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2.
J Oncol ; 2019: 5879616, 2019.
Article in English | MEDLINE | ID: mdl-31827511

ABSTRACT

The recent introduction of the "precision medicine" concept in oncology pushed cancer research to focus on dynamic measurable biomarkers able to predict responses to novel anticancer therapies in order to improve clinical outcomes. Recently, the involvement of extracellular vesicles (EVs) in cancer pathophysiology has been described, and given their release from all cell types under specific stimuli, EVs have also been proposed as potential biomarkers in cancer. Among the techniques used to study EVs, flow cytometry has a high clinical potential. Here, we have applied a recently developed and simplified flow cytometry method for circulating EV enumeration, subtyping, and isolation from a large cohort of metastatic and locally advanced nonhaematological cancer patients (N = 106); samples from gender- and age-matched healthy volunteers were also analysed. A large spectrum of cancer-related markers was used to analyse differences in terms of peripheral blood circulating EV phenotypes between patients and healthy volunteers, as well as their correlation to clinical outcomes. Finally, EVs from patients and controls were isolated by fluorescence-activated cell sorting, and their protein cargoes were analysed by proteomics. Results demonstrated that EV counts were significantly higher in cancer patients than in healthy volunteers, as previously reported. More interestingly, results also demonstrated that cancer patients presented higher concentrations of circulating CD31+ endothelial-derived and tumour cancer stem cell-derived CD133 + CD326- EVs, when compared to healthy volunteers. Furthermore, higher levels of CD133 + CD326- EVs showed a significant correlation with a poor overall survival. Additionally, proteomics analysis of EV cargoes demonstrated disparities in terms of protein content and function between circulating EVs in cancer patients and healthy controls. Overall, our data strongly suggest that blood circulating cancer stem cell-derived EVs may have a role as a diagnostic and prognostic biomarker in cancer.

3.
Stem Cell Rev Rep ; 10(3): 429-46, 2014 Jun.
Article in English | MEDLINE | ID: mdl-24619862

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.


Subject(s)
Cryopreservation , Mesenchymal Stem Cells , Proteome/metabolism , Adipogenesis , Antigens, CD/metabolism , Cell Separation , Cells, Cultured , Humans , Osteogenesis , Protein Interaction Maps , Telomere/genetics , Umbilical Cord/cytology
4.
Int J Immunopathol Pharmacol ; 21(1): 23-33, 2008.
Article in English | MEDLINE | ID: mdl-18336728

ABSTRACT

The protein kinase C (PKC) family of enzymes is a regulator of transmembrane signal transduction. There is evidence demonstrating altered activity of some PKC isoforms (PKC-alpha, PKC-delta and PKC-zeta) in the neurons of brains of Alzheimers Disease (AD) sufferers, but little is known about their involvement in the intracellular machinery of amyloid beta protein-reactive T lymphocytes in AD. By applying a modified, split-well culture system, for Abeta(1-42) reactivity, we carried out flow cytometry analysis and biochemical investigations on the possible involvement of PKC-alpha, PKC-delta and PKC-zeta in the signalling system activated in Abeta-reactive T cells purified from peripheral blood mononucleate cells (PBMC) from healthy subjects and patients with AD. Flow cytometry analysis of Abeta(1-42) activated T lymphocytes in the majority of AD patients highlighted a distinct cellular cluster highly expressing phospho-PKC-delta (P-PKC-delta), while most full-blown AD patients highly expressed two distinct P-PKC-delta and phospho-PKC-zeta (P-PKC-zeta) bright sub-populations. The same investigation performed in freshly purified peripheral T lymphocytes, did not highlight any subpopulation, suggesting that the detection of P-PKC-delta and P-PKC-zeta bright subpopulations is specifically linked to Abeta(1-42) activated T lymphocytes. The data presented here, therefore, suggest possible novel hallmarks to discriminate between healthy elderly subjects and beginning or full-blown Alzheimers Disease patients.


