RP-HPLC, provided most of the amino acid sequence of PhSOD. However, with this first set of data, various amino acid residues were not determined. In addition, there was the need to confirm the presence, in some positions, of seryl and threonyl residues which were obtained in low yield by automatic Edman degradation. Therefore, to complete and confirm the amino acid sequence, we decided to map the entire sequence by mass spectrometry, analysing a new set of peptides derived from trypsin hydrolysis. These peptides were separated by CapLC and analysed on-line by Q-Tof, which provided their molecular masses and the "de novo sequencing data" when it was necessary. However, the sequence of PhSOD is not complete. It remains to be assigned residue 57, which was not identified during the automatic Edman degradation. Indeed, no canonic PTH-amino acid was present. This implicates the presence of post-modification, which is likely in this class of enzymes. Future research plan includes the determination of such residue using Q-Tof mass spectrometry.
- Published
- 2005
12. THE THIOREDOXIN SYSTEM IN THE ARCHAEON SULFOLOBUS SOLFATARICUS
- Author
-
Masullo, Mariorosario, Ruocco, Mr, Ruggiero, A, Grimaldi, P, and Arcari, P.
- Published
- 2005
13. A 35 kDa NAD(P)H OXIDASE PREVIOUSLY ISOLATED FROM THE ARCHAOEN SULFOLOBUS SOLFATARICUS IS INSTEAD A THIOREDOXIN REDUCTASE
- Author
-
Ruocco, Mr, Ruggiero, A, Arcari, P, and Masullo, Mariorosario
- Published
- 2004
14. Hemodialysis related induction of interleukin 6 production by peripheral blood mononuclear cells
- Author
-
MEMOLI B, LIBETTA C, RAMPINO T, DAL CANTON A, CONTE G, SCALA G, RUOCCO MR, ANDREUCCI, VITTORIO EMANUELE, Memoli, B, Libetta, C, Rampino, T, DAL CANTON, A, Conte, G, Scala, G, Ruocco, Mr, and Andreucci, VITTORIO EMANUELE
- Published
- 1992
15. Impaired generation of bone marrow B lymphocytes in mice deficient in C/EBPbeta
- Author
-
Chen X, Liu W, Ambrosino C, Ruocco MR, Valeria Poli, Romani L, Quinto I, Barbieri S, Kl, Holmes, Venuta S, and Scala G
- Subjects
DNA-Binding Proteins ,B-Lymphocytes ,Mice ,Bone Marrow ,Interleukin-7 ,CCAAT-Enhancer-Binding Proteins ,Animals ,Nuclear Proteins ,Cell Count ,Flow Cytometry ,Cells, Cultured ,Mice, Mutant Strains - Abstract
CAAT/enhancer binding proteins (C/EBP) are a family of transcription factors that mediates adipocyte differentiation and the regulation of genes expressed in immune responses and inflammation, such as interleukin-6 (IL-6), IL-8, and granulocyte colony-stimulating factor (G-CSF). We investigated the role of C/EBPbeta (NF-IL6) in the generation of bone marrow B lymphocytes by taking advantage of C/EBPbeta-/- mice. We found that the expansion of bone marrow (BM) B lymphocytes was impaired in long-term lymphoid cultures from C/EBPbeta-/- mice. Consistent with this finding, the number of BM B cells was decreased in C/EBPbeta-/- mice. Both the levels of IL-7 gene expression and bioactive IL-7 from BM stromal cells were decreased in C/EBPbeta-/- mice. Furthermore, the proliferative responsiveness of BM B-cell precursors to IL-7 was also reduced as compared to wild-type mice, indicating that C/EBPbeta is required for the generation of BM B cells induced by IL-7. Accordingly, IL-7 stimulates the C/EBPbeta DNA-binding activity of normal BM pre-B lymphocytes as well as of 70Z/3 pre-B cells. These results point to C/EBPbeta as a critical signaling molecule in BM B lymphopoiesis.
16. Influence of Fibroblasts on Mammary Gland Development, Breast Cancer Microenvironment Remodeling, and Cancer Cell Dissemination
- Author
-
Daniela Russo, Alessandro Arcucci, Stefania Masone, Antonello Accurso, Eleonora Vecchio, Nunzia Martucci, Stefania Montagnani, Maria Rosaria Ruocco, Giuseppe Fiume, Luigi Insabato, Angelica Avagliano, Avagliano, Angelica, Fiume, G, Ruocco, Mr, Martucci, Nunzia, Vecchio, Eleonora, Insabato, Luigi, Russo, Daniela, Accurso, Antonello, Masone, Stefania, Montagnani, Stefania, and Arcucci, Alessandro
- Subjects
0301 basic medicine ,mammary gland ,Cancer Research ,Stromal cell ,Mammary gland ,Review ,Biology ,medicine.disease_cause ,breast cancer microenvironment ,lcsh:RC254-282 ,Metastasis ,Extracellular matrix ,ECM remodeling ,03 medical and health sciences ,0302 clinical medicine ,fibroblasts ,medicine ,metastasis ,Fibroblast ,Cancer ,lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,medicine.disease ,Cell biology ,030104 developmental biology ,medicine.anatomical_structure ,Oncology ,030220 oncology & carcinogenesis ,Cancer cell ,Carcinogenesis ,breast cancer associated fibroblasts (BCAFs) - Abstract
The stromal microenvironment regulates mammary gland development and tumorigenesis. In normal mammary glands, the stromal microenvironment encompasses the ducts and contains fibroblasts, the main regulators of branching morphogenesis. Understanding the way fibroblast signaling pathways regulate mammary gland development may offer insights into the mechanisms of breast cancer (BC) biology. In fact, the unregulated mammary fibroblast signaling pathways, associated with alterations in extracellular matrix (ECM) remodeling and branching morphogenesis, drive breast cancer microenvironment (BCM) remodeling and cancer growth. The BCM comprises a very heterogeneous tissue containing non-cancer stromal cells, namely, breast cancer-associated fibroblasts (BCAFs), which represent most of the tumor mass. Moreover, the different components of the BCM highly interact with cancer cells, thereby generating a tightly intertwined network. In particular, BC cells activate recruited normal fibroblasts in BCAFs, which, in turn, promote BCM remodeling and metastasis. Thus, comparing the roles of normal fibroblasts and BCAFs in the physiological and metastatic processes, could provide a deeper understanding of the signaling pathways regulating BC dissemination. Here, we review the latest literature describing the structure of the mammary gland and the BCM and summarize the influence of epithelial-mesenchymal transition (EpMT) and autophagy in BC dissemination. Finally, we discuss the roles of fibroblasts and BCAFs in mammary gland development and BCM remodeling, respectively.
- Published
- 2020
17. Rat Mitochondrial Manganese Superoxide Dismutase: Amino Acid Positions Involved in Covalent Modifications, Activity, and Heat Stability
- Author
-
Maria Rosaria Ruocco, Francesca Cecere, Emmanuele De Vendittis, Alberto De Vendittis, Giuseppe Parlato, Angela Chambery, Roberta Cotugno, Mariorosario Masullo, Andzelika Michniewicz, Enrico V. Avvedimento, Immacolata Castellano, Antimo Di Maro, Castellano, I, Cecere, F, DE VENDITTIS, A, Cotugno, R, Chambery, Angela, DI MARO, Antimo, Michniewicz, A, Parlato, G, Masullo, M, Avvedimento, Ev, DE VENDITTIS, E, Ruocco, Mr, De Vendittis, A, Chambery, A, Di Maro, A, DE VENDITTIS, Emmanuele, and Ruocco, MARIA ROSARIA
- Subjects
Spectrometry, Mass, Electrospray Ionization ,Hot Temperature ,Glutamine ,Biophysics ,Superoxide dismutase ,Biochemistry ,Cofactor ,Rat SOD2 ,Mutagenic analysis ,Post-translational modifications ,Thermostability ,Mitochondrial Proteins ,Biomaterials ,chemistry.chemical_compound ,Enzyme Stability ,Serine ,Animals ,Sulfhydryl Compounds ,Amino Acids ,Phosphorylation ,chemistry.chemical_classification ,Manganese ,biology ,Kinase ,Organic Chemistry ,Mutagenesis ,General Medicine ,Glutathione ,Superoxide dismutase Rat SOD2 Mutagenic analysis Post–translational modifications Thermostability ,Recombinant Proteins ,Rats ,Amino acid ,Kinetics ,Enzyme ,chemistry ,Mutagenic analysi ,Mutagenesis, Site-Directed ,biology.protein ,Tyrosine ,Post-translational modification ,Protein Processing, Post-Translational ,Protein Binding - Abstract
The role of three amino acid residues (Q143, Y34, S82) of rat mitochondrial superoxide dismutase (ratSOD2) in the enzymatic activity, thermostability, and post-translational modification of the enzyme was investigated through site-directed mutagenesis studies. Six recombinant forms of the enzyme were produced, carrying the Q143 or H143 residue with or without the Y34F or S82A replacement. All proteins bound manganese as active cofactor and were organized as homotetramers. The greatest effect on the activity (sixfold reduction) was observed in ratSOD2 forms containing the H143 variant, whereas Y34F and S82A substitutions moderately reduced the enzymatic activity compared to the Q143 form. Heat inactivation studies showed the high thermo-tolerance of ratSOD2 and allowed an evaluation of the related activation parameters of the heat inactivation process. Compared to Q143, the H143 variant was significantly less heat stable and displayed moderately lower enthalpic and entropic factors; the Y34F substitution caused a moderate reduction of heat stability, whereas the S82A replacement slightly improved the thermo-tolerance of the Q143 variant; both substitutions significantly increased enthalpic and entropic factors of heat inactivation, the greatest effect being observed with S82A substitution. All recombinant forms of ratSOD2 were glutathionylated in Escherichia coli, a feature pointing to the high reactivity of ratSOD2 toward glutathione. Moreover, the S82 position of the enzyme was phosphorylated in an in vitro system containing human mitochondrial protein extracts as source of protein kinases. These data highlight the role played by some residues in ratSOD2 and suggest a fine regulation of the enzyme occurring in vivo. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 1215–1226, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com
- Published
- 2009
18. Glutathionylation of the iron superoxide dismutase from the psychrophilic eubacterium Pseudoalteromonas haloplanktis
- Author
-
Antimo Di Maro, Angela Chambery, Giuseppe Parlato, Francesca Cecere, Immacolata Castellano, Maria Rosaria Ruocco, Mariorosario Masullo, Andzelika Michniewicz, Emmanuele De Vendittis, Castellano, I, Ruocco, MARIA ROSARIA, Cecere, F, DI MARO, A, Chambery, A, Michniewicz, A, Parlato, G, Masullo, M, DE VENDITTIS, Emmanuele, Ruocco, Mr, DI MARO, Antimo, Chambery, Angela, and DE VENDITTIS, E.
