A multidisciplinary functional proteomics-aided strategy as a tool for the profiling of a novel cytotoxic thiadiazolopyrimidone.

In recent years, thiadiazolopyrimidine derivatives have been acknowledged for their striking poly-pharmacological framework, thus representing an interesting scaffold for the development of new therapeutic candidates. This paper examines the synthesis and the interactome characterization of a novel bioactive thiadiazolopyrimidone (compound 1), endowed with cytotoxic activity on HeLa cancer cells. In detail, starting from a small set of synthesized thiadiazolopyrimidones, a multi-disciplinary strategy has been carried out on the most bioactive one to disclose its potential biological targets by functional proteomics, using a label-free mass spectrometry based platform coupling Drug Affinity Responsive Target Stability and targeted Limited Proteolysis-Multiple Reaction Monitoring. The identification of Annexin A6 (ANXA6) as compound 1 most reliable cellular partner paved the way to deepen the protein-ligand interaction through bio-orthogonal approaches and to prove compound 1 action on migration and invasion processes governed by ANXA6 modulation. The identification of compund 1 as the first ANXA6 protein modulator represents a relevant tool to further explore the biological role of ANXA6 in cancer, as well as to develop novel anticancer candidates.

Simvastatin Reduces Doxorubicin-Induced Cardiotoxicity: Effects beyond Its Antioxidant Activity.

This study aimed to evaluate if Simvastatin can reduce, and/or prevent, Doxorubicin (Doxo)-induced cardiotoxicity. H9c2 cells were treated with Simvastatin (10 µM) for 4 h and then Doxo (1 µM) was added, and the effects on oxidative stress, calcium homeostasis, and apoptosis were evaluated after 20 h. Furthermore, we evaluated the effects of Simvastatin and Doxo co-treatment on Connexin 43 (Cx43) expression and localization, since this transmembrane protein forming gap junctions is widely involved in cardioprotection. Cytofluorimetric analysis showed that Simvastatin co-treatment significantly reduced Doxo-induced cytosolic and mitochondrial ROS overproduction, apoptosis, and cytochrome c release. Spectrofluorimetric analysis performed by means of Fura2 showed that Simvastatin co-treatment reduced calcium levels stored in mitochondria and restored cytosolic calcium storage. Western blot, immunofluorescence, and cytofluorimetric analyses showed that Simvastatin co-treatment significantly reduced Doxo-induced mitochondrial Cx43 over-expression and significantly increased the membrane levels of Cx43 phosphorylated on Ser368. We hypothesized that the reduced expression of mitochondrial Cx43 could justify the reduced levels of calcium stored in mitochondria and the consequent induction of apoptosis observed in Simvastatin co-treated cells. Moreover, the increased membrane levels of Cx43 phosphorylated on Ser368, which is responsible for the closed conformational state of the gap junction, let us to hypothesize that Simvastatin leads to cell-to-cell communication interruption to block the propagation of Doxo-induced harmful stimuli. Based on these results, we can conclude that Simvastatin could be a good adjuvant in Doxo anticancer therapy. Indeed, we confirmed its antioxidant and antiapoptotic activity, and, above all, we highlighted that Simvastatin interferes with expression and cellular localization of Cx43 that is widely involved in cardioprotection.

Targeting USP-7 by a Novel Fluorinated 5-Pyrazolyl-Urea Derivative.

The impact of innovative technologies on the target discovery has been employed here to characterize the interactome of STIRUR 41, a promising 3-fluoro-phenyl-5-pyrazolyl-urea derivative endowed with anti-cancer activity, on neuroblastoma-related cells. A drug affinity responsive target stability-based proteomic platform has been optimized to elucidate the molecular mechanism at the basis of STIRUR 41 action, together with immunoblotting analysis and in silico molecular docking. Ubiquitin Specific Protease 7 (USP-7), one of the deubiquitinating enzymes which protect substrate proteins from proteasomal degradation, has been identified as the most affine STIRUR 41 target. As further demonstrated by in vitro and in-cell assays, STIRUR 41 was able to inhibit both the enzymatic activity of USP-7 and its expression levels in neuroblastoma-related cells, thus laying an encouraging base for the blockade of USP-7 downstream signaling.

