[Show abstract][Hide abstract] ABSTRACT: The ErbB tyrosine kinase receptor family has been shown to have an important role in tumorigenesis, and the expression of its receptor members is frequently deregulated in many types of solid tumors. Various drugs targeting these receptors have been approved for cancer treatment. Particularly, in breast cancer, anti-Her2/EGFR molecules represent the standard therapy for Her2-positive malignancies. However, in a number of cases, the tumor relapses or progresses thus suggesting that not all cancer cells have been targeted. One possibility is that a subset of cells capable of regenerating the tumor, such as cancer stem cells (CSCs), may not respond to these therapeutic agents. Accumulating evidences indicate that miR-205-5p is significantly downregulated in breast tumors compared with normal breast tissue and acts as a tumor suppressor directly targeting oncogenes such as Zeb1 and ErbB3. In this study, we report that miR-205-5p is highly expressed in BCSCs and represses directly ERBB2 and indirectly EGFR leading to resistance to targeted therapy. Furthermore, we show that miR-205-5p directly regulates the expression of p63 which is in turn involved in the EGFR expression suggesting a miR-205/p63/EGFR regulation.
[Show abstract][Hide abstract] ABSTRACT: Multiple Sclerosis (MuS) is a disease caused due to an autoimmune attack against myelin components in which non proteic mediators may play a role. Recent research in metabolomics and lipidomics has been driven by rapid advances in technologies such as mass spectrometry and computational methods. They can be used to study multifactorial disorders like MuS, highlighting the effects of disease on metabolic profiling, regardless of the multiple trigger factors. We coupled MALDI-TOF-MS untargeted lipidomics and targeted LC-MS/MS analysis of acylcarnitines and aminoacids to compare cerebrospinal fluid metabolites in 13 MuS subjects and in 12 patients with Other Neurological Diseases (OND). After data processing and statistical evaluation, we found 10 metabolites that significantly (p < 0.05) segregate the two clinical groups. The most relevant result was the alteration of phospholipids levels in MuS and the correlation between some of them with clinical data. In particular lysophosphatidylcholines (m/z = 522.3 Da, 524.3 Da) and an unidentified peak at m/z = 523.0 Da correlated to the Link index, lysophosphatidylinositol (m/z = 573.3 Da) correlated to EDSS and phosphatidylinositol (m/z = 969.6 Da) correlated to disease duration. We also found high levels of glutamate in MuS. In conclusion, our integrated mass spectrometry approach showed high potentiality to find metabolic alteration in cerebrospinal fluid. These data, if confirmed in a wider clinical study, could open the door for the discovery of novel candidate biomarkers of MuS.
[Show abstract][Hide abstract] ABSTRACT: Proteomics and metabolomics investigations of body fluids represent the most challenge for biomarker discovery of several diseases. The search for biomarkers is actually conducted in different body fluids, even if the ideal biomarker should be found in a easily accessible biological fluid, because, if validated, the biomarker could be sought in the healthy population. In this vision tears could be considered an optimum material obtainable by non invasive procedures. In the last years, there was more interest from the scientific community in the study of tears for the research of new biomarkers not only for ocular diseases. In this review we provide a discussion on the current state of biomarkers research in tears and their relevance for clinical practice, reporting the main results of clinical proteomics studies on systemic and eye diseases. We summarize the main methods for tear samples analyses reporting recent advances in "omics" platforms for tears investigations. Moreover, we want to take stock of the emerging field of metabolomics and lipidomics as a new and integrated approach to study protein-metabolites interplay for biomarkers research, where tears represent a still unexplored and attractive field. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: Programmed cell death is a process known to have a crucial role in many aspects of eukaryotes physiology and is clearly essential to their life. As a consequence, the underlying molecular mechanisms have been extensively studied in eukaryotes and we now know that different signalling pathways leading to functionally and morphologically different forms of death exist in these organisms. Similarly, mono-cellular organism can activate signalling pathways leading to death of a number of cells within a colony. The reason why a single-cell organism would activate a program leading to its death is apparently counterintuitive and probably for this reason cell death in prokaryotes has received a lot less attention in the past years. However, as summarized in this review there are many reasons leading to prokaryotic cell death, for the benefit of the colony. Indeed, single-celled organism can greatly benefit from multicellular organization. Within this forms of organization, regulation of death becomes an important issue, contributing to important processes such as: stress response, development, genetic transformation, and biofilm formation.