- Subjects
Antioxidant ,medicine.medical_treatment ,Biophysics ,S-glutathionylation ,Superoxide dismutase ,Biochemistry ,Mass Spectrometry ,Peroxynitrite ,Analytical Chemistry ,Pseudoalteromonas haloplanktis ,chemistry.chemical_compound ,Peroxynitrous Acid ,medicine ,Escherichia coli ,Cysteine ,S-Glutathionylation ,Molecular Biology ,Chromatography, High Pressure Liquid ,Psychrophile ,biology ,Chemistry ,Eubacterium ,Nitrotyrosine ,Pseudoalteromonas haloplankti ,Glutathione ,biology.organism_classification ,Recombinant Proteins ,Enzyme Activation ,Pseudoalteromonas ,biology.protein ,Tyrosine ,Electrophoresis, Polyacrylamide Gel ,Mutant Proteins - Abstract
Our previous work showed that the adduct between beta-mercaptoethanol and the single cysteine residue (Cys57) in superoxide dismutase from the psychrophilic eubacterium Pseudoalteromonas haloplanktis (PhSOD) reduces the enzyme inactivation by peroxynitrite. In this work, immunoblotting experiments prove that peroxynitrite inactivation of PhSOD involves formation of nitrotyrosine residue(s). In order to study the role of Cys57 as a redox-sensor residue modifiable by cellular thiols, a recombinant PhSOD and two Cys57 mutants were produced and characterized. Recombinant and mutant enzymes share similar activity and peroxynitrite inactivation, but different reactivity towards three glutathione forms. Indeed, oxidized glutathione and S-nitrosoglutathione, but reduced glutathione, lead to S-glutathionylation of recombinant PhSOD. This new covalent modification for a Fe-SOD does not occur in both Cys57 mutants, thus indicating that its target is Cys57. Moreover, mass spectrometry analysis confirmed that S-glutathionylation of Cys57 takes place also with endogenous PhSOD. Formation of this mixed disulfide in PhSOD protects the enzyme from tyrosine nitration and peroxynitrite inactivation. PhSOD undergoes S-glutathionylation during its overproduction in E. coli cells and in a growing culture of P. haloplanktis. In both cases the extent of glutathionylated PhSOD is enhanced upon cell exposure to oxidative agents. We suggest that S-glutathionylation of PhSOD could represent a further cold-adaptation strategy to improve the antioxidant cellular defence mechanism. (c) 2008 Elsevier B.V All rights reserved.
- Published
- 2008
19. Psychrophilic superoxide dismutase from Pseudoalteromonas haloplanktis: biochemical characterization and identification of a highly reactive cysteine residue
- Author
-
Giuseppe Parlato, Mariorosario Masullo, Augusto Parente, Maria Rosaria Ruocco, A. Di Maro, M.T. Di Martino, Angela Chambery, E De Vendittis, Immacolata Castellano, Castellano, I, DI MARO, A, Ruocco, MARIA ROSARIA, Chambery, A, Parente, A, DI MARTINO M., T, Parlato, G, Masullo, M, DE VENDITTIS, Emmanuele, DI MARO, Antimo, Ruocco, Mr, Chambery, Angela, DI MARTINO, Mt, and DE VENDITTIS, E.
- Subjects
Models, Molecular ,Molecular Sequence Data ,Superoxide dismutase ,Biochemistry ,Mass Spectrometry ,Pseudoalteromonas haloplanktis ,Residue (chemistry) ,chemistry.chemical_compound ,psychrophilic enzyme ,sulfhydryl reactivity ,Pseudoalteromonas haloplankti ,Enzyme Stability ,Amino Acid Sequence ,Cysteine ,Enzyme Inhibitors ,chemistry.chemical_classification ,biology ,Edman degradation ,Molecular mass ,Chemistry ,covalent modification ,Temperature ,General Medicine ,biology.organism_classification ,Amino acid ,Molecular Weight ,Pseudoalteromonas ,biology.protein ,Sodium azide ,Pseudoalteromonas haloplanktis, Psychrophilic enzyme ,Covalent modification ,Sequence Alignment - Abstract
A psychrophilic superoxide dismutase (SOD) has been characterized from the Antarctic eubacterium Pseudoalteromonas haloplanktis (Ph). PhSOD is a homodimeric iron-containing enzyme and displays a high specific activity, even at low temperature. The enzyme is inhibited by sodium azide and inactivated by hydrogen peroxide; it is also very sensitive to peroxynitrite, a physiological inactivator of the human mitochondrial Mn-SOD. Even though PhSOD is isolated from a cold-adapted micro-organism, its heat stability is well above the maximum growth temperature of P. haloplanktis, a feature common to other Fe- and Mn-SODs. The primary structure of PhSOD was determined by a combination of mass spectrometry and automated Edman degradation. The polypeptide chain is made of 192 amino acid residues, corresponding to a molecular mass of 21251 Da. The alignment with other Fe- and Mn-SODs showed a high amino acid identity with Fe-SOD from Vibrio cholerae (79%) and Escherichia coli (70%). A significant similarity is also shared with human mitochondrial Mn-SOD. PhSOD has the unique and highly reactive Cys57 residue, located in a variable region of the protein. The three-dimensional model of the PhSOD monomer indicates that Cys57 is included in a region, whose structural organization apparently discriminates between dimeric and tetrameric SODs. This residue forms a disulfide adduct with β-mercaptoethanol, when this reducing agent is added in the purification procedure. The reactivity of Cys57 leads also to the formation of a disulfide bridge between two PhSOD subunits in specific denaturing conditions. The possible modification of Cys57 by physiological thiols, eventually regulating the PhSOD functioning, is discussed. © 2006 Elsevier Masson SAS. All rights reserved.
- Published
- 2006
20. Superoxide dismutase from the psychrophilic Antarctic eubacterium Pseudoalteromonas haloplanktis
- Author
-
CASTELLANO, IMMACOLATA, RUOCCO, MARIA ROSARIA, DE VENDITTIS, EMMANUELE, MASULLO M, DI MARO A, CHAMBERY A, Castellano, I, Ruocco, Mr, Masullo, M, DI MARO, Antimo, Chambery, Angela, De Vendittis, E., Castellano, I., Ruocco, MARIA ROSARIA, Masullo, M., Di Maro, A., Chambery, A., DE VENDITTIS, Emmanuele, Castellano, Immacolata, DI MARO, A, and Chambery, A
- Subjects
Pseudoalteromonas haloplanktis ,psychrophile ,Superoxide dismutase - Abstract
The antioxidant function of Fe- and Mn-containing superoxide dismutases (SOD) observed under constraints from extreme rather than mild cellular conditions could reflect an adaptive evolution to oxygen tolerance in the structural organisation of this class of enzymes. For instance, the mitochondrial human Mn-SOD and the hyperthermophilic archaeal Fe-SOD from Sulfolobus solfataricus (SsSOD) share a similar structural organisation. Further studies on members of this ubiquitous enzyme isolated from differently adapted micro–organisms could give useful information on possible adaptive mechanisms in the structure-function relationships of this SOD family. For this reason, this enzyme has been purified and characterised from Pseudoalteromonas haloplanktis, a psychrophilic eubacterium isolated from marine Antarctic sediments. Two chromatographic steps on DEAE-Sepharose and HTP allowed to purify SOD from P. haloplanktis (PhSOD) to homogeneity. The relative molecular weight of the purified enzyme estimated by SDS-PAGE is about 20,000. As SsSOD, also PhSOD shows a homotetrameric structure, as determined by gel filtration. PhSOD has an unusual thermal stability for a psycrophilic enzyme, as evaluated by its half-life of 10 min at 52°C. Similar results were obtained by UV-melting curves. Enzymatic assays showed that PhSOD has a specific activity of 6500 U/mg. The enzyme is inactivated by hydrogen peroxide and it is inhibited by sodium azide, whereas PMSF, a specific inactivator of the archaeal SsSOD, has no effect. Future research plan includes the determination of the metal content and the cloning of the gene encoding PhSOD. To this aim, a molecular probe has been designed on the basis of the amino acid sequence of some fragments of the purified protein.