Supercritical Impregnation of Mesoglycan and Lactoferrin on Polyurethane Electrospun Fibers for Wound Healing Applications.

Fibrous membranes of thermoplastic polyurethane (TPU) were fabricated through a uni-axial electrospinning process. Fibers were then separately charged with two pharmacological agents, mesoglycan (MSG) and lactoferrin (LF), by supercritical CO2 impregnation. Scanning Electron Microscopy (SEM) and Energy Dispersive X-ray Spectroscopy (EDS) analysis proved the formation of a micrometric structure with a homogeneous distribution of mesoglycan and lactoferrin. Besides, the degree of retention is calculated in four liquid media with different pHs. At the same time, angle contact analysis proved the formation of a hydrophobic membrane loaded with MSG and a hydrophilic LF-loaded one. The impregnation kinetics demonstrated a maximum loaded amount equal to 0.18 ± 0.20% and 0.07 ± 0.05% for MSG and LT, respectively. In vitro tests were performed using a Franz diffusion cell to simulate the contact with the human skin. The release of MSG reaches a plateau after about 28 h while LF release leveled off after 15 h. The in vitro compatibility of electrospun membranes has been evaluated on HaCaT and BJ cell lines, as human keratinocytes and fibroblasts, respectively. The reported data proved the potential application of fabricated membranes for wound healing.

Production of Mesoglycan/PCL Based Composites through Supercritical Impregnation.

The development of targeted therapies for wound repair is knowing a growing interest due to the increasing aging of the population and the incidence of chronic pathologies, mainly pressure ulcers. Among molecules recruiting cell populations and promoting the formation of new vital tissue, sodium mesoglycan (MSG) has been proven to be effective in wound healing. In this work, MSG impregnation of polymer matrices has been attempted by a supercritical carbon dioxide-based process. Polymeric matrices are composed of polycaprolactone blends, where water-soluble polymers, polyethylene glycol, polyvinyl pyrrolidone, gelatin, and thermoplastic starch, have been employed to modulate the MSG release, making the devices potentially suitable for topical administrations. Two different techniques have been used to obtain the films: the first one is compression molding, producing compact and continuous structures, and the second one is electrospinning, producing membrane-like designs. A higher amount of MSG can be loaded into the polymeric matrix in the membrane-like structures since, in these films, the impregnation process is faster than in the case of compression molded films, where the carbon dioxide has firstly diffused and then released the active molecule. The type of water-soluble polymer influences the drug release rate: the blend polycaprolactone-gelatin gives a prolonged release potentially suitable for topical administration.

Mesoglycan exerts its fibrinolytic effect through the activation of annexin A2.

Mesoglycan is a drug based on a mixture of glycosaminoglycans mainly used for the treatment of blood vessel diseases acting as antithrombotic and profibrinolytic drugs. Besides the numerous clinical studies, there is no information about its function on the fibrinolytic cascade. Here, we have elucidated the mechanism of action by which mesoglycan induces the activation of plasmin from endothelial cells. Surprisingly, by a proteomic analysis, we found that, following mesoglycan treatment, these cells show a notable amount of annexin A2 (ANXA2) at the plasma membrane. This protein has been widely associated with fibrinolysis and appears able to move to the membrane when phosphorylated. In our model, this translocation has proven to enhance cell migration, invasion, and angiogenesis. Furthermore, the interaction of mesoglycan with syndecan 4 (SDC4), a coreceptor belonging to the class of heparan sulfate proteoglycans, represents the upstream event of the ANXA2 behavior. Indeed, the activation of SDC4 triggers the motility of endothelial cells culminating in angiogenesis. Interestingly, mesoglycan can induce the release of plasmin in endothelial cell supernatants only in the presence of ANXA2. This evaluation suggests that mesoglycan triggers the formation of a chain mechanism starting from the activation of SDC4, and the related cascade of events, including src complex and PKCα activation, promoting the phosphorylation of ANXA2 and its translocation to plasma membrane. This indicates a connection among mesoglycan, SDC4-(PKCα-src), and ANXA2 which, in turn, links the tissue plasminogen activator bringing it closer to plasminogen. This latter is so cleaved to release the plasmin and degrade fibrin sleeves.