[Show abstract][Hide abstract] ABSTRACT: Brought to you by the editorial team of Cell Death and Differentiation, Cell Death and Disease is a peer-reviewed author-pays online journal in the field of translational cell death. It seeks to promote diverse and integrated areas of Experimental and Internal Medicine with its specialties, including Cancer, Immunity and Neuroscience.
[Show abstract][Hide abstract] ABSTRACT: A number of studies suggest that cancer stem cells are essential for tumour growth, and failure to target these cells can result in tumour relapse. As this population of cells has been shown to be resistant to radiation and chemotherapy, it is essential to understand their biology and identify new therapeutic approaches. Targeting cancer metabolism is a potential alternative strategy to counteract tumour growth and recurrence. Here we applied a proteomic and targeted metabolomic analysis in order to point out the main metabolic differences between breast cancer cells grown as spheres and thus enriched in cancer stem cells were compared with the same cells grown in adherent differentiating conditions. This integrated approach allowed us to identify a metabolic phenotype associated with the stem-like condition and shows that breast cancer stem cells (BCSCs) shift from mitochondrial oxidative phosphorylation towards fermentative glycolysis. Functional validation of proteomic and metabolic data provide evidences for increased activities of key enzymes of anaerobic glucose fate such as pyruvate kinase M2 isoform, lactate dehydrogenase and glucose 6-phopshate dehydrogenase in cancer stem cells as well as different redox status. Moreover, we show that treatment with 2-deoxyglucose, a well known inhibitor of glycolysis, inhibits BCSC proliferation when used alone and shows a synergic effect when used in combination with doxorubicin. In conclusion, we suggest that inhibition of glycolysis may be a potentially effective strategy to target BCSCs.
[Show abstract][Hide abstract] ABSTRACT: Cryopreservation is the only method for long-term storage of viable cells and tissues used for cellular therapy, stem cell transplantation and/or tissue engineering. However, the freeze-thaw process strongly contributes to cell and tissue damage through several mechanisms, including oxidative stress, cell injury from intracellular ice formation and altered physical cellular properties. Our previous proteomics investigation was carried out on Wharton's Jelly Stem Cells (WJSCs) having similar properties to adult mesenchymal stem cells and thus representing a rich source of primitive cells to be potentially used in regenerative medicine. The aim of the present work was to investigate molecular changes that occur in WJSCs proteome in different experimental conditions: fresh primary cell culture and frozen cell. To analyze changes in protein expression of WJSCs undergoing different culturing procedures, we performed a comparative proteomic analysis (2DE followed by MALDI-TOF MS/MS nanoESI-Q-TOF MS coupled with nanoLC) between WJSCs from fresh and frozen cell culturing, respectively. Frozen WJSCs showed qualitative and quantitative changes compared to cells from fresh preparation, expressing proteins involved in replication, cellular defence mechanism and metabolism, that could ensure freeze-thaw survival. The results of this study could play a key role in elucidating possible mechanisms related to maintaining active proliferation and maximal cellular plasticity and thus making the use of WJSCs in cell therapy safe following bio-banking.
[Show abstract][Hide abstract] ABSTRACT: p63 is an important regulator of epithelial development expressed in different variants, containing (TA) or lacking (ΔN) the N-terminal transactivation domain. The different isoforms regulate stem cell renewal and differentiation as well as cell senescence. Several studies indicate that p63 isoforms also play a role in cancer development, however very little is known on the role played by p63 in regulating the Cancer Stem phenotype. Here we investigate the cellular signals regulated by TAp63 andΔNp63 in a model of epithelial-cancer stem cells. To this end we used colon cancer stem cells, over-expressing either TAp63 or ΔNp63 isoforms, to carry out a proteomic study by chemical labeling approach coupled to network analysis. Our results indicate that p63 is implicated in a wide range of biological processes, including metabolism. This was further investigated by a targeted strategy, both at protein and metabolite level. The overall data show that TAp63 over-expressing cells are more glycolytic active than ΔNp63 cells, indicating that the two isoforms may regulate the key steps of glycolysis in an opposite manner. The mass spectrometry proteomics data of the study have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository with dataset identifiers PXD000769 and PXD000768.
