Reduction of mdx mouse muscle degeneration by low-intensity endurance exercise: a proteomic analysis in quadriceps muscle of exercised versus sedentary mdx mice

In our recent study was shown a significant recovery of damaged skeletal muscle of mice with x-linked muscular dystrophy (mdx) following low-intensity endurance exercise, probably by reducing the degeneration of dystrophic muscle. Consequently, in the present work we aimed to identify proteins involved in the observed reduction of degenerating fibers. To this end, we used proteomic analysis to evaluate changes in the protein profile of quadriceps dystrophic muscles of exercised versus sedentary mdx mice. Four protein spots were found to be significantly changed and were identified as three isoforms of Carbonic anhydrase 3 (CA3) and superoxide dismutase [Cu-Zn] (SODC). Protein levels of CA3 isoforms were significantly up-regulated in quadriceps of sedentary mdx mice and were completely restored to wild type mice values, both sedentary and exercised, in quadriceps of exercised mdx mice. Protein levels of SODC were down-regulated in quadriceps of sedentary mdx mice and were significantly restored to wild type mice values, both sedentary and exercised, in quadriceps of exercised mdx mice. Western blot data were in agreement with those obtained using proteomic analysis and revealed the presence of one more CA3 isoform that was significantly changed. Based on data found in the present study, it seems that low-intensity endurance exercise may in part contribute to reduce cell degeneration process in mdx muscles, by counteracting oxidative stress

Involvement of multiple myeloma cell-derived exosomes in osteoclast differentiation

Bone disease is the most frequent complication in multiple myeloma (MM) resulting in osteolytic lesions, bone pain, hypercalcemia and renal failure. In MM bone disease the perfect balance between bone-resorbing osteoclasts (OCs) and bone-forming osteoblasts (OBs) activity is lost in favour of OCs, thus resulting in skeletal disorders. Since exosomes have been described for their functional role in cancer progression, we here investigate whether MM cell-derived exosomes may be involved in OCs differentiation. We show that MM cells produce exosomes which are actively internalized by Raw264.7 cell line, a cellular model of osteoclast formation. MM cell-derived exosomes positively modulate pre-osteoclast migration, through the increasing of CXCR4 expression and trigger a survival pathway. MM cell-derived exosomes play a significant pro-differentiative role in murine Raw264.7 cells and human primary osteoclasts, inducing the expression of osteoclast markers such as Cathepsin K (CTSK), Matrix Metalloproteinases 9 (MMP9) and Tartrate-resistant Acid Phosphatase (TRAP). Pre-osteoclast treated with MM cell-derived exosomes differentiate in multinuclear OCs able to excavate authentic resorption lacunae. Similar results were obtained with exosomes derived from MM patient's sera. Our data indicate that MM-exosomes modulate OCs function and differentiation. Further studies are needed to identify the OCs activating factors transported by MM cell-derived exosomes

Metastatic colon cancer exosomes spread malignant properties in tumor microenvironment affecting the behavior of both tumor and endothelial cells

