In humans, two main metabolic enzymes synthesize hydrogen sulfide (H2 S): cystathionine γ lyase (CSE) and cystathionine β synthase (CBS). A third enzyme, 3-mercaptopyruvate sulfurtransferase (3-MST), synthesizes H2 S in the presence of the substrate 3-mercaptopyruvate (3-MP). The immunohistochemistry analysis performed on human melanoma samples demonstrated that CSE expression was highest in primary tumors, decreased in the metastatic lesions and was almost silent in non-lymph node metastases. The primary role played by CSE was confirmed by the finding that the overexpression of CSE induced spontaneous apoptosis of human melanoma cells. The same effect was achieved using different H2 S donors, the most active of which was diallyl trisulfide (DATS). The main pro-apoptotic mechanisms involved were suppression of nuclear factor-κB activity and inhibition of AKT and extracellular signal-regulated kinase pathways. A proof of concept was obtained in vivo using a murine melanoma model. In fact, either l-cysteine, the CSE substrate, or DATS inhibited tumor growth in mice. In conclusion, we have determined that the l-cysteine/CSE/H2 S pathway is involved in melanoma progression.

Multiple myeloma (MM) is a disease with an adverse outcome and new therapeutic strategies are urgently awaited. A rising body of evidence supports the notion that microRNAs (miRNAs), master regulators of eukaryotic gene expression, may exert anti-MM activity. Here, we evaluated the activity of synthetic miR-34a in MM cells. We found that transfection of miR-34a mimics in MM cells induces a significant change of gene expression with relevant effects on multiple signal transduction pathways. We detected early inactivation of pro-survival and proliferative kinases Erk-2 and Akt followed at later time points by caspase-6 and -3 activation and apoptosis induction. To improve the in vivo delivery, we encapsulated miR-34a mimics in stable nucleic acid lipid particles (SNALPs). We found that SNALPs miR-34a were highly efficient in vitro in inhibiting growth of MM cells. Then, we investigated the activity of the SNALPs miR-34a against MM xenografts in SCID mice. We observed significant tumor growth inhibition (p<0.05) which translated in mice survival benefits (p = 0.0047). Analysis of miR-34a and NOTCH1 expression in tumor retrieved from animal demonstrated efficient delivery and gene modulation induced by SNALPs miR-34a in the absence of systemic toxicity. We here therefore provide evidence that SNALPs miR-34a may represent a promising tool for miRNA-therapeutics in MM.

T he pentraxin (PTX) family includes 2 subgroups of proteins that are structurally divided into short and long forms, are encoded by different genes, and are produced by different cells. [1][2][3] PTX3 is the prototype of the long PTX group, which differs from short PTXs by the presence of an unrelated long N-terminal domain giving it a different ligand recognition capacity. 4 PTX3 is highly conserved from mouse to humans (82% identical and 92% conserved amino acids) and is induced by primary inflammatory stimuli in a variety of cell types, 5,6 including mononuclear phagocytes, fibroblasts, adipocytes, dendritic, and endothelial and smooth muscle cells. 5,7 Marked as an innate immunity protein, PTX3 not only regulates inflammatory responses but also is involved in a range of important biological mechanisms, including vascular pathology. In fact, blood vessels produce large amounts of PTX3 during inflammation, and the level of circulating PTX3 increases in several pathological conditions affecting the cardiovascular system. 8,9 Moreover, in advanced atherosclerotic lesions and in patients with vasculitis, the protein is abundantly present in endothelial cells. 10,11 Thus, PTX3 seems to be a rapid marker of primary local innate immunity and inflammation activation and a novel diagnostic tool for vascular disorders. Clinical Perspective on p 1505In addition, high plasma PTX3 has been linked to vascular endothelial dysfunction in several human diseases, including Background-Pentraxin 3 (PTX3), the prototype of long pentraxins, has been described to be associated with endothelial dysfunction in different cardiovascular disorders. No study has yet evaluated the possible direct effect of PTX3 on vascular function. Methods and Results-Through in vitro experiments of vascular reactivity and ultrastructural analyses, we demonstrate that PTX3 induces dysfunction and morphological changes in the endothelial layer through a P-selectin/matrix metalloproteinase-1 pathway. The latter hampered the detachment of endothelial nitric oxide synthase from caveolin-1, leading to an impairment of nitric oxide signaling. In vivo studies showed that administering PTX3 to wild-type mice induced endothelial dysfunction and increased blood pressure, an effect absent in P-selectin-deficient mice. In isolated human umbilical vein endothelial cells, PTX3 significantly blunted nitric oxide production through the matrix metalloproteinase-1 pathway. Finally, using ELISA, we found that hypertensive patients (n=31) have higher plasma levels of PTX3 and its mediators P-selectin and matrix metalloproteinase-1 than normotensive subjects (n=21). Conclusions-Our data show for the first time a direct role of PTX3 on vascular function and blood pressure homeostasis, identifying the molecular mechanisms involved. The findings in humans suggest that PTX3, P-selectin, and matrix metalloproteinase-1 may be novel biomarkers that predict the onset of vascular dysfunction in hypertensive patients. Here, we demonstrate that (1) PTX3 induces vascular dysfuncti...