Novel insights on the molecular mechanism of action of the anti-angiogenic pyrazolyl-urea GeGe-3 by functional proteomics.

In recent years, 5-pyrazolyl-ureas have mostly been known for their attractive poly-pharmacological outline and, in particular, ethyl 1-(2-hydroxypentyl)-5-(3-(3-(trifluoromethyl) phenyl) ureido)-1H-pyrazole-4-carboxylate (named GeGe-3) has emerged as a capable anti-angiogenic compound. This paper examines its interactome by functional proteomics using a label-free mass spectrometry based platform, coupling Drug Affinity Responsive Target Stability and targeted Limited Proteolysis-Multiple Reaction Monitoring. Calreticulin has been recognized as the GeGe-3 principal target and this evidence has been supported by immunoblotting and in silico molecular docking. Furthermore, cell studies have shown that GeGe-3 lowers cell calcium mobilization, cytoskeleton organization and focal adhesion kinase expression, thus linking its biological potential to calreticulin binding and, ultimately, shedding light on the reasonable action mechanism of this molecule as an anti-angiogenic factor.

ANXA1 Contained in EVs Regulates Macrophage Polarization in Tumor Microenvironment and Promotes Pancreatic Cancer Progression and Metastasis.

The tumor microenvironment (TME) is a dynamic system where nontumor and cancer cells intercommunicate through soluble factors and extracellular vesicles (EVs). The TME in pancreatic cancer (PC) is critical for its aggressiveness and the annexin A1 (ANXA1) has been identified as one of the oncogenic elements. Previously, we demonstrated that the autocrine/paracrine activities of extracellular ANXA1 depend on its presence in EVs. Here, we show that the complex ANXA1/EVs modulates the macrophage polarization further contributing to cancer progression. The EVs isolated from wild type (WT) and ANXA1 knock-out MIA PaCa-2 cells have been administrated to THP-1 macrophages finding that ANXA1 is crucial for the acquisition of a protumor M2 phenotype. The M2 macrophages activate endothelial cells and fibroblasts to induce angiogenesis and matrix degradation, respectively. We have also found a significantly increased presence of M2 macrophage in mice tumor and liver metastasis sections previously obtained by orthotopic xenografts with WT cells. Taken together, our data interestingly suggest the relevance of ANXA1 as potential diagnostic/prognostic and/or therapeutic PC marker.

Mesoglycan induces keratinocyte activation by triggering syndecan-4 pathway and the formation of the annexin A1/S100A11 complex.

Wound healing is a dynamic process comprising multiple events, such as inflammation, re-epithelialization, and tissue remodeling. Re-epithelialization phase is characterized by the engagement of several cell populations, mainly of keratinocytes that sequentially go through cycles of migration, proliferation, and differentiation to restore skin functions. Troubles can arise during the re-epithelialization phase of skin wound healing particularly in keratinocyte migration, resulting in chronic non-healing lesions, which represent a serious clinical problem. Over the last decades, the efforts aimed to find new pharmacological approaches for wound care were made, yet almost all current therapeutic strategies used remain inadequate or even ineffective. As such, it is crucial to identify new drugs that can enable a proper regeneration of the epithelium in wounded skin. Here, we have investigated the effects of the fibrinolytic drug mesoglycan, a glycosaminoglycans mixture derived from porcine intestinal mucosa on HaCaT human keratinocytes that were used as in vitro experimental model of skin re-epithelialization. We found that mesoglycan induces keratinocyte migration and early differentiation by triggering the syndecan-4/PKCα pathway and that these effects were at least in part, because of the formation of the annexin A1/S100A11 complex. Our data suggest that mesoglycan may be useful as a new pro-healing drug for skin wound care.

TFF1 Promotes EMT-Like Changes through an Auto-Induction Mechanism.