Subject(s)
Alzheimer Disease/immunology , Amyloid beta-Peptides/pharmacology , Isoenzymes/metabolism , Lymphocyte Activation , Peptide Fragments/pharmacology , Protein Kinase C/metabolism , T-Lymphocytes/enzymology , Adult , Aged , Aged, 80 and over , Alzheimer Disease/enzymology , Cells, Cultured , Flow Cytometry , Humans , Middle Aged , Phosphorylation , Signal Transduction
5.
Int J Immunopathol Pharmacol ; 18(3): 445-55, 2005.
Article in English | MEDLINE | ID: mdl-16164827

ABSTRACT

Release of vascular endothelial growth factor (VEGF) and other candidate angiogenic factors such as basic fibroblast growth factor and transforming growth factor beta, may play a role in sustaining neoplastic cell proliferation and tumor growth. We evaluated VEGF expression and synthesis in the two erythromegakaryocytic cell lines B1647, HEL and one megakaryocytic cell line MO7 expressing erythroid markers. In this study RT-PCR was performed to evaluate VEGF expression and that of its receptor KDR; VEGF production was assayed by Elisa test and western blot analysis; sensitivity to VEGF was tested by thymidine incorporation. VEGF and its receptor KDR were expressed in B1647 and HEL, both as mRNAs and as proteins, while only KDR transcript was found in MO7 cells. Only B1647 and HEL cells showed a strong spontaneous proliferating activity. In fact, measurable amounts of VEGF were present in the unstimulated cell medium, thus suggesting an autocrine production of VEGF by B1647 and HEL cells, but not by MO7, which was inhibited in mRNA-silencing conditions. This production could not be further boosted by other growth factors, whereas it was inhibited by TGF-beta1. Finally, analysis of Shc signal transduction proteins following stimulation with VEGF indicated that only p46 was tyrosine phosphorylated. These data indicate that leukemic cells may be capable of autocrine production of VEGF which, in turn, maintains cell proliferation, possibly mediated by Shc p46 phosphorylation.


Subject(s)
Cell Proliferation/drug effects , Megakaryocytes/metabolism , Signal Transduction/drug effects , Vascular Endothelial Growth Factor A/biosynthesis , Vascular Endothelial Growth Factor A/pharmacology , Adaptor Proteins, Signal Transducing/chemistry , Adaptor Proteins, Signal Transducing/metabolism , Biomarkers/blood , Biomarkers/metabolism , Blotting, Western , Cell Line, Tumor , Dose-Response Relationship, Drug , Enzyme-Linked Immunosorbent Assay , Humans , Megakaryocytes/drug effects , Phosphorylation , Precipitin Tests , RNA, Messenger/metabolism , RNA, Small Interfering/pharmacology , Reverse Transcriptase Polymerase Chain Reaction , Shc Signaling Adaptor Proteins , Src Homology 2 Domain-Containing, Transforming Protein 1 , Transforming Growth Factor beta/pharmacology , Transforming Growth Factor beta1 , Tyrosine/chemistry , Vascular Endothelial Growth Factor A/metabolism , Vascular Endothelial Growth Factor Receptor-2/metabolism
6.
J Appl Toxicol ; 24(4): 289-96, 2004.
Article in English | MEDLINE | ID: mdl-15300717

ABSTRACT

Recent findings have suggested that oxidative damage might contribute to the cytotoxicity and carcinogenicity of aflatoxin B(1) (AFB(1)). The induction of oxidative stress also plays an important role in the toxicity of another mycotoxin: ochratoxin A (OTA). In this study, the protective effect of rosmarinic acid (Ros A) against AFB(1) and OTA-induced cytotoxicity was investigated in a human hepatoma-derived cell line (Hep G2). Rosmarinic acid, a natural phenolic compound contained in many Lamiaceae herbs such as Perilla frutescens, sage, basil and mint, inhibits complement-dependent inflammatory processes and may have therapeutic potential. The ability of Ros A to reduce radical oxygen species (ROS) production, protein and DNA synthesis inhibition and apoptosis caused by the two mycotoxins was also investigated. Our experiments proved the significant cytoprotective effect of Ros A in vitro from OTA- and AFB(1)-induced cell damage. In particular, 24-h pretreatment with 50 micro M Ros A inhibited the cytotoxicity of 10 micro M AFB(1) (by 45%) and 10 micro M OTA (by 35%) in Hep G2 cells (P < 0.001). Moreover, Ros A dose dependently attenuated ROS production and DNA and protein synthesis inhibition induced by both of the toxins. Similarly, apoptosis cell death was prevented, as demonstrated by reduction of DNA fragmentation and inhibition of caspase-3 activation (P < 0.001).


Subject(s)
Aflatoxin B1/toxicity , Antioxidants/pharmacology , Apoptosis/drug effects , Cinnamates/pharmacology , Ochratoxins/toxicity , Carcinoma, Hepatocellular/pathology , Caspase 3 , Caspases/metabolism , Cell Line, Tumor , Cell Survival/drug effects , DNA/biosynthesis , DNA Fragmentation/drug effects , Depsides , Dose-Response Relationship, Drug , Humans , Liver Neoplasms/pathology , Protein Biosynthesis , Reactive Oxygen Species/metabolism , Rosmarinic Acid
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