Journal of Proteome Research 03/2014; 13(4). DOI:10.1021/pr4012574 · 4.25 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Metal ions are necessary for the proper functioning of the immune system, and, therefore, they might have a significant influence on the interaction between bacteria and host. Ionic dyshomeostasis has been recently observed also in cystic fibrosis (CF) patients, whose respiratory tract is frequently colonized by Stenotrophomonas maltophilia. For the first time, here we used an inductively mass spectrometry method to perform a spatial and temporal analysis of the pattern of changes in a broad range of major trace elements in response to pulmonary infection by S. maltophilia. To this, DBA/2 mouse lungs were comparatively infected by a CF strain and by an environmental one. Our results showed that pulmonary ionomic profile was significantly affected during infection. Infected mice showed increased lung levels of Mg, P, S, K, Zn, Se, and Rb. To the contrary, Mn, Fe, Co, and Cu levels resulted significantly decreased. Changes of element concentrations were correlated with pulmonary bacterial load and markers of inflammation, and occurred mostly on day 3 post-exposure, when severity of infection culminated. Interestingly, CF strain - significantly more virulent than the environmental one in our murine model - provoked a more significant impact in perturbing pulmonary metal homeostasis. Particularly, exposure to CF strain exclusively increased P and K levels, while decreased Fe and Mn ones. Overall, our data clearly indicate that S. maltophilia modulates pulmonary metal balance in a concerted and virulence-dependent manner highlighting the potential role of the element dyshomeostasis during the progression of S. maltophilia infection, probably exacerbating the harmful effects of the loss of CF transmembrane conductance regulator function. Further investigations are required to understand the biological significance of these alterations and to confirm they are specifically caused by S. maltophilia.
PLoS ONE 02/2014; 9(2):e88769. DOI:10.1371/journal.pone.0088769 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Multidrug resistance (MDR) in cancer cells is often caused by the high expression of the plasma membrane drug transporter P-glycoprotein (Pgp) associated with an elevated intracellular glutathione (GSH) content in various human tumors. Several chemosensitizers reverse MDR but have significant toxicities. Antiemetic medications are often used for controlling chemotherapy-induced nausea and vomiting in cancer patient. In this in vitro study we investigated if the effects of two common antiemetic drugs such as dimenhydrinate (dime) and ondansentron (onda) and a natural compound (6)-gingerol (ginger), the active principle of ginger root, interfere on Pgp activity and intracellular GSH content in order to evaluate their potential use as chemosensitizing agents in anticancer chemotherapy. The human doxorubicin (doxo) resistant uterine sarcoma cells (MES-SA/Dx5) that overexpress Pgp, were treated with each antiemetic alone (1, 10 and 20 microM) or in combination with different doxo concentrations (2, 4, and 8 microM). We measured the intracellular accumulation and cytotoxicity of doxo (MTT assay), the cellular GSH content (GSH assay) and ROS production (DFC-DA assay), in comparison with verapamil (Ver), a specific inhibitor for Pgp, used as reference molecule. We found that exposure at 2, 4 and 8 microM doxo concentrations in the presence of dime, onda and ginger enhanced significantly doxo accumulation and cytotoxicity on resistant MES-SA/Dx5 cells when compared with doxo alone. Moreover, treatment with ginger (20 microM) increased cellular GSH content (greater than 10 percent) in resistant cells, while ROS production remained below the control values for all antiemetic compounds at all concentrations. These findings provide the rationale for innovative clinical trials of antiemetics or their derivatives as a new potential generation of chemosensitizers to improve effectiveness of the anticancer drugs in MDR human tumours.
Journal of biological regulators and homeostatic agents 01/2014; 27(4):1029-37. · 2.04 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Escherichia coli remains one of the most frequent causes of several common bacterial infections in humans and animals. E. coli is the prominent cause of enteritis, urinary tract infection, septicaemia and other clinical infections, such as neonatal meningitis. E. coli is also prominently associated with diarrhoea in pet and farm animals. The therapeutic treatment of E. coli infections is threatened by the emergence of antimicrobial resistance. The prevalence of multidrug-resistant E. coli strains is increasing worldwide principally due to the spread of mobile genetic elements, such as plasmids. The rise of multidrug-resistant strains of E. coli also occurs in Europe. Therefore, the spread of resistance in E. coli is an increasing public health concern in European countries. This paper summarizes the current status of E. coli strains clinically relevant in European countries. Furthermore, therapeutic interventions and strategies to prevent and control infections are presented and discussed. The article also provides an overview of the current knowledge concerning promising alternative therapies against E. coli diseases.