Trefoil factor 1 (TFF1) is a small secreted protein expressed in the gastrointestinal tract where, together with the other two members of its family, it plays an essential role in mucosal protection and repair against injury. The molecular mechanisms involved in the protective function of all three TFF proteins are not fully elucidated. In this paper, we investigated the role of TFF1 in epithelial to mesenchymal transition (EMT) events. The effects of TFF1 on cellular models in normoxia and/or hypoxia were evaluated by western blot, immunofluorescence, qRT-PCR and trans-well invasion assays. Luciferase reporter assays were used to assess the existence of an auto-regulatory mechanism of TFF1. The methylation status of TFF1 promoter was measured by high-resolution melting (HRM) analysis. We demonstrate a TFF1 auto-induction mechanism with the identification of a specific responsive element located between -583 and -212 bp of its promoter. Our results suggest that TFF1 can regulate its own expression in normoxic, as well as in hypoxic, conditions acting synergistically with the hypoxia-inducible factor 1 (HIF-1α) pathway. Functionally, this auto-induction mechanism seems to promote cell invasion and EMT-like modifications in vitro. Additionally, exogenously added human recombinant TFF1 protein was sufficient to observe similar effects. Together, these findings suggest that the hypoxic conditions, which can be induced by gastric injury, promote TFF1 up-regulation, strengthened by an auto-induction mechanism, and that the trefoil peptide takes part in the epithelial-mesenchymal transition events eventually triggered to repair the damage.

miR-196a Is Able to Restore the Aggressive Phenotype of Annexin A1 Knock-Out in Pancreatic Cancer Cells by CRISPR/Cas9 Genome Editing.

Annexin A1 (ANXA1) is a Ca2+-binding protein that is involved in pancreatic cancer (PC) progression. It is able to mediate cytoskeletal organization maintaining a malignant phenotype. Our previous studies showed that ANXA1 Knock-Out (KO) MIA PaCa-2 cells partially lost their migratory and invasive capabilities and also the metastatization process appeared affected in vivo. Here, we investigated the microRNA (miRNA) profile in ANXA1 KO cells finding that the modification in miRNA expression suggests the significant involvement of ANXA1 in PC development. In this study, we focused on miR-196a which appeared down modulated in absence of ANXA1. This miRNA is a well known oncogenic factor in several tumour models and it is able to trigger the agents of the epithelial to mesenchymal transition (EMT), like ANXA1. Our results show that the reintroduction in ANXA1 KO cells of miR-196a through the mimic sequence restored the early aggressive phenotype of MIA PaCa-2. Then, ANXA1 seems to support the expression of miR-196a and its role. On the other hand, this miRNA is able to mediate cytoskeletal dynamics and other protein functions promoting PC cell migration and invasion. This work describes the correlation between ANXA1 and specific miRNA sequences, particularly miR-196a. These results could lead to further information on ANXA1 intracellular role in PC, explaining other aspects that are apart from its tumorigenic behaviour.

Annexin A1 May Induce Pancreatic Cancer Progression as a Key Player of Extracellular Vesicles Effects as Evidenced in the In Vitro MIA PaCa-2 Model System.

Pancreatic Cancer (PC) is one of the most aggressive malignancies worldwide. As annexin A1 (ANXA1) is implicated in the establishment of tumour metastasis, the role of the protein in PC progression as a component of extracellular vesicles (EVs) has been investigated. EVs were isolated from wild type (WT) and ANXA1 knock-out (KO) PC cells and then characterised by multiple approaches including Western blotting, Field Emission-Scanning Electron Microscopy, and Dynamic Light Scattering. The effects of ANXA1 on tumour aggressiveness were investigated by Wound-Healing and invasion assays and microscopic analysis of the Epithelial to Mesenchymal Transition (EMT). The role of ANXA1 on angiogenesis was also examined in endothelial cells, using similar approaches. We found that WT cells released more EVs enriched in exosomes than those from cells lacking ANXA1. Notably, ANXA1 KO cells recovered their metastatic potential only when treated by WT EVs as they underwent EMT and a significant increase of motility. Similarly, human umbilical vein endothelial cells (HUVEC) migrated and invaded more rapidly when treated by WT EVs whereas ANXA1 KO EVs weakly induced angiogenesis. This study suggests that EVs-related ANXA1 is able to promote cell migration, invasion, and angiogenesis, confirming the relevance of this protein in PC progression.