- Published
- 2005
21. Hemodialysis related induction of interleukin-6 production by peripheral blood mononuclear cells
- Author
-
Bruno Memoli, Antonio Dal Canton, Teresa Rampino, Giuseppe Scala, Carmelo Libetta, Maria Rosaria Ruocco, Giuseppe Conte, Vittorio E. Andreucci, B., Memoli, C., Libetta, T., Rampino, A., Dalcanton, G., Conte, S., Scala, Ruocco, MARIA ROSARIA, V., Andreucci, Memoli, B, Libetta, C, Rampino, T, Dal Canton, A, Conte, Giuseppe, Scala, G, Ruocco, Mr, and Andreucci, Ve
- Subjects
Adult ,Male ,medicine.medical_specialty ,medicine.medical_treatment ,Peripheral blood mononuclear cell ,Renal Dialysis ,Internal medicine ,medicine ,Humans ,Methylmethacrylates ,Interleukin 6 ,Cellulose ,Incubation ,Dialysis ,Uremia ,IL-6 ,biology ,business.industry ,Beta-2 microglobulin ,Interleukin-6 ,PBMC ,Membranes, Artificial ,Amyloidosis ,Middle Aged ,Endocrinology ,Membrane ,Cytokine ,Nephrology ,Immunology ,biology.protein ,Leukocytes, Mononuclear ,Female ,Hemodialysis ,Hemodialysi ,business ,beta 2-Microglobulin ,Kidneys, Artificial - Abstract
Hemodialysis related induction of interleukin-6 production by peripheral blood mononuclear cells. Interleukin-6 (IL-6) has a complex spectrum of biological activities, for example, growth and differentiation of B cells and synthesis of acute-phase proteins by the liver. To evaluate the role of this cytokine in the inflammatory response induced by blood interaction with hemodialysis membranes, we have investigated the IL-6 synthesis and release in supernatant of 24-hour cultured peripheral blood mononuclear cells (PBMC) isolated from: (a) 10 hemodialyzed patients, (b) seven patients with advanced chronic renal failure (GFR ≤10 ml/min), and (c) eight healthy control subjects. In the same groups of subjects we evaluated the relationship between IL-6 synthesis and release and beta-2-microglobulin (β2m) production. Before and after dialytic treatment hemodialysis patient blood samples were drawn using the following criteria: (1) after two months of dialysis with cuprophan membranes, (2) after one and two months of dialysis with polymethylmethacrylate (PMMA) membranes, and finally, (3) after one further month of dialysis with cuprophan membranes. IL-6 was determined after 72 hours of incubation of PBMC supernatant serial dilutions with IL-6-dependent hybridoma cell line, 7TD1. Compared to IL-6 synthesis in control subjects (6.0 ± 5.6 U/3 × 106 PBMC/24 hr), hemodialyzed patients, when treated with cuprophan membranes, showed significantly higher value of IL-6 production both before (23 ± 13 U/3 × 106 PBMC/24 hr) and after (26.2 ± 11.3 U/3 × 106 PBMC/24 hr) the dialytic session. When patients were hemodialyzed with PMMA membranes, at the start of dialysis IL-6 levels were not significantly different from values observed in healthy controls (10.6 ± 4 U/3 × 106 PBMC/24 hr, after 1 month of dialysis and 7.8 U/3 × 106 PBMC/24 hr, after 2 months, respectively). When the patients were switched back to cuprophan membranes, IL-6 production was greatly increased after one month of dialysis (CU2, 44.6 ± 9.4 U/3 × 106 PBMC/24 hr, at the start of dialysis) reaching values significantly higher than those obtained in the first period with cuprophan membranes. No difference was observed between the values of IL-6 production obtained pre- and post-dialysis with cuprophan or PMMA membranes. IL-6 production values in uremic non-dialyzed patients were similar to values found in control subjects (8.6 ± 6.4 U/3 × 106 PBMC/24 hr). β2m release showed a behavior quite similar to IL-6 throughout the study. In fact, a statistically significant linear relationship was obtained between β2m and IL-6 values of production (r = 0.8296, P < 0.001). In conclusion, our results show higher levels of IL-6 production in hemodialyzed patients treated with cuprophan membranes, thereby suggesting a chronic stimulation. β2m production is highly related to IL-6 production; this relationship suggests a possible implication for this cytokine in the pathogenesis of dialysis amyloidosis.
- Published
- 1992
22. Guardians and Mediators of Metastasis: Exploring T Lymphocytes, Myeloid-Derived Suppressor Cells, and Tumor-Associated Macrophages in the Breast Cancer Microenvironment.
- Author
-
Ruocco MR, Gisonna A, Acampora V, D'Agostino A, Carrese B, Santoro J, Venuta A, Nasso R, Rocco N, Russo D, Cavaliere A, Altobelli GG, Masone S, Avagliano A, Arcucci A, and Fiume G
- Subjects
- Humans, Female, T-Lymphocytes immunology, Animals, Tumor Microenvironment immunology, Breast Neoplasms pathology, Breast Neoplasms immunology, Myeloid-Derived Suppressor Cells immunology, Myeloid-Derived Suppressor Cells metabolism, Myeloid-Derived Suppressor Cells pathology, Tumor-Associated Macrophages immunology, Tumor-Associated Macrophages metabolism, Tumor-Associated Macrophages pathology, Lymphocytes, Tumor-Infiltrating immunology, Lymphocytes, Tumor-Infiltrating metabolism, Neoplasm Metastasis
- Abstract
Breast cancers (BCs) are solid tumors composed of heterogeneous tissues consisting of cancer cells and an ever-changing tumor microenvironment (TME). The TME includes, among other non-cancer cell types, immune cells influencing the immune context of cancer tissues. In particular, the cross talk of immune cells and their interactions with cancer cells dramatically influence BC dissemination, immunoediting, and the outcomes of cancer therapies. Tumor-infiltrating lymphocytes (TILs), tumor-associated macrophages (TAMs), and myeloid-derived suppressor cells (MDSCs) represent prominent immune cell populations of breast TMEs, and they have important roles in cancer immunoescape and dissemination. Therefore, in this article we review the features of TILs, TAMs, and MDSCs in BCs. Moreover, we highlight the mechanisms by which these immune cells remodel the immune TME and lead to breast cancer metastasis.
- Published
- 2024
- Full Text
- View/download PDF
23. Enhanced pro-apoptotic activity of rituximab through IBTK silencing in non-Hodgkin lymphoma B-cells.
- Author
-
Vecchio E, Marino R, Mimmi S, Canale C, Caiazza C, Arcucci A, Ruocco MR, Schiavone M, Santamaria G, Palmieri C, Iaccino E, Mallardo M, Quinto I, and Fiume G
- Abstract
Rituximab is a commonly used chemotherapeutic drug for patients with aggressive lymphomas, such as non-Hodgkin's lymphoma (NHL). Currently, the combination of Rituximab and chemotherapy (R-CHOP) stands as the most prevalent first-line therapy for NHL. Nevertheless, the development of new therapeutic approaches remains imperative. An increasing body of evidence highlights a novel role for IBTK in tumorigenesis and cancer growth. In this study, we aim to broaden our understanding of IBTK's function in B-lymphoma, with a particular focus on its impact on the expression of the oncogene MYC. Here, we assessed the effects of combining Rituximab with IBTK silencing on cell viability through cell cycle analysis and Annexin V assays in vitro . Furthermore, we leveraged the transplantability of Eμ-myc lymphomas to investigate whether the inhibition of IBTK could elicit anti-tumor effects in the treatment of lymphomas in vivo . Our data suggests that IBTK silencing may serve as an effective anti-tumor agent for aggressive B-Lymphomas, underscoring its role in promoting apoptosis when used in combination with Rituximab, both in in vitro and in vivo settings., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Vecchio, Marino, Mimmi, Canale, Caiazza, Arcucci, Ruocco, Schiavone, Santamaria, Palmieri, Iaccino, Mallardo, Quinto and Fiume.)
- Published
- 2024
- Full Text
- View/download PDF
24. Celecoxib, a Non-Steroidal Anti-Inflammatory Drug, Exerts a Toxic Effect on Human Melanoma Cells Grown as 2D and 3D Cell Cultures.
- Author
-
Venuta A, Nasso R, Gisonna A, Iuliano R, Montesarchio S, Acampora V, Sepe L, Avagliano A, Arcone R, Arcucci A, and Ruocco MR
- Abstract
Cutaneous melanoma (CM) remains one of the leading causes of tumor mortality due to its high metastatic spread. CM growth is influenced by inflammation regulated by prostaglandins (PGs) whose synthesis is catalyzed by cyclooxygenases (COXs). COX inhibitors, including non-steroidal anti-inflammatory drugs (NSAIDs), can inhibit tumor development and growth. In particular, in vitro experiments have shown that celecoxib, a NSAID, inhibits the growth of some tumor cell lines. However, two-dimensional (2D) cell cultures, used in traditional in vitro anticancer assays, often show poor efficacy due to a lack of an in vivo like cellular environment. Three-dimensional (3D) cell cultures, such as spheroids, are better models because they can mimic the common features displayed by human solid tumors. Hence, in this study, we evaluated the anti-neoplastic potential of celecoxib, in both 2D and 3D cell cultures of A2058 and SAN melanoma cell lines. In particular, celecoxib reduced the cell viability and migratory capability and triggered the apoptosis of melanoma cells grown as 2D cultures. When celecoxib was tested on 3D melanoma cell cultures, the drug exerted an inhibitory effect on cell outgrowth from spheroids and reduced the invasiveness of melanoma cell spheroids into the hydrogel matrix. This work suggests that celecoxib could represent a new potential therapeutic approach in melanoma therapy., Competing Interests: The authors declare no conflict of interest.
