Involvement of thyroid hormones in brain development and cancer

The development and maturation of the mammalian brain are regulated by thyroid hormones (THs). Both hypothyroidism and hyperthyroidism cause serious anomalies in the organization and function of the nervous system. Most importantly, brain development is sensitive to TH supply well before the onset of the fetal thyroid function, and thus depends on the trans-placental transfer of maternal THs during pregnancy. Although the mechanism of action of THs mainly involves direct regulation of gene expression (genomic effects), mediated by nuclear receptors (THRs), it is now clear that THs can elicit cell responses also by binding to plasma membrane sites (non-genomic effects). Genomic and non-genomic effects of THs cooperate in modeling chromatin organization and function, thus controlling proliferation, maturation, and metabolism of the nervous system. However, the complex interplay of THs with their targets has also been suggested to impact cancer proliferation as well as metastatic processes. Herein, after discussing the general mechanisms of action of THs and their physiological effects on the nervous system, we will summarize a collection of data showing that thyroid hormone levels might influence cancer proliferation and invasion

FROM EPIGENETICS TO ANTI-DOPING APPLICATION: A NEW TOOL OF DETECTION

Eukaryotic genomes transcribe up to 90% of the genomic DNA but only 1\u20132% of these transcripts encode for proteins, whereas the vast majority are transcribed as non-coding RNAs (ncRNAs). They are divided into short ncRNA, particularly microRNA (miRNA) and small interference RNA (siRNA), and long ncRNAs. Noteworthy, they are unexpectedly stable since they are protected from degradation through different mechanisms: package in exosomes/microvesicles structures, in apoptotic bodies, in HDL lipoprotein, or by RNA binding proteins. For several years already, biomarkers have been used to detect biological disease; in the last years, a requirement appeared to find some of them to unearth the signs of doping. The potential of ncRNAs as a biological candidate is strongly debated and it seems to have become the right tool in the anti-doping hands. In the recent years, the next-generation sequencing (NGS) technology was used by the World Anti-Doping Agency to draft the athlete biological passport (ABP), measuring the circulating miRNAs and applying these new biomarkers in anti-doping. NGS technology does not require any prior knowledge of ncRNAs, but the limit to employ this biomarker to detect performance-enhancing drug use must consider the intrinsic and extrinsic factors that might affect measurements. Key words: pbiomarkers, doping, HDL, ncRNA, exosom

RNA-binding activity of the rat calmodulin-binding PEP-19 protein and of the long PEP-19 isoform

Synthesis of H1\u2da histone protein, in the developing rat brain, seems to be regulated mainly at the post-transcriptional level. Since regulation of RNA metabolism depends on a series of RNA-binding proteins, we have been searching for RNA-binding proteins involved in the post-transcriptional regulation of the H1\u2da gene. We recently reported isolation, from a cDNA expression library, of an insert encoding a novel protein, the C-terminal half of which is identical to that of PEP-19, a brain-specific protein involved in calcium metabolism. The novel protein was called long PEP-19 isoform (LPI). Herein we show that LPI, as well as PEP-19, can bind H1\u2da RNA. Moreover, in order to improve production of functional LPI/PEP-19, we modified the protocol normally adopted for preparing histidine tagged-proteins from bacteria, by adding an additional purification step. We also found that both LPI and PEP can compete for H1\u2da RNA binding with PIPPin (CSD-C2), another RNA-binding protein previously discovered in our laboratory. Since PEP19/LPI contain a calmodulin binding domain, we finally investigated whether their ability to bind RNA is affected by calmodulin. Our results show that calmodulin interferes with binding of H1\u2da RNA to both PEP-19 and LPI, while it is not able to bind RNA on its own. This finding suggests that calcium/calmodulin may have a role in controlling H1\u2da mRNA metabolism in the developing brain

Extracellular vesicles shed by melanoma cells contain a modified form of H1.0 linker histone and H1.0 mRNA-binding proteins

