New orphan disease therapies from the proteome of industrial plasma processing waste- a treatment for aceruloplasminemia.

Plasma-derived therapeutic proteins are produced through an industrial fractionation process where proteins are purified from individual intermediates, some of which remain unused and are discarded. Relatively few plasma-derived proteins are exploited clinically, with most of available plasma being directed towards the manufacture of immunoglobulin and albumin. Although the plasma proteome provides opportunities to develop novel protein replacement therapies, particularly for rare diseases, the high cost of plasma together with small patient populations impact negatively on the development of plasma-derived orphan drugs. Enabling therapeutics development from unused plasma fractionation intermediates would therefore constitute a substantial innovation. To this objective, we characterized the proteome of unused plasma fractionation intermediates and prioritized proteins for their potential as new candidate therapies for human disease. We selected ceruloplasmin, a plasma ferroxidase, as a potential therapy for aceruloplasminemia, an adult-onset ultra-rare neurological disease caused by iron accumulation as a result of ceruloplasmin mutations. Intraperitoneally administered ceruloplasmin, purified from an unused plasma fractionation intermediate, was able to prevent neurological, hepatic and hematological phenotypes in ceruloplasmin-deficient mice. These data demonstrate the feasibility of transforming industrial waste plasma fraction into a raw material for manufacturing of new candidate proteins for replacement therapies, optimizing plasma use and reducing waste generation.

Post-translational modifications and antioxidant properties of different therapeutic human serum albumins.

Human serum albumin (HSA) is widely used for the treatment of diverse clinical conditions to restore plasma volume, manage burns and treat hypoproteinemia.Although the HSA preparations should ideally preserve its functionality, the structural integrity and antioxidant properties of HSA may be compromised as a result of the manufacturing process. The present study examined seven commercially available HSA preparations for clinical use to investigate their post-translational modifications (PTMs) and antioxidant activity, including DPPH radical-scavenging, peroxyl radical antioxidant and metal binding activities, by means of mass spectrometry and Ellman's assay. The results confirmed that most of the PTMs of HSA and especially the oxidation of the free thiol residue varied between the different commercial albumins and the percentage of these PTMs were higher than those of physiological HSA. Moreover, HSA-DA isoform was increased at the end of the stability time and new oxidative modifications occurred in these samples. In conclusion, the bioprocesses for production of commercial albumins are responsible of their wide heterogeneity, being the ethanol fractionation and their storage conditions the more critical phases. Nonetheless, the Kedrion albumin shows a high content of free thiol and a lower concentration of PTMs than other commercial albumins.

Negative effects of a high tumour necrosis factor-α concentration on human gingival mesenchymal stem cell trophism: the use of natural compounds as modulatory agents.

BACKGROUND: Adult mesenchymal stem cells (MSCs) play a crucial role in the maintenance of tissue homeostasis and in regenerative processes. Among the different MSC types, the gingiva-derived mesenchymal stem cells (GMSCs) have arisen as a promising tool to promote the repair of damaged tissues secreting trophic mediators that affect different types of cells involved in regenerative processes. Tumour necrosis factor (TNF)-α is one of the key mediators of inflammation that could affect tissue regenerative processes and modify the MSC properties in in-vitro applications. To date, no data have been reported on the effects of TNF-α on GMSC trophic activities and how its modulation with anti-inflammatory agents from natural sources could modulate the GMSC properties. METHODS: GMSCs were isolated and characterized from healthy subjects. The effects of TNF-α were evaluated on GMSCs and on the well-being of endothelial cells. The secretion of cytokines was measured and related to the modification of GMSC-endothelial cell communication using a conditioned-medium method. The ability to modify the inflammatory response was evaluated in the presence of Ribes nigrum bud extract (RBE). RESULTS: TNF-α differently affected GMSC proliferation and the expression of inflammatory-related proteins (interleukin (IL)-6, IL-10, transforming growth factor (TGF)-ß, and cyclooxygenase (COX)-2) dependent on its concentration. A high TNF-α concentration decreased the GMSC viability and impaired the positive cross-talk between GMSCs and endothelial cells, probably by enhancing the amount of pro-inflammatory cytokines in the GMSC secretome. RBE restored the beneficial effects of GMSCs on endothelial viability and motility under inflammatory conditions. CONCLUSIONS: A high TNF-α concentration decreased the well-being of GMSCs, modifying their trophic activities and decreasing endothelial cell healing. These data highlight the importance of controlling TNF-α concentrations to maintain the trophic activity of GMSCs. Furthermore, the use of natural anti-inflammatory agents restored the regenerative properties of GMSCs on endothelial cells, opening the way to the use and development of natural extracts in wound healing, periodontal regeneration, and tissue-engineering applications that use MSCs.