- Published
- 2023
- Full Text
- View/download PDF
25. Thyroid Cancer and Fibroblasts.
- Author
-
Avagliano A, Fiume G, Bellevicine C, Troncone G, Venuta A, Acampora V, De Lella S, Ruocco MR, Masone S, Velotti N, Carotenuto P, Mallardo M, Caiazza C, Montagnani S, and Arcucci A
- Abstract
Thyroid cancer is the most common type of endocrine cancer, and its prevalence continue to rise. Non-metastatic thyroid cancer patients are successfully treated. However, looking for new therapeutic strategies is of great importance for metastatic thyroid cancers that still lead to death. With respect to this, the tumor microenvironment (TME), which plays a key role in tumor progression, should be considered as a new promising therapeutic target to hamper thyroid cancer progression. Indeed, thyroid tumors consist of cancer cells and a heterogeneous and ever-changing niche, represented by the TME, which contributes to establishing most of the features of cancer cells. The TME consists of extracellular matrix (ECM) molecules, soluble factors, metabolites, blood and lymphatic tumor vessels and several stromal cell types that, by interacting with each other and with tumor cells, affect TME remodeling, cancer growth and progression. Among the thyroid TME components, cancer-associated fibroblasts (CAFs) have gained more attention in the last years. Indeed, recent important evidence showed that thyroid CAFs strongly sustain thyroid cancer growth and progression by producing soluble factors and ECM proteins, which, in turn, deeply affect thyroid cancer cell behavior and aggressiveness. Hence, in this article, we describe the thyroid TME, focusing on the desmoplastic stromal reaction, which is a powerful indicator of thyroid cancer progression and an invasive growth pattern. In addition, we discuss the origins and features of the thyroid CAFs, their influence on thyroid cancer growth and progression, their role in remodeling the ECM and their immune-modulating functions. We finally debate therapeutic perspectives targeting CAFs.
- Published
- 2022
- Full Text
- View/download PDF
26. Generation and Characterization of a Tumor Stromal Microenvironment and Analysis of Its Interplay with Breast Cancer Cells: An In Vitro Model to Study Breast Cancer-Associated Fibroblast Inactivation.
- Author
-
Romano V, Ruocco MR, Carotenuto P, Barbato A, Venuta A, Acampora V, De Lella S, Vigliar E, Iaccarino A, Troncone G, Calì G, Insabato L, Russo D, Franco B, Masone S, Velotti N, Accurso A, Pellegrino T, Fiume G, Belviso I, Montagnani S, Avagliano A, and Arcucci A
- Subjects
- Cell Line, Tumor, Culture Media, Conditioned metabolism, Cyclooxygenase 2 genetics, Cyclooxygenase 2 metabolism, Female, Fibroblasts metabolism, Humans, Inflammation pathology, Stromal Cells metabolism, Tumor Microenvironment, Breast Neoplasms metabolism, Cancer-Associated Fibroblasts metabolism
- Abstract
Breast cancer-associated fibroblasts (BCAFs), the most abundant non-cancer stromal cells of the breast tumor microenvironment (TME), dramatically sustain breast cancer (BC) progression by interacting with BC cells. BCAFs, as well as myofibroblasts, display an up regulation of activation and inflammation markers represented by α-smooth muscle actin (α-SMA) and cyclooxygenase 2 (COX-2). BCAF aggregates have been identified in the peripheral blood of metastatic BC patients. We generated an in vitro stromal model consisting of human primary BCAFs grown as monolayers or 3D cell aggregates, namely spheroids and reverted BCAFs, obtained from BCAF spheroids reverted to 2D cell adhesion growth after 216 h of 3D culture. We firstly evaluated the state of activation and inflammation and the mesenchymal status of the BCAF monolayers, BCAF spheroids and reverted BCAFs. Then, we analyzed the MCF-7 cell viability and migration following treatment with conditioned media from the different BCAF cultures. After 216 h of 3D culture, the BCAFs acquired an inactivated phenotype, associated with a significant reduction in α-SMA and COX-2 protein expression. The deactivation of the BCAF spheroids at 216 h was further confirmed by the cytostatic effect exerted by their conditioned medium on MCF-7 cells. Interestingly, the reverted BCAFs also retained a less activated phenotype as indicated by α-SMA protein expression reduction. Furthermore, the reverted BCAFs exhibited a reduced pro-tumor phenotype as indicated by the anti-migratory effect exerted by their conditioned medium on MCF-7 cells. The deactivation of BCAFs without drug treatment is possible and leads to a reduced capability of BCAFs to sustain BC progression in vitro. Consequently, this study could be a starting point to develop new therapeutic strategies targeting BCAFs and their interactions with cancer cells.
- Published
- 2022
- Full Text
- View/download PDF
27. Editorial: Tumor Microenvironment and Cancer Cell Interactions in Solid Tumor Growth and Therapy Resistance.
- Author
-
Ruocco MR, Lamberti A, Serrano MJ, Fiume G, and Arcucci A
- Abstract
Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
- Published
- 2022
- Full Text
- View/download PDF
28. Influence of Tumor Microenvironment and Fibroblast Population Plasticity on Melanoma Growth, Therapy Resistance and Immunoescape.
- Author
-
Romano V, Belviso I, Venuta A, Ruocco MR, Masone S, Aliotta F, Fiume G, Montagnani S, Avagliano A, and Arcucci A
- Subjects
- Cancer-Associated Fibroblasts metabolism, Cell Communication, Cell Plasticity physiology, Extracellular Matrix metabolism, Humans, Melanoma pathology, Melanoma physiopathology, Signal Transduction, Skin Neoplasms pathology, Stromal Cells metabolism, Melanoma, Cutaneous Malignant, Fibroblasts physiology, Melanoma metabolism, Tumor Microenvironment physiology
- Abstract
Cutaneous melanoma (CM) tissue represents a network constituted by cancer cells and tumor microenvironment (TME). A key feature of CM is the high structural and cellular plasticity of TME, allowing its evolution with disease and adaptation to cancer cell and environmental alterations. In particular, during melanoma development and progression each component of TME by interacting with each other and with cancer cells is subjected to dramatic structural and cellular modifications. These alterations affect extracellular matrix (ECM) remodelling, phenotypic profile of stromal cells, cancer growth and therapeutic response. The stromal fibroblast populations of the TME include normal fibroblasts and melanoma-associated fibroblasts (MAFs) that are highly abundant and flexible cell types interacting with melanoma and stromal cells and differently influencing CM outcomes. The shift from the normal microenvironment to TME and from normal fibroblasts to MAFs deeply sustains CM growth. Hence, in this article we review the features of the normal microenvironment and TME and describe the phenotypic plasticity of normal dermal fibroblasts and MAFs, highlighting their roles in normal skin homeostasis and TME regulation. Moreover, we discuss the influence of MAFs and their secretory profiles on TME remodelling, melanoma progression, targeted therapy resistance and immunosurveillance, highlighting the cellular interactions, the signalling pathways and molecules involved in these processes.
- Published
- 2021
- Full Text
- View/download PDF
29. Quinoa as source of type 1 ribosome inactivating proteins: A novel knowledge for a revision of its consumption.
- Author
-
Landi N, Ruocco MR, Ragucci S, Aliotta F, Nasso R, Pedone PV, and Di Maro A
- Subjects
- Humans, Seeds enzymology, Chenopodium quinoa enzymology, Diet, Ribosome Inactivating Proteins, Type 1 analysis
- Abstract
This study investigates on the presence of toxic proteins in quinoa seeds. To this aim, a plethora of biochemical approaches were adopted for the purification and characterization of quinoin, a type 1 ribosome-inactivating protein (RIP) contained in quinoa seeds. We determined its melting temperature (68.2 ± 0.6 °C) and thermostability (loss of activity after 10-min incubation at 70 °C). Considering that quinoa seeds are used as a food, we found that quinoin is cytotoxic against BJ-5ta (human fibroblasts) and HaCaT (human keratinocytes) in a dose- and time-dependent manner. Moreover, in an in vitro digestive pepsin-trypsin treatment, 30% of quinoin is resistant to enzymatic cleavage. This toxin was found in seeds (0.23 mg/g of seeds) and in sprouted seeds obtained after 24-h (0.12 mg/g of sprout) and 48-h (0.09 mg/g of sprout). We suggest a thermal treatment of quinoa seeds before consumption in order to inactivate the toxin, particularly in sprouts, generally consumed raw., (Copyright © 2020 Elsevier Ltd. All rights reserved.)
- Published
- 2021
- Full Text
- View/download PDF
30. Inhibition mechanism of naphthylphenylamine derivatives acting on the CDC25B dual phosphatase and analysis of the molecular processes involved in the high cytotoxicity exerted by one selected derivative in melanoma cells.
- Author
-
Aliotta F, Nasso R, Rullo R, Arcucci A, Avagliano A, Simonetti M, Sanità G, Masullo M, Lavecchia A, Ruocco MR, and Vendittis E
- Subjects
- Amino Acid Sequence, Antineoplastic Agents pharmacology, Apoptosis drug effects, Caspase 3 metabolism, Caspase 9 metabolism, Catalytic Domain, Cell Line, Tumor, Drug Screening Assays, Antitumor, Enzyme Inhibitors pharmacology, Humans, Mutation, Optical Imaging, Structure-Activity Relationship, Aniline Compounds chemistry, Antineoplastic Agents chemistry, Enzyme Inhibitors chemistry, Melanoma drug therapy, cdc25 Phosphatases antagonists & inhibitors
- Abstract
The dual phosphatases CDC25 are involved in cell cycle regulation and overexpressed in many tumours, including melanoma. CDC25 is a promising target for discovering anticancer drugs, and several studies focussed on characterisation of quinonoid CDC25 inhibitors, frequently causing undesired side toxic effects. Previous work described an optimisation of the inhibition properties by naphthylphenylamine (NPA) derivatives of NSC28620, a nonquinonoid CDC25 inhibitor. Now, the CDC25B•inhibitor interaction was investigated through fluorescence studies, shedding light on the different inhibition mechanism exerted by NPA derivatives. Among the molecular processes, mediating the specific and high cytotoxicity of one NPA derivative in melanoma cells, we observed decrease of phosphoAkt, increase of p53, reduction of CDC25 forms, cytochrome c cytosolic translocation and increase of caspase activity, that lead to the activation of an apoptotic programme. A basic knowledge on CDC25 inhibitors is relevant for discovering potent bioactive molecules, to be used as anticancer agents against the highly aggressive melanoma.
- Published
- 2020
- Full Text
- View/download PDF
31. Influence of Fibroblasts on Mammary Gland Development, Breast Cancer Microenvironment Remodeling, and Cancer Cell Dissemination.