International Journal of Environmental Research and Public Health 12/2013; 10(12):6235-54. DOI:10.3390/ijerph10126235 · 2.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Many adult tissues contain a population of stem cells with the ability to regenerate structures similar to the microenvironments from which they are derived in vivo and represent a promising therapy for the regeneration of complex tissues in the clinical disorder. Human adult stem cells (SCs) including bone marrow stem cells (BMSCs), dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) have been characterized for their high proliferative potential, expression of characteristic SC-associated markers and for the plasticity to differentiate in different lineage in vitro.
The aim of this study is to define the molecular features of stem cells from oral tissue by comparing the proteomic profiles obtained with 2-DE followed by MALDI-TOF/TOF of ex-vivo cultured human PDLSCs, DPSCs and BMSCs. Our results showed qualitative similarities in the proteome profiles among the SCs examined including some significant quantitative differences. To enrich the knowledge of oral SCs proteome we performed an analysis in narrow range pH 4-7 and 6-9, and we found that DPSCs vs PDLSCs express differentially regulated proteins that are potentially related to growth, regulation and genesis of neuronal cells, suggesting that SCs derived from oral tissue source populations may possess the potential ability of neuronal differentiation which is very consistent with their neural crest origin.
This study identifies some differentially expressed proteins by using comparative analysis between DPSCs and PDLSCs and BMSCs and suggests that stem cells from oral tissue could have a different cell lineage potency compared to BMSCs.
PLoS ONE 08/2013; 8(8):e71101. DOI:10.1371/journal.pone.0071101 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Blastoschizomyces capitatus is an uncommon, opportunistic pathogenic fungus, which causes invasive and disseminated infections. This microorganism is normally present in both environmental and normal human flora. Within a host, B. capitatus is able to grow in both unicellular yeast and multicellular filamentous growth forms. In this study, we obtained in vitro morphological conversion of B. capitatus from yeast-to-mycelial phase to investigate the presence and expression of glutathione transferase (GST) enzymes in both cell forms. A protein with GST activity using the model substrate 1-chloro-2,4-dinitrobenzene was detected in both morphologies and identified by tandem mass spectrometry as a eukaryotic elongation factor 1Bγ (eEF1Bγ) protein, a member of the GST superfamily. No significant difference in GST-specific activity and kinetic constants were observed between mycelial and yeast forms, indicating that eEF1Bγ protein did not show differential expression between the two phases.
[Show abstract][Hide abstract] ABSTRACT: Mitochondria carry maternally inherited genetic material, called the mitochondrial genome (mtDNA), which can be defined as the 25th human chromosome. The chromosome-centric Human Proteome Project (c-HPP) has initially focused its activities addressing the characterization and quantification of the nuclear encoded proteins. Following the last International HUPO Congress in Boston (September 2012) it was clear that however small the mitochondrial chromosome is, it plays an important role in many biological and physiopathological functions. Mutations in the mtDNA have been shown to be associated with dozens of unexplained disorders and the information contained in the mtDNA should be of major relevance to the understanding of many human diseases. Within this paper we describe the Italian initiative of the Human Proteome Project dedicated to mitochondria as part of both programs: chromosome-centric (c-HPP) and Biology/Disease (B/D-HPP). The mt-HPP has finally shifted the attention of the HUPO community outside the nuclear chromosomes with the general purpose to highlight the mitochondrial processes influencing the human health. Following this vision and considering the large interest and evidence collected on the non-Mendelian heredity of Homo sapiens associated with mt-chromosome and with the microbial commensal ecosystem constituting our organism we may speculate that this program will represent an initial step toward other HPP initiatives focusing on human phenotypic heredity.
[Show abstract][Hide abstract] ABSTRACT: Recent studies have shown that type 2 diabetes mellitus (T2DM) is a risk factor for cognitive dysfunction or dementia. Insulin resistance is often associated with T2DM and can induce defective insulin signaling in the central nervous system as well as increase the risk of cognitive impairment in the elderly. Glucagone like peptide-1 (GLP-1) is an incretin hormone and, like GLP-1 analogs, stimulates insulin secretion and has been employed in the treatment of T2DM. GLP-1 and GLP-1 analogs also enhance synaptic plasticity and counteract cognitive deficits in mouse models of neuronal dysfunction and/or degeneration. In this study, we investigated the potential neuroprotective effects of long-term treatment with exenatide, a GLP-1 analog, in two animal models of neuronal dysfunction: the PS1-KI and 3xTg-AD mice. We found that exenatide promoted beneficial effects on short- and long-term memory performances in PS1-KI but not in 3xTg-AD animals. In PS1-KI mice, the drug increased brain lactate dehydrogenase activity leading to a net increase in lactate levels, while no effects were observed on mitochondrial respiration. On the contrary, exenatide had no effects on brain metabolism of 3xTg-AD mice. In summary, our data indicate that exenatide improves cognition in PS1-KI mice, an effect likely driven by increasing the brain anaerobic glycolysis rate.