Secreted phospholipases A2 (sPLA2s) are extracellular enzymes that catalyze the release of free fatty acids and lysophospholipids from membrane phospholipids and also bind to different receptors (e.g., PLA2R1 or integrins). To date, 12 mammalian sPLA2s have been identified, which play a critical role in pathophysiological processes including inflammation and cancer. sPLA2s activate immune cells such as human neutrophils (PMNs) by enzymatic activity- or receptor-mediated mechanisms. In addition, human PMNs synthesize and store human group V (hGV) and human group X (hGX) sPLA2s in their granules, but only the former is released upon cellular activation. We investigated the effects of sPLA2s on the release of proangiogenic and antiangiogenic factors by PMNs. We found that exogenous hGV and hGX sPLA2s induce the release of vascular endothelial growth factor (VEGF)-A, angiopoietin 1 (Ang1), and CXCL8/IL-8. Only hGV induces the secretion of the antiangiogenic isoform of VEGF-A, namely, VEGF-A165b. While the release of VEGF-A, Ang1, and CXCL8/IL-8 was likely mediated by hGV enzymatic activity and/or binding to PLA2R1 and heparan sulfate proteoglycans, the release of VEGF-A165b requires the interaction with αVβ3 and α4β1 integrins. We also provide evidence that endogenous hGV released by N-formyl-met-leu-phe (fMLF)-activated PMNs is involved in the release of angiogenic factors. The translational relevance of these data is supported by our findings that hGV expression is increased in human samples of lung cancer which are infiltrated by PMNs. Overall, our results suggest that the hGV–neutrophil axis may play a relevant role in the modulation of cancer-related inflammation and angiogenesis.

Anaplastic thyroid carcinoma (ATC) is one of the most aggressive human solid tumor and current treatments are ineffective in increasing patients' survival. Thus, the development of new therapeutic approaches for ATC is needed. We have previously shown that the oncolytic adenovirus dl922-947 induces ATC cell death in vitro and tumor regression in vivo. However, the impact of dl922-947 on the pro-tumorigenic ATC microenvironment is still unknown. Since viruses are able to regulate cytokine and chemokine production from infected cells, we sought to investigate whether dl922-947 virotherapy has such effect on ATC cells, thereby modulating ATC microenvironment. dl922-947 decreased IL-8/CXCL8 and MCP-1/CCL2 production by the ATC cell lines 8505-c and BHT101-5. These results correlated with dl922-947-mediated reduction of NF-κB p65 binding to IL8 promoter in 8505-c and BHT101-5 cells and CCL2 promoter in 8505-c cells. IL-8 stimulates cancer cell proliferation, survival and invasion, and also angiogenesis. dl922-947-mediated reduction of IL-8 impaired ATC cell motility in vitro and ATC-induced angiogenesis in vitro and in vivo. We also show that dl922-947-mediated reduction of the monocyte-attracting chemokine CCL2 decreased monocyte chemotaxis in vitro and tumor macrophage density in vivo. Interestingly, dl922-947 treatment induced the switch of tumor macrophages toward a pro-inflammatory M1 phenotype, likely by increasing the expression of the pro-inflammatory cytokine interferon-γ. Altogether, we demonstrate that dl922-947 treatment re-shape the pro-tumorigenic ATC microenvironment by modulating cancer-cell intrinsic factors and the immune response. An in-depth knowledge of dl922-947-mediated effects on ATC microenvironment may help to refine ATC virotherapy in the context of cancer immunotherapy.