I tumori sono caratterizzati da eterogeneità intratumorale e questo rende difficile lo sviluppo di nuovi trattamenti. Diversi studi indicano gli esosomi come importanti componenti che agiscono sulla eterogeneità tumorale. In questo contesto, il nostro scopo è di capire se gli esosomi derivanti da cellule ad alto potenziale metastatico possono influenzare il comportamento delle cellule tumorali meno aggressive e le proprietà dell’endotelio, promuovendo la disseminazione delle cellule tumorali. La nostra strategia sperimentale è basata sull’uso di un modello isogenico. Abbiamo usato cellule di carcinoma del colon, SW620 e SW480, isolate rispettivamente dal tumore primario e dal sito di metastasi dello stesso paziente. In particolare noi abbiamo valutato, a livello morfologico e funzionale, come gli esosomi derivanti dalla linea ad alto potenziale metastatico influenzino il comportamento biologico sia delle cellule meno aggressive che delle cellule endoteliali. Inoltre abbiamo condotto un’analisi proteomica SWATH-MS per caratterizzare gli esosomi . Abbiamo trovato che gli esosomi rilasciati dalle cellule ameboidi, SW620, sono capaci di indurre una transizione nelle SW480 fibroblastoidi, dal fenotipo mesenchimale a quello ameboide. Abbiamo osservato che dopo il trattamento con gli esosomi delle SW620, le cellule SW480 mostrano un blebbing non apoptotico, associato a una maggiore invasione e motilità delle cellule. Inoltre, il trattamento delle HUVECs con gli esosomi delle SW620 induce, rispetto al trattamento con gli esosomi delle SW480, una maggiore permeabilità dell’endotelio, dovuta alla destabilizzazione dei sistemi giunzionali delle cellule endoteliali. I nostri dati indicano che gli effetti del trattamento con gli esosomi derivanti dalla linea metastatica , sono mediati da RhoA, responsabile del rimodelllamento del citoscheletro. Inoltre, questo dato è stato fortemente supportato dai risultati di proteomica quantitativa che indicano un arricchimento di RacGap1, attivatore di RhoA, negli esosomi delle SW620. In conclusione, questo lavoro di tesi dimostra come, attraverso gli esosomi , le cellule ad alto potenziale metastatico possano influenzare il fenotipo delle cellule meno aggressive e del microambiente tumorale.Tumors are characterized by intratumoral heterogeneity that makes the development of new treatments difficult. Several studies suggest that exosomes may be an important microenvironmental factor affecting tumor heterogeneity. In this context our goal is to understand if exosomes derived from cell line with highly metastatic potential, may affect the behavior of less aggressive cells and the properties of tumor microenvironment. Our experimental strategy is based on the use of an isogenic model. We used SW480 and SW620 colon carcinoma cell lines derived from, respectively, primary and secondary tumors resected from a single patient, thus showing different metastatic potential. Specifically we evaluated, at morphological and functional level, how exosomes derived from highly metastatic cells influence the biological behavior of both less aggressive and endothelial cells. Finally we performed a proteomic characterization of exosomes by SWATH-MS method. We found that exosomes released by amoeboid SW620 cells are able to induce a mesenchymal-amoeboid transition in SW480 fibroblast-like cells. We observed that after treatment with SW620 exosomes, SW480 cells show non apoptotic membrane blebbing, associated with higher migratory and invasive capabilities. Moreover, the treatment of the HUVECs with SW620 exosomes induce the monolayer permeability, due to destabilization of endothelial junctional systems. Our data indicate that the effects of SW620 exosomes treatment is mediated by RhoA, responsible of cytoskeletal remodeling. Moreover, this data was strongly supported by quantitative proteomic analysis showing an enrichment in SW620 exosomes of RacGap1, activator of RhoA. We demonstrated that, through exosomes, highly metastatic cells spread their malignant behavior to less aggressive cells and affect tumor microenvironment

Evolutionary insight on localization of 18S, 28S rDNA genes on homologous chromosomes in Primates genomes

We explored the topology of 18S and 28S rDNA units by fluorescence in situ hybridization (FISH) in the karyotypes of thirteen species representatives from major groups of Primates and Tupaia minor (Günther, 1876) (Scandentia), in order to expand our knowledge of Primate genome reshuffling and to identify the possible dispersion mechanisms of rDNA sequences. We documented that rDNA probe signals were identified on one to six pairs of chromosomes, both acrocentric and metacentric ones. In addition, we examined the potential homology of chromosomes bearing rDNA genes across different species and in a wide phylogenetic perspective, based on the DAPI-inverted pattern and their synteny to human. Our analysis revealed an extensive variability in the topology of the rDNA signals across studied species. In some cases, closely related species show signals on homologous chromosomes, thus representing synapomorphies, while in other cases, signal was detected on distinct chromosomes, leading to species specific patterns. These results led us to support the hypothesis that different mechanisms are responsible for the distribution of the ribosomal DNA cluster in Primates

Proteomic profiling and functional characterization of metastatic colon cancer exosomes spreading malignant properties in tumor microenvironment

Human tumors display a remarkable intratumor heterogeneity affecting clinically relevant phenotypes such as ability to metastasize or to tolerate cytotoxic drugs. Recent published data indicate that tumor derived exosomes (TDEs) can have a pivotal role in regulating tumor heterogeneity by transferring functional biomolecules between various populations of tumor cells and between tumor cells and nontumor cells with consequences for whole tumor microenvironment. In this context, our goal was to understand if exosomes derived from highly metastatic cell line may influence the behaviour of less aggressive tumor cells and the properties of endhothelium

EXOSOMES DERIVED FROM METASTATIC COLON CANCER CELLS TRANSFER MALIGNANT PHENOTYPIC TRAITS TO SURROUNDING CELLS: THEIR EMERGING ROLE IN TUMOR HETEROGENEITY