- Author
-
Avagliano A, Fiume G, Ruocco MR, Martucci N, Vecchio E, Insabato L, Russo D, Accurso A, Masone S, Montagnani S, and Arcucci A
- Abstract
The stromal microenvironment regulates mammary gland development and tumorigenesis. In normal mammary glands, the stromal microenvironment encompasses the ducts and contains fibroblasts, the main regulators of branching morphogenesis. Understanding the way fibroblast signaling pathways regulate mammary gland development may offer insights into the mechanisms of breast cancer (BC) biology. In fact, the unregulated mammary fibroblast signaling pathways, associated with alterations in extracellular matrix (ECM) remodeling and branching morphogenesis, drive breast cancer microenvironment (BCM) remodeling and cancer growth. The BCM comprises a very heterogeneous tissue containing non-cancer stromal cells, namely, breast cancer-associated fibroblasts (BCAFs), which represent most of the tumor mass. Moreover, the different components of the BCM highly interact with cancer cells, thereby generating a tightly intertwined network. In particular, BC cells activate recruited normal fibroblasts in BCAFs, which, in turn, promote BCM remodeling and metastasis. Thus, comparing the roles of normal fibroblasts and BCAFs in the physiological and metastatic processes, could provide a deeper understanding of the signaling pathways regulating BC dissemination. Here, we review the latest literature describing the structure of the mammary gland and the BCM and summarize the influence of epithelial-mesenchymal transition (EpMT) and autophagy in BC dissemination. Finally, we discuss the roles of fibroblasts and BCAFs in mammary gland development and BCM remodeling, respectively.
- Published
- 2020
- Full Text
- View/download PDF
32. Metabolic Plasticity of Melanoma Cells and Their Crosstalk With Tumor Microenvironment.
- Author
-
Avagliano A, Fiume G, Pelagalli A, Sanità G, Ruocco MR, Montagnani S, and Arcucci A
- Abstract
Cutaneous melanoma (CM) is a highly aggressive and drug resistant solid tumor, showing an impressive metabolic plasticity modulated by oncogenic activation. In particular, melanoma cells can generate adenosine triphosphate (ATP) during cancer progression by both cytosolic and mitochondrial compartments, although CM energetic request mostly relies on glycolysis. The upregulation of glycolysis is associated with constitutive activation of BRAF/MAPK signaling sustained by BRAF
V600E kinase mutant. In this scenario, the growth and progression of CM are strongly affected by melanoma metabolic changes and interplay with tumor microenvironment (TME) that sustain tumor development and immune escape. Furthermore, CM metabolic plasticity can induce a metabolic adaptive response to BRAF/MEK inhibitors (BRAFi/MEKi), associated with the shift from glycolysis toward oxidative phosphorylation (OXPHOS). Therefore, in this review article we survey the metabolic alterations and plasticity of CM, its crosstalk with TME that regulates melanoma progression, drug resistance and immunosurveillance. Finally, we describe hallmarks of melanoma therapeutic strategies targeting the shift from glycolysis toward OXPHOS., (Copyright © 2020 Avagliano, Fiume, Pelagalli, Sanità, Ruocco, Montagnani and Arcucci.)- Published
- 2020
- Full Text
- View/download PDF
33. Metabolic flexibility in melanoma: A potential therapeutic target.
- Author
-
Ruocco MR, Avagliano A, Granato G, Vigliar E, Masone S, Montagnani S, and Arcucci A
- Subjects
- Animals, Disease Progression, Disease Susceptibility, Glycolysis, Humans, Melanoma drug therapy, Melanoma etiology, Melanoma pathology, Mitochondria drug effects, Mitochondria genetics, Mitochondria metabolism, Molecular Targeted Therapy, Treatment Outcome, Tumor Microenvironment drug effects, Energy Metabolism drug effects, Melanoma metabolism
- Abstract
Cutaneous melanoma (CM) represents one of the most metastasizing and drug resistant solid tumors. CM is characterized by a remarkable metabolic plasticity and an important connection between oncogenic activation and energetic metabolism. In fact, melanoma cells can use both cytosolic and mitochondrial compartments to produce adenosine triphosphate (ATP) during cancer progression. However, the CM energetic demand mainly depends on glycolysis, whose upregulation is strictly linked to constitutive activation of BRAF/MAPK pathway affected by BRAF
V600E kinase mutant. Furthermore, the impressive metabolic plasticity of melanoma allows the development of resistance mechanisms to BRAF/MEK inhibitors (BRAFi/MEKi) and the adaptation to microenvironmental changes. The metabolic interaction between melanoma cells and tumor microenvironment affects the immune response and CM growth. In this review article, we describe the regulation of melanoma metabolic alterations and the metabolic interactions between cancer cells and microenvironment that influence melanoma progression and immune response. Finally, we summarize the hallmarks of melanoma therapies and we report BRAF/MEK pathway targeted therapy and mechanisms of metabolic resistance., (Copyright © 2019 Elsevier Ltd. All rights reserved.)- Published
- 2019
- Full Text
- View/download PDF
34. Development of a Stromal Microenvironment Experimental Model Containing Proto-Myofibroblast Like Cells and Analysis of Its Crosstalk with Melanoma Cells: A New Tool to Potentiate and Stabilize Tumor Suppressor Phenotype of Dermal Myofibroblasts.
- Author
-
Avagliano A, Ruocco MR, Nasso R, Aliotta F, Sanità G, Iaccarino A, Bellevicine C, Calì G, Fiume G, Masone S, Masullo M, Montagnani S, and Arcucci A
- Subjects
- Adult, Cell Communication drug effects, Cell Line, Tumor, Cell Movement drug effects, Cell Survival drug effects, Cells, Cultured, Cellular Microenvironment, Female, Humans, Melanoma metabolism, Middle Aged, Myofibroblasts metabolism, Phenotype, Spheroids, Cellular cytology, Spheroids, Cellular drug effects, Spheroids, Cellular metabolism, Tumor Microenvironment, Actins metabolism, Culture Media, Conditioned pharmacology, Cyclooxygenase 2 metabolism, Melanoma pathology, Myofibroblasts cytology, Vimentin metabolism
- Abstract
Melanoma is one of the most aggressive solid tumors and includes a stromal microenvironment that regulates cancer growth and progression. The components of stromal microenvironment such as fibroblasts, fibroblast aggregates and cancer-associated fibroblasts (CAFs) can differently influence the melanoma growth during its distinct stages. In this work, we have developed and studied a stromal microenvironment model, represented by fibroblasts, proto-myofibroblasts, myofibroblasts and aggregates of inactivated myofibroblasts, such as spheroids. In particular, we have generated proto-myofibroblasts from primary cutaneous myofibroblasts. The phenotype of proto-myofibroblasts is characterized by a dramatic reduction of α-smooth muscle actin (α-SMA) and cyclooxygenase-2 (COX-2) protein levels, as well as an enhancement of cell viability and migratory capability compared with myofibroblasts. Furthermore, proto-myofibroblasts display the mesenchymal marker vimentin and less developed stress fibers, with respect to myofibroblasts. The analysis of crosstalk between the stromal microenvironment and A375 or A2058 melanoma cells has shown that the conditioned medium of proto-myofibroblasts is cytotoxic, mainly for A2058 cells, and dramatically reduces the migratory capability of both cell lines compared with the melanoma-control conditioned medium. An array analysis of proto-myofibroblast and melanoma cell-conditioned media suggests that lower levels of some cytokines and growth factors in the conditioned medium of proto-myofibroblasts could be associated with their anti-tumor activity. Conversely, the conditioned media of melanoma cells do not influence the cell viability, outgrowth, and migration of proto-myofibroblasts from spheroids. Interestingly, the conditioned medium of proto-myofibroblasts does not alter the cell viability of both BJ-5ta fibroblast cells and myofibroblasts. Hence, proto-myofibroblasts could be useful in the study of new therapeutic strategies targeting melanoma.
- Published
- 2019
- Full Text
- View/download PDF
35. Ageritin from poplar mushrooms: scale-up purification and cytotoxicity towards undifferentiated and differentiated SH-SY5Y cells.
- Author
-
Ragucci S, Pacifico S, Ruocco MR, Crescente G, Nasso R, Simonetti M, Masullo M, Piccolella S, Pedone PV, Landi N, and Di Maro A
- Subjects
- Apoptosis drug effects, Caspase 3 genetics, Caspase 3 metabolism, Cell Line, Tumor, Humans, Neurons cytology, Neurons drug effects, Neurons metabolism, Plant Extracts chemistry, Plant Extracts isolation & purification, Ribonucleases chemistry, Ribonucleases isolation & purification, Agaricales chemistry, Cell Differentiation drug effects, Plant Extracts pharmacology, Ribonucleases pharmacology
- Abstract
Ageritin is the first reported ribotoxin-like protein from basidiomycetes fungi. It can induce ribosomal integrity damage and translation block, and interferes with mitochondrial redox activity of some glioma and neuroblastoma cell lines. Herein, Ageritin has been investigated as a valuable neurotoxin towards either undifferentiated or retinoic acid (RA)-differentiated SH-SY5Y neuroblastoma cells showing a selective cell toxicity against undifferentiated cells. MTT and sulforhodamine B (SRB) assays highlighted that Ageritin markedly decreases the mitochondrial redox activity and viability of undifferentiated cells, meanwhile inducing evident morphological changes eliciting neuronal-like appearance in these cells. Data from lactate dehydrogenase release assay, cytofluorimetric analysis and caspase-3 enzymatic activity measurement suggest that Ageritin promotes cell death through a caspase-dependent apoptotic pathway. The Z-VAD-FMK caspase inhibitor was able to prevent this apoptotic pathway activation. Based on the interesting behaviour of Ageritin vs. SH-SY5Y cells, the development of a scale-up procedure to obtain the purified protein in larger amounts (yield 2.5 mg per 100 g) has been optimized.