Background Molecular markers for prostate cancer (PCa) are required to improve the early definition of patient outcomes. Atypically large extracellular vesicles (EVs), referred as “Large Oncosomes” (LO), have been identified in highly migratory and invasive PCa cells. We recently developed and characterized the DU145R80 subline, selected from parental DU145 cells as resistant to inhibitors of mevalonate pathway. DU145R80 showed different proteomic profile compared to parental DU145 cells, along with altered cytoskeleton dynamics and a more aggressive phenotype. Methods Immunofluorescence staining and western blotting were used to identify blebbing and EVs protein cargo. EVs, purified by gradient ultra-centrifugations, were analyzed by tunable resistive pulse sensing and multi-parametric flow cytometry approach coupled with high-resolution imaging technologies. LO functional effects were tested in vitro by adhesion and invasion assays and in vivo xenograft model in nude mice. Xenograft and patient tumor tissues were analyzed by immunohistochemistry. Results We found spontaneous blebbing and increased shedding of LO from DU145R80 compared to DU145 cells. LO from DU145R80, compared to those from DU145, carried increased amounts of key-molecules involved in PCa progression including integrin alpha V (αV-integrin). By incubating DU145 cells with DU145R80-derived LO we demonstrated that αV-integrin on LO surface was functionally involved in the increased adhesion and invasion of recipient cells, via AKT. Indeed either the pre-incubation of LO with an αV-integrin blocking antibody, or a specific AKT inhibition in recipient cells are able to revert the LO-induced functional effects. Moreover, DU145R80-derived LO also increased DU145 tumor engraftment in a mice model. Finally, we identified αV-integrin positive LO-like structures in tumor xenografts as well as in PCa patient tissues. Increased αV-integrin tumor expression correlated with high Gleason score and lymph node status. Conclusions Overall, this study is the first to demonstrate the critical role of αV-integrin positive LO in PCa aggressive features, adding new insights in biological function of these large EVs and suggesting their potential use as PCa prognostic markers. Electronic supplementary material The online version of this article (10.1186/s13046-019-1317-6) contains supplementary material, which is available to authorized users.

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Background: Malignant pleural mesothelioma (MPM) is an aggressive cancer associated with asbestos exposure that urgently requires effective therapeutic strategies. Current treatments are unable to increase significantly patient survival, which is often limited to <1 year from diagnosis. Virotherapy, based on the use of oncolytic viruses that exert anti-cancer effects by direct cell lysis and through the induction of anti-tumor immune response, represents an alternative therapeutic option for rare tumors with limited life expectancy. In this study, we propose the use of the adenovirus dl 922-947, engineered to allow selective replication in cancer cells, to counteract MPM. Methods: We performed a thorough preclinical assessment of dl 922-947 effects in a set of MPM cell lines and xenografts. Cytotoxicity of dl 922-947 alone and in combination assays was evaluated by sulforhodamine B assay. Cell cycle, calreticulin expression, and high mobility group box protein 1 (HMGB1) secretion were determined by flow cytometry, whereas ATP content was determined by a luminescence-based bioassay. The modulation of angiogenic factors in MPM-infected cells was evaluated through ELISA. Results: We found that dl 922-947 infection exhibits cytotoxic effects in MPM cell lines, affecting cell viability, cell cycle progression, and regulating main hallmarks of immunogenic cell death inducing calreticulin surface exposure, HMGB1 and ATP release. Our results also suggest that dl 922-947 may affect angiogenic signals by regulation of VEGF-A and IL-8 secretion. Furthermore, dl 922-947 shows anti-tumor efficacy in murine xenograft models reducing tumor growth and enhancing survival. Finally, the combination with cisplatin potentiated the cytotoxic effect of dl 922-947. Conclusions: Overall our data identify virotherapy, based on the use of dl 922-947, as a new possible therapeutic strategy against MPM, which could be used alone, in combination with standard chemotherapy drugs, as shown here, or other approaches also aimed at enhancing the antitumoral immune response elicited by the virus.