The Pharmaceutical Device Prisma® Skin Promotes in Vitro Angiogenesis through Endothelial to Mesenchymal Transition during Skin Wound Healing.

Glycosaminoglycans are polysaccharides of the extracellular matrix supporting skin wound closure. Mesoglycan is a mixture of glycosaminoglycans such as chondroitin-, dermatan-, heparan-sulfate and heparin and is the main component of Prisma® Skin, a pharmaceutical device developed by Mediolanum Farmaceutici S.p.a. Here, we show the in vitro effects of this device in the new vessels formation by endothelial cells, since angiogenesis represents a key moment in wound healing. We found a strong increase of migration and invasion rates of these cells treated with mesoglycan and Prisma® Skin which mediate the activation of the pathway triggered by CD44 receptor. Furthermore, endothelial cells form longer capillary-like structures with a great number of branches, in the presence of the same treatments. Thus, the device, thanks to the mesoglycan, leads the cells to the Endothelial-to-Mesenchymal Transition, suggesting the switch to a fibroblast-like phenotype, as shown by immunofluorescence assays. Finally, we found that mesoglycan and Prisma® Skin inhibit inflammatory reactions such as nitric oxide secretion and NF-κB nuclear translocation in endothelial cells and Tumor Necrosis Factor-α production by macrophages. In conclusion, based on our data, we suggest that Prisma® Skin may be able to accelerate angiogenesis in skin wound healing, and regulate inflammation avoiding chronic, thus pathological, responses.

Role of intracellular and extracellular annexin A1 in migration and invasion of human pancreatic carcinoma cells.

BACKGROUND: Annexin A1 (ANXA1), a 37 kDa multifunctional protein, is over-expressed in tissues from patients of pancreatic carcinoma (PC) where the protein seems to be associated with malignant transformation and poor prognosis. METHODS: The expression and localization of ANXA1 in MIA PaCa-2, PANC-1, BxPC-3 and CAPAN-2 cells were detected by Western Blotting and Immunofluorescence assay. Expression and activation of Formyl Peptide Receptors (FPRs) were shown through flow cytometry/PCR and FURA assay, respectively. To investigate the role of ANXA1 in PC cell migration and invasion, we performed in vitro wound-healing and matrigel invasion assays. RESULTS: In all the analyzed PC cell lines, a huge expression and a variable localization of ANXA1 in sub-cellular compartments were observed. We confirmed the less aggressive phenotype of BxPC-3 and CAPAN-2 compared with PANC-1 and MIA PaCa-2 cells, through the evaluation of Epithelial-Mesenchymal Transition (EMT) markers. Then, we tested MIA PaCa-2 and PANC-1 cell migration and invasiveness rate which was inhibited by specific ANXA1 siRNAs. Both the cell lines expressed FPR-1 and -2. Ac2-26, an ANXA1 mimetic peptide, induced intracellular calcium release, consistent with FPR activation, and significantly increased cell migration/invasion rate. Interestingly, in MIA PaCa-2 cells we found a cleaved form of ANXA1 (33 kDa) that localizes at cellular membranes and is secreted outside the cells, as confirmed by MS analysis. The importance of the secreted form of ANXA1 in cellular motility was confirmed by the administration of ANXA1 blocking antibody that inhibited migration and invasion rate in MIA PaCa-2 but not in PANC-1 cells that lack the 33 kDa ANXA1 form and show a lower degree of invasiveness. Finally, the treatment of PANC-1 cells with MIA PaCa-2 supernatants significantly increased the migration rate of these cells. CONCLUSION: This study provides new insights on the role of ANXA1 protein in PC progression. Our findings suggest that ANXA1 protein could regulate metastasis by favouring cell migration/invasion intracellularly, as cytoskeleton remodelling factor, and extracellularly like FPR ligand.

Production of mono and bilayer devices for wound dressing by coupling of electrospinning and supercritical impregnation techniques.