- Published
- 2019
- Full Text
- View/download PDF
36. Discovery of Novel Naphthylphenylketone and Naphthylphenylamine Derivatives as Cell Division Cycle 25B (CDC25B) Phosphatase Inhibitors: Design, Synthesis, Inhibition Mechanism, and in Vitro Efficacy against Melanoma Cell Lines.
- Author
-
Cerchia C, Nasso R, Mori M, Villa S, Gelain A, Capasso A, Aliotta F, Simonetti M, Rullo R, Masullo M, De Vendittis E, Ruocco MR, and Lavecchia A
- Subjects
- Aniline Compounds pharmacology, Aniline Compounds therapeutic use, Antineoplastic Agents pharmacology, Antineoplastic Agents therapeutic use, Cell Line, Tumor, Cell Survival drug effects, Cell Survival physiology, Enzyme Inhibitors chemical synthesis, Enzyme Inhibitors therapeutic use, Humans, Ketones pharmacology, Ketones therapeutic use, Melanoma drug therapy, Protein Structure, Tertiary, Treatment Outcome, Aniline Compounds chemical synthesis, Antineoplastic Agents chemical synthesis, Drug Design, Drug Discovery methods, Ketones chemical synthesis, cdc25 Phosphatases antagonists & inhibitors
- Abstract
CDC25 phosphatases play a critical role in the regulation of the cell cycle and thus represent attractive cancer therapeutic targets. We previously discovered the 4-(2-carboxybenzoyl)phthalic acid (NSC28620) as a new CDC25 inhibitor endowed with promising anticancer activity in breast, prostate, and leukemia cells. Herein, we report a structure-based optimization of NSC28620, leading to the identification of a series of novel naphthylphenylketone and naphthylphenylamine derivatives as CDC25B inhibitors. Compounds 7j , 7i , 6e , 7f , and 3 showed higher inhibitory activity than the initial lead, with K
i values in the low micromolar range. Kinetic analysis, intrinsic fluorescence studies, and induced fit docking simulations provided a mechanistic understanding of the activity of these derivatives. All compounds were tested in the highly aggressive human melanoma cell lines A2058 and A375. Compound 4a potently inhibited cell proliferation and colony formation, causing an increase of the G2/M phase and a reduction of the G0/G1 phase of the cell cycle in both cell lines.- Published
- 2019
- Full Text
- View/download PDF
37. Mitochondrial Flexibility of Breast Cancers: A Growth Advantage and a Therapeutic Opportunity.
- Author
-
Avagliano A, Ruocco MR, Aliotta F, Belviso I, Accurso A, Masone S, Montagnani S, and Arcucci A
- Subjects
- Breast Neoplasms metabolism, Female, Humans, Models, Biological, Breast Neoplasms pathology, Breast Neoplasms therapy, Mitochondria metabolism
- Abstract
Breast cancers are very heterogeneous tissues with several cell types and metabolic pathways together sustaining the initiation and progression of disease and contributing to evasion from cancer therapies. Furthermore, breast cancer cells have an impressive metabolic plasticity that is regulated by the heterogeneous tumour microenvironment through bidirectional interactions. The structure and accessibility of nutrients within this unstable microenvironment influence the metabolism of cancer cells that shift between glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) to produce adenosine triphosphate (ATP). In this scenario, the mitochondrial energetic pathways of cancer cells can be reprogrammed to modulate breast cancer's progression and aggressiveness. Moreover, mitochondrial alterations can lead to crosstalk between the mitochondria and the nucleus, and subsequently affect cancer tissue properties. This article reviewed the metabolic plasticity of breast cancer cells, focussing mainly on breast cancer mitochondrial metabolic reprogramming and the mitochondrial alterations influencing nuclear pathways. Finally, the therapeutic strategies targeting molecules and pathways regulating cancer mitochondrial alterations are highlighted.
- Published
- 2019
- Full Text
- View/download PDF
38. Metabolic Reprogramming of Cancer Associated Fibroblasts: The Slavery of Stromal Fibroblasts.
- Author
-
Avagliano A, Granato G, Ruocco MR, Romano V, Belviso I, Carfora A, Montagnani S, and Arcucci A
- Subjects
- Fibroblasts, Stromal Cells, Cancer-Associated Fibroblasts metabolism, Tumor Microenvironment
- Abstract
Cancer associated fibroblasts (CAFs) are the main stromal cell type of solid tumour microenvironment and undergo an activation process associated with secretion of growth factors, cytokines, and paracrine interactions. One of the important features of solid tumours is the metabolic reprogramming that leads to changes of bioenergetics and biosynthesis in both tumour cells and CAFs. In particular, CAFs follow the evolution of tumour disease and acquire a catabolic phenotype: in tumour tissues, cancer cells and tumour microenvironment form a network where the crosstalk between cancer cells and CAFs is associated with cell metabolic reprogramming that contributes to CAFs activation, cancer growth, and progression and evasion from cancer therapies. In this regard, the study of CAFs metabolic reprogramming could contribute to better understand their activation process, the interaction between stroma, and cancer cells and could offer innovative tools for the development of new therapeutic strategies able to eradicate the protumorigenic activity of CAFs. Therefore, this review focuses on CAFs metabolic reprogramming associated with both differentiation process and cancer and stromal cells crosstalk. Finally, therapeutic responses and potential anticancer strategies targeting CAFs metabolic reprogramming are reviewed.
- Published
- 2018
- Full Text
- View/download PDF
39. Involvement of Breast Cancer-Associated Fibroblasts in Tumor Development, Therapy Resistance and Evaluation of Potential Therapeutic Strategies.
- Author
-
Ruocco MR, Avagliano A, Granato G, Imparato V, Masone S, Masullo M, Nasso R, Montagnani S, and Arcucci A
- Subjects
- Antineoplastic Agents chemistry, Antineoplastic Agents therapeutic use, Breast Neoplasms diagnosis, Cell Proliferation drug effects, Female, Humans, Molecular Structure, Antineoplastic Agents pharmacology, Breast Neoplasms drug therapy, Breast Neoplasms pathology, Drug Resistance, Neoplasm drug effects, Fibroblasts drug effects, Fibroblasts pathology
- Abstract
Breast cancer is the most common cancer in women, which incidence has increased in recent years. It is constituted by very heterogeneous tissue characterized by an abnormal microenvironment regulating tumor progression and providing evasion from cancer therapies. Breast cancer-associated fibroblasts (BCAFs) are the main cell type of breast cancer microenvironment and can represent up to 80% of the tumor mass. In particular, BCAFs induce cancer initiation, proliferation, invasion and metastasis by undergoing an activation process associated with the secretion of growth factors, cytokines, and paracrine interactions. Therapy resistance is the main cause of poor therapeutic results or even failure in breast cancer patients. Despite recent advances in breast cancer management, there is a need for new prognostic markers and novel agents for targeting key signalling pathways to either improve the efficacy of the current therapies, or reduce toxicity. In this view, BCAFs represent markers useful to clinical diagnosis, therapy, and prognosis of breast cancer. This review focuses on the role of BCAFs in cancer, and describes the processes of endocrine/chemotherapy resistance linked to BCAFs action. Moreover, it points to molecules and pathways regulating therapy resistance induced by BCAFs. Finally, potential therapeutic strategies targeting BCAFs and offering new tools in breast cancer therapy are highlighted., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)
- Published
- 2018
- Full Text
- View/download PDF
40. Generation and analysis of spheroids from human primary skin myofibroblasts: an experimental system to study myofibroblasts deactivation.
- Author
-
Granato G, Ruocco MR, Iaccarino A, Masone S, Calì G, Avagliano A, Russo V, Bellevicine C, Di Spigna G, Fiume G, Montagnani S, and Arcucci A
- Abstract
Myofibroblasts are activated fibroblasts involved in tissue repair and cancer. They are characterized by de novo expression of α -smooth muscle actin ( α -SMA), immunoregulatory phenotype and paracrine interaction with normal and tumorigenic cells leading to cell proliferation. At the end of wound-healing myofibroblasts undergo apoptotic cell death, whereas in vitro -activated fibroblasts are also subjected to a programmed necrosis-like cell death, termed nemosis, associated with cyclooxygenase-2 (COX-2) expression induction and inflammatory response. Furthermore, myofibroblasts form clusters during wound healing, fibrotic states and tumorigenesis. In this study, we generated and analysed clusters such as spheroids from human primary cutaneous myofibroblasts, which represent a part of stromal microenvironment better than established cell lines. Therefore, we evaluated apoptotic or necrotic cell death, inflammation and activation markers during myofibroblasts clustering. The spheroids formation did not trigger apoptosis, necrotic cell death and COX-2 protein induction. The significant decrease of α -SMA in protein extracts of spheroids, the cytostatic effect exerted by spheroids conditioned medium on both normal and cancer cell lines and the absence of proliferation marker Ki-67 after 72 h of three-dimensional culture indicated that myofibroblasts have undergone a deactivation process within spheroids. The cells of spheroids reverted to adhesion growth preserved their proliferation capability and can re-acquire a myofibroblastic phenotype. Moreover, the spontaneous formation of clusters on plastic and glass substrates suggests that aggregates formation could be a physiological feature of cutaneous myofibroblasts. This study represents an experimental model to analyse myofibroblasts deactivation and suggests that fibroblast clusters could be a cell reservoir regulating tissues turnover., Competing Interests: The authors declare no conflict of interest.