The Urokinase Receptor Induces a Mesenchymal Gene Expression Signature in Glioblastoma Cells and Promotes Tumor Cell Survival in Neurospheres.

PLAUR encodes the urokinase receptor (uPAR), which promotes cell survival, migration, and resistance to targeted cancer therapeutics in glioblastoma cells in culture and in mouse model systems. Herein, we show that patient survival correlates inversely with PLAUR mRNA expression in gliomas of all grades, in glioblastomas, and in the subset of glioblastomas that demonstrate the mesenchymal gene expression signature. PLAUR clusters with genes that define the more aggressive mesenchymal subtype in transcriptome profiles of glioblastoma tissue and glioblastoma cells in neurospheres, which are enriched for multipotent cells with stem cell-like qualities. When PLAUR was over-expressed or silenced in glioblastoma cells, neurosphere growth and expression of mesenchymal subtype biomarkers correlated with uPAR abundance. uPAR also promoted glioblastoma cell survival in neurospheres. Constitutively-active EGF Receptor (EGFRvIII) promoted neurosphere growth; however, unlike uPAR, EGFRvIII did not induce the mesenchymal gene expression signature. Immunohistochemical analysis of human glioblastomas showed that uPAR is typically expressed by a small sub-population of the cancer cells; it is thus reasonable to conclude that this subpopulation of cells is responsible for the effects of PLAUR on patient survival. We propose that uPAR-expressing glioblastoma cells demonstrate a mesenchymal gene signature, an increased capacity for cell survival, and stem cell-like properties.

Carnosol controls the human glioblastoma stemness features through the epithelial-mesenchymal transition modulation and the induction of cancer stem cell apoptosis.

A high cell proliferation rate, invasiveness and resistance to chemotherapy are the main features of glioblastoma (GBM). GBM aggressiveness has been widely associated both with a minor population of cells presenting stem-like properties (cancer stem-like cells, CSCs) and with the ability of tumor cells to acquire a mesenchymal phenotype (epithelial-mesenchymal transition, EMT). Carnosol (CAR), a natural inhibitor of MDM2/p53 complex, has been attracted attention for its anti-cancer effects on several tumor types, including GBM. Herein, the effects of CAR on U87MG-derived CSC viability and stemness features were evaluated. CAR decreased the rate of CSC formation and promoted the CSC apoptotic cell death through p53 functional reactivation. Moreover, CAR was able to control the TNF-α/TGF-ß-induced EMT, counteracting the effects of the cytokine on EMT master regulator genes (Slug, Snail, Twist and ZEB1) and modulating the activation of miR-200c, a key player in the EMT process. Finally, CAR was able to increase the temozolomide (TMZ) anti-proliferative effects. These findings demonstrate that CAR affected the different intracellular mechanism of the complex machinery that regulates GBM stemness. For the first time, the diterpene was highlighted as a promising lead for the development of agents able to decrease the stemness features, thus controlling GBM aggressiveness.

Osteogenesis Is Improved by Low Tumor Necrosis Factor Alpha Concentration through the Modulation of Gs-Coupled Receptor Signals.