Several studies have clearly demonstrated that within a heterogeneous tumor mass the inter-clonal cooperation between metastatic and non-metastatic cells can facilitate the tumor progression. Recent accumulating evidence has highlighted that tumor-derived exosomes (TDEs) play a relevant role as mediator of the inter-clonal collaborative cooperation affecting the properties of both tumor and non-tumor component. In this context, our goal was to understand if exosomes derived from highly metastatic cells may influence the behaviour of less aggressive tumor cells and the properties of endothelium. We found that metastatic SW620 cells transfer through exosomes (SW620Exos) their round/amoeboid phenotype to elongated non-metastatic SW480 cells also inducing the increase of the motile and invasive activities. Moreover, SW620Exos caused endothelial hyperpermeability by altering the junctional complexes in HUVECs. The SWATH-based quantitative proteomic analysis highlighted that SW620Exos were significant enriched in several proteins related to the RhoA/ROCK sig¬naling, known to induce the amoeboid motility as well as the destabilization of endothelial junctional complexes. According to this data, we found that the treatment with ExoSW620 elicited in both SW480 cells and HUVECs the increase of RhoA activity, while the induced morphological and functional effects were reverted by co-treatment with a specific ROCK inhibitor. RacGap1 and thrombin were identified as putative key mediators of the effects induced by SW620Exos in target cells. Taken together our data indicates that within a heterogeneous tumor mass exosomes released by metastatic cells affect the features of both tumor and non-tumor cell components, thus contributing to accelerate the metastatic cascade

SFPQ and NONO suppress RNA:DNA-hybrid-related telomere instability

LncRNA TERRA forms RNA-DNA hybrids at telomere sites leading to telomere instability. Here the authors identify the RNA interacting factors NONO and SFPQ as proteins that interact with TERRA and telomere chromatin and reveal putative roles for these factors in telomere integry maintenance by interfering with RNA:DNA hybrid formation

In vitro antitumor effects of the cold-water extracts of Pleurotus eryngii var ferulae and Pleurotus nebrodensis on human colon cancer cells

For centuries, mushrooms have been used as folk medicines especially in Asian countries where their medicinal properties are well known. On the basis of numerous experimental evidences collected in the last decades, the immunomodulatory and anti-neoplastic properties of substances extracted from various species belonging to genera of edible mushrooms [Agaricus L., Auricularia Bull. ex Juss., Ganoderma P. Karst., Grifola Gray, Lentinus Fr., Schizophyllum Fr., Tremella Dill ex L., etc.] are extensively recognized also at scientific levels. Several works have demonstrate that anti-cancer property of these molecules is due to their ability to enhance immune system activity and/or to act directly on cancer cells. Polysaccharides are responsible of a remarkable antitumor activity in vivo and many authors demonstrated their action through screening against sarcoma 180 in mice with an intraperitoneal or oral methods of administration (Ikekawa et al., 1969; Mori et al., 1986; Mizuno et al., 1995, Wasser & Weis, 1997). Among the well-studied medicinal mushrooms there are those belonging to the genus Pleurotus (Fr.) P. Kumm., one of the widely cultivated edible mushroom. Most of them are cultivated and provide a relatively cheap food of high dietetic value through rather simple solid-state fermentation processes. In addition, Pleurotus biomass demonstrates significant medicinal effects and its bioactive compounds possess antibiotic, antitumor, hypocholesterolemic and immunomodulation properties (Gunde-Cimerman, 1999; Babitskatya et al., 2000; Gunde-Cimerman & Plemenitas, 2001; Wasser, 2002; Zhang et al., 2004; Chang, 2006; Tsai et al., 2009). Most of researches concerning the anti-tumor properties of Pleurotus spp. have been carried out by testing the methanol/ethanol/ hot water extracts of Pleurotus ostreatus (Jacq.) P. Kumm. in several in vivo and in vitro cancer models. These studies provided interesting data supporting the possibility to isolate new therapeutic agents from this mushroom, but much less is known about the biological activities of extracts from other species of genus Pleurotus. Species like P. eryngii (DC.) Quél. var. ferulae (Lanzi) Sacc. and P. nebrodensis (Inzenga) Quél. are typical Mediterranean mushrooms and are particularly popular as choice edible mushrooms (Gargano et al., 2011 Zervakis, 2001). The aim of the present research was to evaluate whether the cold-water extract of P. eryngii var. ferulae (CWE-Pef) and P. nebrodensis (CWE-Pn) can affect the tumor phenotype of human colon cancer cells. Our result demonstrate that CWE-Pef and CWE-Pn inhibited the growth of HCT116 colon cancer cells by inducing apoptosis, promoted cancer cells aggregation and inhibited the adhesion of tumor cells to endothelial cells. Furthermore, both extracts inhibited tyrosine phosphorylation signaling as well as ERK phosphorylation. These effects were specific for tumor cells since no effects were observed on non-tumor cells. Finally, our data indicated that the active compounds may be protein/peptide compounds since high temperature treatment of CWE-Pef and CWE-Pn completely eliminated their anti-proliferative activity