- Published
- 2017
- Full Text
- View/download PDF
41. Cancer: An Oxidative Crosstalk between Solid Tumor Cells and Cancer Associated Fibroblasts.
- Author
-
Arcucci A, Ruocco MR, Granato G, Sacco AM, and Montagnani S
- Subjects
- Cell Communication, Cell Differentiation, Fibroblasts metabolism, Fibroblasts pathology, Humans, Myofibroblasts metabolism, Myofibroblasts pathology, Oxidation-Reduction, Reactive Oxygen Species metabolism, Signal Transduction, Tumor Microenvironment, Neoplasms metabolism, Neoplasms pathology
- Abstract
Redox balance is associated with the regulation of several cell signalling pathways and functions. In fact, under physiological conditions, cells maintain a balance between oxidant and antioxidant systems, and reactive oxygen species (ROS) can act as second messengers to regulate cell proliferation, cell death, and other physiological processes. Cancer tissues usually contain higher levels of ROS than normal tissues, and this ROS overproduction is associated with tumor development. Neoplastic tissues are very heterogeneous systems, composed of tumor cells and microenvironment that has a critical role in tumor progression. Cancer associated fibroblasts (CAFs) represent the main cell type of tumor microenvironment, and they contribute to tumor growth by undergoing an irreversible activation process. It is known that ROS can be transferred from cancer cells to fibroblasts. In particular, ROS affect the behaviour of CAFs by promoting the conversion of fibroblasts to myofibroblasts that support tumor progression and dissemination. Furthermore, the wrecking of redox homeostasis in cancer cells and tumor microenvironment induces a metabolic reprogramming in tumor cells and cancer associated fibroblasts, giving advantage to cancer growth. This review describes the role of ROS in tumor growth, by focusing on CAFs activation and metabolic interactions between cancer cells and stromal fibroblasts.
- Published
- 2016
- Full Text
- View/download PDF
42. Ligand-based chemoinformatic discovery of a novel small molecule inhibitor targeting CDC25 dual specificity phosphatases and displaying in vitro efficacy against melanoma cells.
- Author
-
Capasso A, Cerchia C, Di Giovanni C, Granato G, Albano F, Romano S, De Vendittis E, Ruocco MR, and Lavecchia A
- Subjects
- Antineoplastic Agents pharmacology, Apoptosis drug effects, Blotting, Western, Cell Line, Tumor, Cell Proliferation drug effects, Dose-Response Relationship, Drug, Dual-Specificity Phosphatases metabolism, Humans, Kinetics, Ligands, Melanoma metabolism, Melanoma pathology, Phosphorylation drug effects, Proto-Oncogene Proteins c-akt metabolism, Reactive Oxygen Species metabolism, Signal Transduction drug effects, Tumor Suppressor Protein p53 metabolism, cdc25 Phosphatases metabolism, Drug Discovery methods, Dual-Specificity Phosphatases antagonists & inhibitors, Enzyme Inhibitors pharmacology, Small Molecule Libraries pharmacology, cdc25 Phosphatases antagonists & inhibitors
- Abstract
CDC25 phosphatases are important regulators of the cell cycle and represent promising targets for anticancer drug discovery. We recently identified NSC 119915 as a new quinonoid CDC25 inhibitor with potent anticancer activity. In order to discover more active analogs of NSC 119915, we performed a range of ligand-based chemoinformatic methods against the full ZINC drug-like subset and the NCI lead-like set. Nine compounds (3, 5-9, 21, 24, and 25) were identified with Ki values for CDC25A, -B and -C ranging from 0.01 to 4.4 μM. One of these analogs, 7, showed a high antiproliferative effect on human melanoma cell lines, A2058 and SAN. Compound 7 arrested melanoma cells in G2/M, causing a reduction of the protein levels of CDC25A and, more consistently, of CDC25C. Furthermore, an intrinsic apoptotic pathway was induced, which was mediated by ROS, because it was reverted in the presence of antioxidant N-acetyl-cysteine (NAC). Finally, 7 decreased the protein levels of phosphorylated Akt and increased those of p53, thus contributing to the regulation of chemosensitivity through the control of downstream Akt pathways in melanoma cells. Taken together, our data emphasize that CDC25 could be considered as a possible oncotarget in melanoma cells and that compound 7 is a small molecule CDC25 inhibitor that merits to be further evaluated as a chemotherapeutic agent for melanoma, likely in combination with other therapeutic compounds.
- Published
- 2015
- Full Text
- View/download PDF
43. Erratum - Analysis of extracellular superoxide dismutase and Akt in ascending aortic aneurysm with tricuspid or bicuspid aortic valve.
- Author
-
Arcucci A, Ruocco MR, Albano F, Granato G, Romano V, Corso G, Bancone C, De Vendittis E, Della Corte A, and Montagnani S
- Abstract
This correct the article published on European Journal of Histochemistry 2014;58:200-206 doi: 10.4081/ejh.2014.2383.
- Published
- 2015
- Full Text
- View/download PDF
44. Analysis of extracellular superoxide dismutase and Akt in ascending aortic aneurysm with tricuspid or bicuspid aortic valve.
- Author
-
Arcucci A, Ruocco MR, Albano F, Granato G, Romano V, Corso G, Bancone C, De Vendittis E, Della Corte A, and Montagnani S
- Subjects
- Aged, Aorta pathology, Aortic Valve enzymology, Bicuspid Aortic Valve Disease, Female, Humans, Immunohistochemistry, Male, Reference Standards, Aortic Aneurysm enzymology, Aortic Aneurysm physiopathology, Aortic Valve abnormalities, Aortic Valve Insufficiency, Extracellular Space enzymology, Heart Valve Diseases enzymology, Proto-Oncogene Proteins c-akt metabolism, Superoxide Dismutase chemistry, Tricuspid Valve enzymology
- Abstract
Ascending aortic aneurysm (AsAA) is a consequence of medial degeneration (MD), deriving from apoptotic loss of smooth muscle cells (SMC) and fragmentation of elastin and collagen fibers. Alterations of extracellular matrix structure and protein composition, typical of medial degeneration, can modulate intracellular pathways. In this study we examined the relevance of superoxide dismutase (SOD3) and Akt in AsAA pathogenesis, evaluating their tissue distribution and protein levels in ascending aortic tissues from controls (n=6), patients affected by AsAA associated to tricuspid aortic valve (TAV, n=9) or bicuspid aortic valve (BAV, n=9). The results showed a significant reduction of SOD3, phospho-Akt and Akt protein levels in AsAA tissues from patients with BAV, compared to controls, whereas the differences observed between controls and patients with TAV were not significant. The decreased levels of SOD3 and Akt in BAV aortic tissues are associated with decreased Erk1/Erk2 phosphorylation and MMP-9 levels increase. The authors suggest a role of decreased SOD3 protein levels in the progression of AsAA with BAV and a link between ECM modifications of aortic media layer and impaired Erk1/Erk2 and Akt signaling in the late stages of the aortopathy associated with BAV.
- Published
- 2014
- Full Text
- View/download PDF
45. Evaluation of cytotoxic effects of 7-dehydrocholesterol on melanoma cells.
- Author
-
Gelzo M, Granato G, Albano F, Arcucci A, Dello Russo A, De Vendittis E, Ruocco MR, and Corso G
- Subjects
- Apoptosis radiation effects, Cell Proliferation radiation effects, Dehydrocholesterols administration & dosage, Humans, Melanoma pathology, Membrane Potential, Mitochondrial drug effects, Membrane Potential, Mitochondrial radiation effects, Neoplasm Staging, Skin drug effects, Skin pathology, Skin radiation effects, Ultraviolet Rays, Apoptosis drug effects, Cell Proliferation drug effects, Dehydrocholesterols adverse effects, Melanoma drug therapy
- Abstract
Ultraviolet radiation is the main cause of skin cancers, and melanoma is the most serious form of tumor. There is no therapy for advanced-stage melanoma and its metastasis because of their high resistance to various anticancer therapies. Human skin is an important metabolic organ in which occurs photoinduced synthesis of vitamin D3 from 7-dehydrocholesterol (7-DHC). 7-DHC, the precursor of cholesterol biosynthesis, is highly reactive and easily modifiable to produce 7-DHC-derived compounds. The intracellular levels of 7-DHC or its derivatives can have deleterious effects on cellular functionality and viability. In this study we evaluated the effects on melanoma cell lines of 7-DHC as such and for this aim we used much care to minimize 7-DHC modifications. We found that from 12 to 72 h of treatment 82-86% of 7-DHC entered the cells, and the levels of 7-DHC-derived compounds were not significant. Simultaneously, reactive oxygen species production was significantly increased already after 2h. After 24 h and up to 72 h, 7-DHC-treated melanoma cells showed a reduction in cell growth and viability. The cytotoxic effect of 7-DHC was associated with an increase in Bax levels, decrease in Bcl-2/Bax ratio, reduction of mitochondrial membrane potential, increase in apoptosis-inducing factor levels, unchanged caspase-3 activity, and absence of cleavage of PARP-1. These findings could explain the mechanism through which 7-DHC exerts its cytotoxic effects. This is the first report in which the biological effects found in melanoma cells are mainly attributable to 7-DHC as such., (Copyright © 2014 Elsevier Inc. All rights reserved.)
- Published
- 2014
- Full Text
- View/download PDF
46. Structural and denaturation studies of two mutants of a cold adapted superoxide dismutase point to the importance of electrostatic interactions in protein stability.
- Author
-
Merlino A, Russo Krauss I, Castellano I, Ruocco MR, Capasso A, De Vendittis E, Rossi B, and Sica F
- Subjects
- Circular Dichroism, Crystallography, X-Ray, Models, Molecular, Protein Conformation, Protein Denaturation, Protein Stability, Spectrometry, Fluorescence, Static Electricity, Superoxide Dismutase genetics, Cold Temperature, Mutation, Superoxide Dismutase chemistry
- Abstract
A peculiar feature of the psychrophilic iron superoxide dismutase from Pseudoalteromonas haloplanktis (PhSOD) is the presence in its amino acid sequence of a reactive cysteine (Cys57). To define the role of this residue, a structural characterization of the effect of two PhSOD mutations, C57S and C57R, was performed. Thermal and denaturant-induced unfolding of wild type and mutant PhSOD followed by circular dichroism and fluorescence studies revealed that C→R substitution alters the thermal stability and the resistance against denaturants of the enzyme, whereas C57S only alters the stability of the protein against urea. The crystallographic data on the C57R mutation suggest an involvement of the Arg side chain in the formation of salt bridges on protein surface. These findings support the hypothesis that the thermal resistance of PhSOD relies on optimization of charge-charge interactions on its surface. Our study contributes to a deeper understanding of the denaturation mechanism of superoxide dismutases, suggesting the presence of a structural dimeric intermediate between the native state and the unfolded state. This hypothesis is supported by the crystalline and solution data on the reduced form of the enzyme., (Copyright © 2014 Elsevier B.V. All rights reserved.)