In this paper, electrospinning and supercritical impregnation were coupled to produce polyurethane fibrous membranes loaded with mesoglycan and lactoferrin. The proposed methodology allowed the production of three skin wound healing bilayer systems: a first system containing mesoglycan loaded through electrospinning and lactoferrin loaded by supercritical impregnation, a second system where the use of the two techniques was reversed, and a third sample where the drugs were both encapsulated through a one-step process. SEM analysis demonstrated the formation of microfibers with a homogeneous drug distribution. The highest loadings were 0.062 g/g for mesoglycan and 0.013 g/g for lactoferrin. Then, hydrophilicity and liquid retention analyses were carried out to evaluate the possibility of using the manufacturers as active patches. The kinetic profiles, obtained through in vitro tests conducted using a Franz diffusion cell, proved that the diffusion of the active drugs followed a double-step release before attaining the equilibrium after about 30 h. When the electrospun membranes were placed in contact with HUVEC, HaCaT, and BJ cell lines, as human endothelial cells, keratinocytes, and fibroblasts, respectively, no cytotoxic events were assessed. Finally, the capacity of the most promising system to promote the healing process was performed by carrying out scratch tests on HaCat cells.

The pro-healing effects of heparan sulfate and growth factors are enhanced by the heparinase enzyme: New association for skin wound healing treatment.

Effective treatment strategies for skin wound repair are the focus of numerous studies. New pharmacological approaches appear necessary to guarantee a correct and healthy tissue regeneration. For these reasons, we purposed to investigate the effects of the combination between heparan sulfate and growth factors further adding the heparinase enzyme. Interestingly, for the first time, we have found that this whole association retains a marked pro-healing activity when topically administered to the wound. In detail, this combination significantly enhances the motility and activation of the main cell populations involved in tissue regeneration (keratinocytes, fibroblasts and endothelial cells), compared with single agents administered without heparinase. Notably, using an experimental C57BL/6 mouse model of skin wounding, we observed that the topical treatment of skin lesions with heparan sulfate + growth factors + heparinase promotes the highest closure of wounds compared to each substance mixed with the other ones in all the possible combinations. Eosin/hematoxylin staining of skin biopsies revealed that treatment with the whole combination allows the formation of a well-structured matrix with numerous new vessels. Confocal analyses for vimentin, FAP1α, CK10 and CD31 have highlighted the presence of activated fibroblasts, differentiated keratinocytes and endothelial cells at the closed region of wounds. Our results encourage defining this combined treatment as a new and appealing therapy expedient in skin wound healing, as it is able to activate cell components and promote a dynamic lesions closure.

The Protecting Activity of RIPACUT®: A New Therapeutic Approach Preserving Epithelial Health Based on the Combination of Iceland Lichen Extract, Silver Salt, and Sodium Hyaluronate.

Epithelial integrity and function must be maintained in a dynamic healthy equilibrium, keeping unaltered the oxidative and inflammatory conditions and the microbiome of the cutaneous layers. Beside the skin, other mucous membranes can be injured, such as the nasal and anal ones, because of the contact with the external environment. Here, we detected the effects of RIPACUT®, a combination of Iceland lichen extract, silver salt and sodium hyaluronate that individually act in diverse biological ways. The findings we obtained on keratinocytes, nasal and intestinal epithelial cells reveal that this combination showed a marked antioxidant activity, further assessed by the DPPH assay. Additionally, by analyzing the release of the IL-1ß, TNF-α and IL-6 cytokines, we proved the anti-inflammatory effect of RIPACUT®. In both cases, the main preserving action was due to Iceland lichen. We also observed a notable antimicrobial activity mediated by the silver compound. These data suggest that RIPACUT® could signify the basis for an attractive pharmacological approach to maintaining healthy epithelial conditions. Interestingly, this may be extended to the nasal and anal areas where it protects against oxidative, inflammatory and infectious insults. Thus, these outcomes encourage the creation of sprays or creams for which sodium hyaluronate can guarantee a surface film-forming effect.