Extracellular vesicles (EVs) are now recognized as a fundamental way for cell-to-cell horizontal transfer of properties, in both physiological and pathological conditions. Most of EV-mediated cross-talk among cells depend on the exchange of proteins, and nucleic acids, among which mRNAs, and non-coding RNAs such as different species of miRNAs. Cancer cells, in particular, use EVs to discard molecules which could be dangerous to them (for example differentiation-inducing proteins such as histone H1.0, or antitumor drugs), to transfer molecules which, after entering the surrounding cells, are able to transform their phenotype, and even to secrete factors, which allow escaping from immune surveillance. Herein we report that melanoma cells not only secrete EVs which contain a modified form of H1.0 histone, but also transport the corresponding mRNA. Given the already known role in tumorigenesis of some RNA binding proteins (RBPs), we also searched for proteins of this class in EVs. This study revealed the presence in A375 melanoma cells of at least three RBPs, with apparent MW of about 65, 45 and 38 kDa, which are able to bind H1.0 mRNA. Moreover, we purified one of these proteins, which by MALDI-TOF mass spectrometry was identified as the already known transcription factor MYEF2

Neurobiology of performance anxiety:A new approach

The aim of this study is to investigate the neurobiology of stress/emotionality, creating a multidisciplinary assessment model, which can help to provide psychological and physiological responses depending on the genetic background related to sport performances, social closeness and performance anxiety management in team sports. We enrolled 20 female volleyball players aged 13 \ub1 1 years old played in two different teams during a regional championship final. Saliva collection was carried out before and after the match. In order to evaluate the neuroendocrine effectors involved in stress and performance, we analyzed cortisol and progesterone levels through Elisa standard kit as well as HSP70 and amylase activity as stress-induced markers. As concern the psychometric assesment, we administrated he CSAI-2 test, Closeness Generating Procedure and STAI test. Genomic DNA was isolated from saliva cells using a QIAamp saliva kit according to the manufacturer\u2019s protocols. The SNP of the 5-HTTLPR, BDNF, DRD4 were analyzed. The results of the T-test performed on the total results showed a statistically significant relationship (p < 0.05) in cortisol levels pre and post match, as well between amylase and HP70 according to the genetic background. The analysis performed using just post match samples show a negative correlation between social closeness, cortisol and progesterone levels, with p < 0.010 for progesterone vs social closeness and p < 0.012 for cortisol vs social closeness. About the winner teams and the looser teams, there is a negative correlation between pre match cortisol levels and performance anxiety (p < 0.042)

Potential roles of extracellular vesicles in brain cell-to-cell communication

Potential roles of extracellular vesicles in brain cell-to-cell communication Extracellular vesicles (EVs) are released into thè extracellular space from both cancer and normal brain cells, and are probably able to modify thè phenotypic properties of receiving cells1. EVs released from astrocytes and neurons contain FGF2 and VEGF2'3 and induce a 'blood-brain barrier' (BBB) phenotype in cultured brain capillary endothelial cells (BCECs, unpublished results), On thè other hand, EVs from G26/24 oligodendroglioma induce apoptosis in neurons and astrocytes4-5. These effects are probably due to Fas Ligand and TRAIL, present in G26/24 vesicles4-5. Moreover, G26/24 EVs contain extracellular matrix remodeling proteases (such as ADAMTS)6, H1.0 histone protein, and H1.0 mRNA7. In particular, we previously hypothesized that G26/24 cells, and tumor cells in generai, can escape differentiation cues, and continue to proliferate by eliminating proteins, such as thè H1° linker histone (and its mRNA)7, which could otherwise block proliferation. To study vesicle release in a System that can better resemble in vivo conditions, astrocytes and BCECs were cultured on poly-L-lactic acid (PLLA) scaffolds and tested for their ability to grow and survive on this three-dimensional structures. We analyzed in parallel thè celi growth in 2D and 3D culture systems and observed thè differences in celi morphology by fluorescence analysis: threedimensional scaffolds have thè ability to guide celi growth, provide support, encourage celi adhesion and proliferation. Astrocytes8 and BCECs (unpublished results) adapted well to these porous matrices, not only remaining on thè surface, but also penetrating inside thè scaffolds. EVs released by astrocytes in these scaffolds are probably exosomes, as suggested by transmission electron microscopy pictures, and by thè presence of intracellular structures resembling multivesicular bodies. This 3D celi culture System could be further enriched to host different brain celi types, in order to set, for example, an in vitro model of BBB, that may be useful for drug delivery studies, and for thè formulation of new therapeutic strategies for thè treatment of neurological diseases. References [1] Schiera, G., Di Liegro, C.M., Di Liegro I. Int J Mol Sci. 2017, 18(12). pii: E2774. [2] Schiera, G., Proia, P., Alberti, C., Mineo, M., Savettieri, G., Di Liegro, I., 2007. J Celi Mol Med. 2007, 111(6), 1384-94. [3] Proia, P., Schiera, G., Mineo, M., Ingrassia, A.M. Santoro, G., Savettieri, G., Di Liegro, I. Int J Mol Med. 2008, 21(1), 63-7. [4] D'Agostino, S., Salamene, M., Di Liegro, I., Vittorelli, ML, Int J Oncol. 2006, 29(5), 1075-85. [5] Lo Cicero, A., Schiera, G., Proia, P., Saladino, P., Savettieri, G., Di Liegro, C.M., Di Liegro, I. Int J Oncol. 2011,39(6): 1353-7. [6] Lo Cicero, A., Majkowska, I., Nagase, H., Di Liegro, I., Troeberg, L., Matrix Biol. 2012, 31(4), 229-33. [7] Schiera, G., Di Liegro, C.M., Saladino, P., Pitti, R., Savettieri, G., Proia, P., Di Liegro, I. Int J Oncol. 2013, 43(6), 1771-6. [8] Carfì Pavia, F., Di Bella, M.A., Brucato, V., Blanda, V., Zummo, F., Vitrano, I., Di Liegro, C.M., Ghersi, G., Di Liegro, I., Schiera, G. Mol Med Rep. 2019 [Epub ahead of print]. [9] Di Bella MA, Zummo F., Carfì Pavia F., Brucato V., Di Liegro I., Schiera G. 2017, In: Microscopy and Imaging Science: practical approaches to applied research and education, pp 260-264. Ed: A. Méndez-Vilas Publisher, Formatex Research Center (Spain), ISBN-13, 978-84-942134-9-6