- Published
- 2014
- Full Text
- View/download PDF
47. Markers of mitochondrial dysfunction during the diclofenac-induced apoptosis in melanoma cell lines.
- Author
-
Albano F, Arcucci A, Granato G, Romano S, Montagnani S, De Vendittis E, and Ruocco MR
- Subjects
- Caspase 9 metabolism, Cell Line, Tumor, Cytochromes c metabolism, Enzyme Activation drug effects, Humans, Reactive Oxygen Species metabolism, Superoxide Dismutase metabolism, Anti-Inflammatory Agents, Non-Steroidal pharmacology, Apoptosis drug effects, Biomarkers, Tumor metabolism, Diclofenac pharmacology, Melanoma pathology, Mitochondria drug effects, Mitochondria metabolism
- Abstract
Melanoma is an aggressive cutaneous cancer, whose incidence is growing in recent years, especially in the younger population. The favorable therapy for this neoplasm consists in its early surgical excision; otherwise, in case of late diagnosis, melanoma becomes very refractory to any conventional therapy. Nevertheless, the acute inflammatory response occurring after excision of the primary melanoma can affect the activation and/or regulation of melanoma invasion and metastasis. Nonsteroidal anti-inflammatory drugs (NSAIDs), widely employed in clinical therapy as cyclooxygenase inhibitors, also display a cytotoxic effect on some cancer cell lines; therefore, their possible usage in combination with conventional chemo- and radio-therapies of tumors is being considered. In particular, diclofenac, one of the most common NSAIDs, displays its anti-proliferative effect in many tumor lines, through an alteration of the cellular redox state. In this study, the possible anti-neoplastic potential of diclofenac on the human melanoma cell lines A2058 and SAN was investigated, and a comparison was made with the results obtained from the nonmalignant fibroblast cell line BJ-5ta. Either in A2058 or SAN, the diclofenac treatment caused typical apoptotic morphological changes, as well as an increase of the number of sub-diploid nuclei; conversely, the same treatment on BJ-5ta had only a marginal effect. The observed decrease of Bcl-2/Bax ratio and a parallel increase of caspase-3 activity confirmed the pro-apoptotic role exerted by diclofenac in melanoma cells; furthermore, the drug provoked an increase of the ROS levels, a decrease of mitochondrial superoxide dismutase (SOD2), the cytosolic translocation of both SOD2 and cytochrome c, and an increase of caspase-9 activity. Finally, the cytotoxic effect of diclofenac was amplified, in melanoma cells, by the silencing of SOD2. These data improve the knowledge on the effects of diclofenac and suggest that new anti-neoplastic treatments should be based on the central role of mitochondrion in cancer development; under this concern, the possible involvement of SOD2 as a novel target could be considered., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)
- Published
- 2013
- Full Text
- View/download PDF
48. Crystallographic and spectroscopic characterizations of Sulfolobus solfataricus TrxA1 provide insights into the determinants of thioredoxin fold stability.
- Author
-
Esposito L, Ruggiero A, Masullo M, Ruocco MR, Lamberti A, Arcari P, Zagari A, and Vitagliano L
- Subjects
- Amino Acid Motifs, Amino Acid Sequence, Circular Dichroism, Crystallography, X-Ray, Hydrogen Bonding, Models, Molecular, Molecular Sequence Data, Protein Denaturation, Protein Stability, Protein Structure, Quaternary, Protein Structure, Tertiary, Structural Homology, Protein, Archaeal Proteins chemistry, Sulfolobus solfataricus, Thioredoxins chemistry
- Abstract
Structural characterizations of thioredoxins (Trxs) are important for their involvement in severe pathologies and for their stable scaffold. Here we report a combined structural and spectroscopic characterization of a Trx isolated from the hyperthermophilic archaeon Sulfolobus solfataricus (SsTrxA1). Thermal denaturation unveils that SsTrxA1 is endowed with a remarkable stability in the explored temperature range 50-105°C. The structure of the oxidized form of SsTrxA1 determined at 1.9Å resolution presents a number of peculiar features. Although the protein was crystallized in a slightly acid medium (pH 6.5) as many as ten intramolecular/intermolecular carboxyl-carboxylate interactions involving glutamic and aspartic acid side chains are found in three independent SsTrxA1 molecules present in the asymmetric unit. Surprisingly for a hyperthermostable protein, the structure of SsTrxA1 is characterized by the presence (a) of a very limited number of intramolecular salt bridges and (b) of a cavity nearby Cys52, a residue that is frequently a phenylananine in other members of the family. Chemical denaturation investigations carried out on SsTrxA1 and SsTrxA2 show that both proteins present a significant stability against guanidine hydrochloride, thus indicating that ionic interactions play a minor role in their stabilization. Compared to Trxs from mesophilic sources, SsTrxA1 displays a longer α-helix 1 and a shorter loop connecting this α-helix with β-strand 2. As these features are shared with Trxs isolated from thermophilic sources, the shortening of this loop may be a general strategy adopted to stabilize this fold. This feature may be exploited for the design of hyperthermostable Trx scaffolds., (Copyright © 2011 Elsevier Inc. All rights reserved.)
- Published
- 2012
- Full Text
- View/download PDF
49. Analysis of extracellular superoxide dismutase in fibroblasts from patients with systemic sclerosis.
- Author
-
Arcucci A, Ruocco MR, Amatruda N, Riccio A, Tarantino G, Albano F, Mele V, and Montagnani S
- Subjects
- Adult, Female, Fluorescent Antibody Technique, Humans, RNA, Messenger analysis, Superoxide Dismutase analysis, Superoxide Dismutase metabolism, Fibroblasts enzymology, Scleroderma, Systemic enzymology, Superoxide Dismutase genetics
- Abstract
Systemic sclerosis (SSc) is a chronic disease of connective tissue characterized by vascular damage, autoantibody production and extensive fibrosis of skin, skeletal muscles, vessels and visceral organs. Fibrosis is a biological process involving inflammatory response and reactive oxygen species (ROS) accumulation leading to fibroblast activation. Extracellular superoxide dismutase (SOD3), a copper and zinc superoxide dismutase, which is expressed in selected tissues, is secreted into the extracellular space and catalyzes the dismutation of superoxide radical to hydrogen peroxide and molecular oxygen. Moreover, SOD3 is associated to inflammatory responses in some experimental models. In this paper we analysed, by RT-PCR and immunofluorescence, SOD3 expression and intracellular localization in dermal fibroblasts from both healthy donors and patients affected by diffuse form of SSc. Moreover, we determined SOD3 enzymatic activity in fibroblast culture medium with the xanthine/xanthine oxidase method. Increased expression of SOD3 mRNA was detected in systemic sclerosis fibroblasts (SScF), as compared to control healthy fibroblasts (HF), and SOD3 immunofluorescence staining displayed a characteristic pattern of secretory proteins in both HF and SScF. Superoxide dismutase assay demonstrated that SOD3 enzymatic activity in SScF culture medium is four times more than in HF culture medium. These data suggest that an alteration in SOD3 expression and activity could be associated to SSc fibrosis.
- Published
- 2011
50. Diclofenac-induced apoptosis in the neuroblastoma cell line SH-SY5Y: possible involvement of the mitochondrial superoxide dismutase.
- Author
-
Cecere F, Iuliano A, Albano F, Zappelli C, Castellano I, Grimaldi P, Masullo M, De Vendittis E, and Ruocco MR
- Subjects
- Antioxidants metabolism, Cell Line, Tumor, Cytochromes c metabolism, Gene Expression Regulation, Neoplastic drug effects, Humans, Intracellular Space drug effects, Intracellular Space metabolism, Membrane Potential, Mitochondrial drug effects, Neuroblastoma enzymology, Neuroblastoma genetics, Protein Transport drug effects, RNA, Messenger genetics, RNA, Messenger metabolism, Reactive Oxygen Species metabolism, Subcellular Fractions drug effects, Subcellular Fractions metabolism, Superoxide Dismutase genetics, Thioredoxins pharmacology, Apoptosis drug effects, Diclofenac pharmacology, Mitochondria drug effects, Mitochondria enzymology, Neuroblastoma pathology, Superoxide Dismutase metabolism
- Abstract
Diclofenac, a nonsteroidal anti-inflammatory drug, induces apoptosis on the neuroblastoma cell line SH-SY5Y through a mitochondrial dysfunction, affecting some antioxidant mechanisms. Indeed, the time- and dose-dependent increase of apoptosis is associated to an early enhancement of the reactive oxygen species (ROS). Mitochondrial superoxide dismutase (SOD2) plays a crucial role in the defence against ROS, thus protecting against several apoptotic stimuli. Diclofenac decreased the protein levels and the enzymatic activity of SOD2, without any significant impairment of the corresponding mRNA levels in the SH-SY5Y extracts. When cells were incubated with an archaeal exogenous thioredoxin, an attenuation of the diclofenac-induced apoptosis was observed, together with an increase of SOD2 protein levels. Furthermore, diclofenac impaired the mitochondrial membrane potential, leading to a release of cytochrome c. These data suggest that mitochondria are involved in the diclofenac-induced apoptosis of SH-SY5Y cells and point to a possible role of SOD2 in this process.
- Published
- 2010
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.