A DR/NIR Hybrid Polymeric Tool for Functional Bio-Coatings: Theoretical Study, Cytotoxicity, and Antimicrobial Activity.

Among modern biomaterials, hybrid tools containing an organic component and a metal cation are recognized as added value, and, for many advanced biomedical applications, synthetic polymers are used as thin protective/functional coatings for medical or prosthetic devices and implants. These materials require specific non-degradability, biocompatibility, antimicrobial, and antiproliferative properties to address safety aspects concerning their use in medicine. Moreover, bioimaging monitoring of the biomedical device and/or implant through biological tissues is a desirable ability. This article reports a novel hybrid metallopolymer obtained by grafting zinc-coordinated fragments to an organic polymeric matrix. This hybrid polymer, owing to its relevant emission in the deep red to near-infrared (DR/NIR) region, is monitorable; therefore, it represents a potential material for biomedical coating. Furthermore, it shows good biocompatibility and adhesion properties and excellent stability in slightly acidic/basic water solutions. Finally, in contact with the superficial layers of human skin, it shows antimicrobial properties against Staphylococcus aureus bacterial strains.

The CD73 is induced by TGF-ß1 triggered by nutrient deprivation and highly expressed in dedifferentiated human melanoma.

CD73 is the key enzyme in the generation of extracellular adenosine, a mediator involved in tumor progression, tumor immune escape and resistance to anti-cancer therapeutics. Microenvironmental conditions influence the expression of CD73 in tumor cells. However how CD73 expression and activity is regulated in a stress condition of lower nutrient availability are largely unknown. Our results indicate that serum starvation leads to a marked up-regulation of CD73 expression on A375 melanoma cells in a time-dependent manner. The cell-surface expression of CD73 is associated with an increased release of TGF-ß1 by starved cells. Blockade of TGF-ß1 receptors or TGFß/SMAD3 signaling pathway significantly reduce the expression of CD73 induced by starvation. Treatment of cells with rTGF-ß1 up-regulates the expression of CD73 in a concentration-dependent manner, confirming the role of this pathway in regulating CD73 in melanoma A375 cells. The increased expression of CD73 is associated with enhanced AMPase activity, which is selectively reduced by inhibitors of CD73 activity, APCP and PSB-12489. Pharmacological blockade of CD73 significantly inhibits invasion of melanoma cells in a transwell system. Furthermore, using multiplex immunofluorescence imaging we found that, within human melanoma metastases, tumor cells at the dedifferentiated stage show the highest CD73 protein expression. In summary, our data provide new insights into the mechanism regulating the expression/activity of CD73 in melanoma cells in a condition of lower availability of nutrients, which is a common feature of the tumor microenvironment. Within human metastatic melanoma tissues elevated protein expression of CD73 is associated with an invasive-like phenotype.

The combination of mesoglycan and VEGF promotes skin wound repair by enhancing the activation of endothelial cells and fibroblasts and their cross-talk.

Skin wound healing requires accurate therapeutic topical managements to accelerate tissue regeneration. Here, for the first time, we found that the association mesoglycan/VEGF has a strong pro-healing activity. In detail, this combination induces angiogenesis in human endothelial cells promoting in turn fibroblasts recruitment. These ones acquire a notable ability to invade the matrigel coating and to secrete an active form of metalloproteinase 2 in presence of endothelial cells treated with mesoglycan/VEGF. Next, by creating intrascapular lesions on the back of C57Bl6 mice, we observed that the topical treatments with the mesoglycan/VEGF promotes the closure of wounds more than the single substances beside the control represented by a saline solution. As revealed by eosin/hematoxylin staining of mice skin biopsies, treatment with the combination mesoglycan/VEGF allows the formation of a well-structured matrix with a significant number of new vessels. Immunofluorescence analyses have revealed the presence of endothelial cells at the closed region of wounds, as evaluated by CD31, VE-cadherin and fibronectin staining and of activated fibroblasts assessed by vimentin, col1A and FAP1α. These results encourage defining the association mesoglycan/VEGF to activate endothelial and fibroblast cell components in skin wound healing promoting the creation of new vessels and the deposition of granulation tissue.