Enzymatic spermine metabolites induce apoptosis associated with increase of p53, caspase-3 and mir-34a in both neuroblastoma cells, SJNKP and the N-Myc-amplified form IMR5

Neuroblastoma (NB) is a common malignant solid tumor in children and accounts for 15% of childhood cancer mortality. Amplification of the N-Myc oncogene is a well-established poor prognostic marker in NB patients and strongly correlates with higher tumor aggression and resistance to treatment. New therapies for patients with N-Myc-amplified NB need to be developed. After treating NB cells with BSAO/SPM, the detection of apoptosis was determined after annexin V-FITC labeling and DNA staining with propidium iodide. The mitochondrial membrane potential activity was checked, labeling cells with the probe JC-1 dye. We analyzed, by real-time RT-PCR, the transcript of genes involved in the apoptotic process, to determine possible down-or upregulation of mRNAs after the treatment on SJNKP and the N-Myc-amplified IMR5 cell lines with BSAO/SPM. The experiments were carried out considering the proapoptotic genes Tp53 and caspase-3. After treatment with BSAO/SPM, both cell lines displayed increased mRNA levels for all these proapoptotic genes. Western blotting analysis with PARP and caspase-3 antibody support that BSAO/SPM treatment induces high levels of apoptosis in cells. The major conclusion is that BSAO/SPM treatment leads to antiproliferative and cytotoxic activity of both NB cell lines, associated with activation of apoptosis

Hsp60 Is Actively Secreted by Human Tumor Cells

Background: Hsp60, a Group I mitochondrial chaperonin, is classically considered an intracellular chaperone with residence in the mitochondria; nonetheless, in the last few years it has been found extracellularly as well as in the cell membrane. Important questions remain pertaining to extracellular Hsp60 such as how generalized is its occurrence outside cells, what are its extracellular functions and the translocation mechanisms that transport the chaperone outside of the cell. These questions are particularly relevant for cancer biology since it is believed that extracellular chaperones, like Hsp70, may play an active role in tumor growth and dissemination. Methodology/Principal Findings: Since cancer cells may undergo necrosis and apoptosis, it could be possible that extracellular Hsps are chiefly the result of cell destruction but not the product of an active, physiological process. In this work, we studied three tumor cells lines and found that they all release Hsp60 into the culture media by an active mechanism independently of cell death. Biochemical analyses of one of the cell lines revealed that Hsp60 secretion was significantly reduced, by inhibitors of exosomes and lipid rafts. Conclusions/Significance: Our data suggest that Hsp60 release is the result of an active secretion mechanism and, since extracellular release of the chaperone was demonstrated in all tumor cell lines investigated, our observations most likel