[Show abstract][Hide abstract] ABSTRACT: Cystic fibrosis (CF) is an autosomal recessive disorder associated with mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene and defective chloride transport across the epithelial cell membranes. Abnormal epithelial ion transport is the primary cause of persistent airway infections and chronic inflammation in CF patients. In order to gain further insight into the mechanisms of epithelial dysfunctions linked to CFTR mutations, we performed and integrated proteomic and ionomic analysis of human bronchial epithelial IB3-1 cells and compared them with a CFTR-complemented isogenic cell line (C38). Aside from changes that were consistent with known effects related to CFTR mutations, such as differences in glycolytic and gluconeogenic pathways and unfolded protein responses, differential proteomics highlighted significant alteration of protein expression and, in particular, of the 14-3-3 signalling pathway that is known to be involved in cellular calcium (Ca) homeostasis. Of note, restoring chloride efflux by acting on Ca cellular homeostasis has been shown to be a promising therapeutic intervention for CF. Ionomic analysis showed significant changes in the IB3-1 element profile compared with C38 cells and in particular we observed an increase of intracellular Ca that significantly correlates with intracellular zinc (Zn) levels, suggesting a synergistic role of Ca and Zn influx. This finding is particularly intriguing because Zn has been reported to be effective in CF treatment increasing Ca influx. Taken together, our proteomic and ionomic data reveal that CFTR mutation sets in motion endogenous mechanisms counteracting impaired chloride transport mainly acting on epithelial ion transport and increasing intracellular Ca, suggesting potential links between protein expression and this response.
[Show abstract][Hide abstract] ABSTRACT: Primary open angle glaucoma (POAG) is one of the main causes of irreversible blindness worldwide. The pathogenesis of POAG is still unclear. Alteration and sclerosis of trabecular meshwork with changes in aqueous humor molecular composition seem to play the key role. Increased intraocular pressure is widely known to be the main risk factor for the onset and progression of the disease. Unfortunately, the early diagnosis of POAG still remains the main challenge. In order to provide insight into the patho-physiology of glaucoma, here we report a shotgun proteomics approach to tears of patients with POAG naïve to therapy. Our proteomics results showed 27 differential tear proteins in POAG vs. CTRL comparison (25 up regulated proteins in the POAG group and two unique proteins in the CTRL group), 16 of which were associated with inflammatory response, free radical scavenging, cell-to-cell signaling and interaction. Overall the protein modulation shown in POAG tears proves the involvement of biochemical networks linked to inflammation. Among all regulated proteins, a sub-group of 12 up-regulated proteins in naïve POAG patients were found to be down-regulated in medically controlled POAG patients treated with prostanoid analogues (PGA), as reported in our previous work (i.e., lipocalin-1, lysozyme C, lactotransferrin, proline-rich-protein 4, prolactin-inducible protein, zinc-alpha-2-glycoprotein, polymeric immunoglobulin receptor, cystatin S, Ig kappa chain C region, Ig alpha-2 chain C region, immunoglobulin J chain, Ig alpha-1 chain C region). In summary, our findings indicate that the POAG tears protein expression is a mixture of increased inflammatory proteins that could be potential biomarkers of the disease, and their regulation may be involved in the mechanism by which PGA are able to decrease the intraocular pressure in glaucoma patients.
[Show abstract][Hide abstract] ABSTRACT: Transthyretin (TTR) is an homotetrameric protein of the Central Nervous System (CNS) that plays the role of the major Thyroxine (T4) carrier from blood to Cerebrospinal Fluid (CSF). T4 physiologically helps oligodendrocyte precursor cells to turn into myelinating oligodendrocytes, enhancing remyelination after myelin sheet damage. We investigated post-translational oxidative modifications of serum and CSF TTR in Multiple Sclerosis (MS) subjects, highlighting high levels of S-sulfhydration and S-sulfonation of cysteine in position ten only in the cerebral TTR, which correlate with an anomalous TTR protein folding as well as with disease duration. Moreover we found low levels of free T4 in CSF of MS patients, suggestive of a potential role of these modifications on T4 transport into the brain.