In the early phase of bone damage, low concentrations of the cytokine tumor necrosis factor alpha (TNF-α) favor osteoblast differentiation. In contrast, chronic high doses of the same cytokine contribute to bone loss, demonstrating opposite effects depending on its concentration and on the time of exposure. In the bone microenvironment, TNF-α modulates the expression/function of different G protein-coupled receptors (GPCRs) and of their regulatory proteins, GPCR-regulated kinases (GRKs), thus dictating their final biological outcome in controlling bone anabolic processes. Here, the effects of TNF-α were investigated on the expression/responsiveness of the A2B adenosine receptor (A2BAR), a Gs-coupled receptor that promotes mesenchymal stem cell (MSC) differentiation into osteoblasts. Low TNF-α concentrations exerted a prodifferentiating effect on MSCs, pushing them toward an osteoblast phenotype. By regulating GRK2 turnover and expression, the cytokine impaired A2BAR desensitization, accelerating receptor-mediated osteoblast differentiation. These data supported the anabolic effect of TNF-α submaximal concentration and demonstrated that the cytokine regulates GPCR responses by interfering with the receptor desensitization machinery, thereby enhancing the anabolic responses evoked by A2BAR ligands. Overall, these results indicated that GPCR desensitization plays a pivotal role in osteogenesis and that its manipulation is an effective strategy to favor bone remodeling.

Pertussis Toxin Is a Robust and Selective Inhibitor of High Grade Glioma Cell Migration and Invasion.

In high grade glioma (HGG), extensive tumor cell infiltration of normal brain typically precludes identifying effective margins for surgical resection or irradiation. Pertussis toxin (PT) is a multimeric complex that inactivates diverse Gi/o G-protein coupled receptors (GPCRs). Despite the broad continuum of regulatory events controlled by GPCRs, PT may be applicable as a therapeutic. We have shown that the urokinase receptor (uPAR) is a major driver of HGG cell migration. uPAR-initiated cell-signaling requires a Gi/o GPCR, N-formyl Peptide Receptor 2 (FPR2), as an essential co-receptor and is thus, PT-sensitive. Herein, we show that PT robustly inhibits migration of three separate HGG-like cell lines that express a mutated form of the EGF Receptor (EGFR), EGFRvIII, which is constitutively active. PT also almost completely blocked the ability of HGG cells to invade Matrigel. In the equivalent concentration range (0.01-1.0 µg/mL), PT had no effect on cell survival and only affected proliferation of one cell line. Neutralization of EGFRvIII expression in HGG cells, which is known to activate uPAR-initiated cell-signaling, promoted HGG cell migration. The increase in HGG cell migration, induced by EGFRvIII neutralization, was entirely blocked by silencing FPR2 gene expression or by treating the cells with PT. When U87MG HGG cells were cultured as suspended neurospheres in serum-free, growth factor-supplemented medium, uPAR expression was increased. HGG cells isolated from neurospheres migrated through Transwell membranes without loss of cell contacts; this process was inhibited by PT by >90%. PT also inhibited expression of vimentin by HGG cells; vimentin is associated with epithelial-mesenchymal transition and worsened prognosis. We conclude that PT may function as a selective inhibitor of HGG cell migration and invasion.

New insights into the anticancer activity of carnosol: p53 reactivation in the U87MG human glioblastoma cell line.

Glioblastoma multiforme (GBM) is an aggressive brain tumour with high resistance to radio- and chemotherapy. As such, increasing attention has focused on developing new therapeutic strategies to improve treatment responses. Recently, attention has been shifted to natural compounds that are able to halt tumour development. Among them, carnosol (CAR), a phenolic diterpene present in rosemary, has become a promising molecule that is able to prevent certain types of solid cancer. However, no data are available on the effects of CAR in GBM. Here, CAR activity decreased the proliferation of different human glioblastoma cell lines, particularly cells that express wild type p53. The p53 pathway is involved in the control of apoptosis and is often impaired in GBM. Notably, CAR, through the dissociation of p53 from its endogenous inhibitor MDM2, was able to increase the intracellular p53 levels in GBM cells. Accordingly, functional reactivation of p53 was demonstrated by the stimulation of p53 target genes' transcription, the induction of apoptosis and cell cycle blockade. Most importantly, CAR produced synergistic effects with temozolomide (TMZ) and reduced the restoration of the tumour cells' proliferation after drug removal. Thus, for the first time, these data highlighted the potential use of the diterpene in the sensitization of GBM cells to chemotherapy through a direct re-activation of p53 pathway. Furthermore, progress has been made in delineating the biochemical mechanisms underlying the pro-apoptotic effects of this molecule.