Binding and Uptake into Human Hepatocellular Carcinoma Cells of Peptide-Functionalized Gold Nanoparticles

One of the most daunting challenges of nanomedicine is the finding of appropriate targeting agents to deliver suitable payloads precisely to cells affected by malignancies. Even more complex is to achieve the ability to ensure the nanosystems enter those cells. Here we use 2 nm (metal core) gold nanoparticles to target human hepatocellular carcinoma (HepG2) cells stably transfected with the SERPINB3 (SB3) protein. The nanoparticles were coated with a 85:15 mixture of thiols featuring, respectively, a phosphoryl choline, to ensure water solubility and biocompatibility, and a 28-mer peptide corresponding to the amino acid sequence 21-47 of the hepatitis B virus-PreS1 protein (PreS1(21-47)). Conjugation of the peptide was performed via the maleimide-thiol reaction in methanol allowing the use of a limited amount of the targeting molecule. This is an efficient procedure also in the perspective of selecting libraries of new targeting agents. The rationale behind the selection of the peptide is that SB3, which is undetectable in normal hepatocytes, is over-expressed in hepatocellular carcinoma and in hepatoblastoma and has been proposed as a target of the hepatitis B virus (HBV). For the latter the key recognition element is the PreS1(21-47) peptide, which is a fragment of one of the proteins composing the viral envelope. The ability of the conjugated nanoparticles to bind the target protein SB3, expressed in liver cancer cells, was investigated by surface plasmon resonance analysis and in vitro via cellular uptake analysis followed by atomic absorption analysis of digested samples. The results showed that the PreS1(21-47) peptide is a suitable targeting agent for cells overexpressing the SB3 protein. Even more important is the evidence that the gold nanoparticles are internalized by the cells. The comparison between the surface plasmon resonance analysis and the cellular uptake studies suggests the presentation of the protein on cell surface is critical for efficient recognition

SerpinB3 promotes pro-fibrogenic responses in activated hepatic stellate cells

SerpinB3 is a hypoxia- and hypoxia-inducible factor-2\u3b1-dependent cystein protease inhibitor that is up-regulated in hepatocellular carcinoma and in parenchymal cells during chronic liver diseases (CLD). SerpinB3 up-regulation in CLD patients has been reported to correlate with the extent of liver fibrosis and the production of transforming growth factor-\u3b21, but the actual role of SerpinB3 in hepatic fibrogenesis is still poorly characterized. In the present study we analyzed the pro-fibrogenic action of SerpinB3 in cell cultures and in two different murine models of liver fibrosis. "In vitro" experiments revealed that SerpinB3 addition to either primary cultures of human activated myofibroblast-like hepatic stellate cells (HSC/MFs) or human stellate cell line (LX2 cells) strongly up-regulated the expression of genes involved in fibrogenesis and promoted oriented migration, but not cell proliferation. Chronic liver injury by CCl4 administration or by feeding a methionine/choline deficient diet to transgenic mice over-expressing human SerpinB3 in hepatocytes confirmed that SerpinB3 over-expression significantly increased the mRNA levels of pro-fibrogenic genes, collagen deposition and \u3b1SMA-positive HSC/MFs as compared to wild-type mice, without affecting parenchymal damage. The present study provides for the first time evidence that hepatocyte release of SerpinB3 during CLD can contribute to liver fibrogenesis by acting on HSC/MFs

SerpinB3 and Yap Interplay Increases Myc Oncogenic Activity

SerpinB3 has been recently described as an early marker of liver carcinogenesis, but the potential mechanistic role of this serpin in tumor development is still poorly understood. Overexpression of Myc often correlates with more aggressive tumour forms, supporting its involvement in carcinogenesis. Yes-associated protein (Yap), the main effector of the Hippo pathway, is a central regulator of proliferation and it has been found up-regulated in hepatocellular carcinomas. The study has been designed to investigate and characterize the interplay and functional modulation of Myc by SerpinB3 in liver cancer. Results from this study indicate that Myc was up-regulated by SerpinB3 through calpain and Hippo-dependent molecular mechanisms in transgenic mice and hepatoma cells overexpressing human SerpinB3, and also in human hepatocellular carcinomas. Human recombinant SerpinB3 was capable to inhibit the activity of Calpain in vitro, likely reducing its ability to cleave Myc in its non oncogenic Myc-nick cytoplasmic form. SerpinB3 indirectly increased the transcription of Myc through the induction of Yap pathway. These findings provide for the first time evidence that SerpinB3 can improve the production of Myc through direct and indirect mechanisms that include the inhibition of generation of its cytoplasmic form and the activation of Yap pathway

Engineered EVs for Oxidative Stress Protection

Extracellular vesicles (EVs) are increasingly studied as vectors for drug delivery because they can transfer a variety of molecules across biological barriers. SerpinB3 is a serine protease inhibitor that has shown a protective anti-apoptotic function in a variety of stressful conditions. The aim of this study was to evaluate protection from oxidative stress-induced damage, using extracellular vesicles that overexpress SerpinB3 (EVs-SB3) in order to enhance the effect of extracellular vesicles on cellular homeostasis. EVs-SB3s were obtained from HepG2 cells engineered to overexpress SerpinB3 and they revealed significant proteomic changes, mostly characterized by a reduced expression of other proteins compared with EVs from non-engineered cells. These EV preparations showed a significantly higher protection from H2O2 induced oxidative stress in both the hepatoma cell line and in primary cardiomyocytes, compared to cells treated with naïve EVs or SerpinB3 alone, used at the same concentration. In conclusion, the induction of SerpinB3 transgene expression results in the secretion of EVs enriched with the protein product that exhibits enhanced cytoprotective activity, compared with naïve EVs or the nude SerpinB3 protein.Fil: Tolomeo, Anna Maria. Università di Padova; ItaliaFil: Quarta, Santina. Università di Padova; ItaliaFil: Biasiolo, Alessandra. Università di Padova; ItaliaFil: Ruvoletto, Mariagrazia. Università di Padova; ItaliaFil: Pozzobon, Michela. Università di Padova; ItaliaFil: De Lazzari, Giada. Università di Padova; ItaliaFil: Malvicini, Ricardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Medicina Traslacional, Trasplante y Bioingeniería. Fundación Favaloro. Instituto de Medicina Traslacional, Trasplante y Bioingeniería; ArgentinaFil: Turato, Cristian. Università di Padova; ItaliaFil: Arrigoni, Giorgio. Università di Padova; ItaliaFil: Pontisso, Patrizia. Università di Padova; ItaliaFil: Muraca, Maurizio. Università di Padova; Itali

Experimental validation of specificity of the squamous cell carcinoma antigen-immunoglobulin M (SCCA-IgM) assay in patients with cirrhosis

Background: Squamous cell carcinoma antigen-immunoglobulin M (SCCA-IgM) is a useful biomarker for the risk of development of hepatocellular carcinoma (HCC) in patients with cirrhosis due to its progressive increase associated to HCC evolution. In patients with cirrhosis, other assays have been affected by interfering reactivities of IgM. In this study, the analytical specificity of the SCCA-IgM assay was assessed by evaluating SCCA-IgM measurement dependence on different capture phases, and by measuring the recovery of SCCA-IgM reactivity following serum fractionation. Methods: Serum samples from 82 patients with cirrhosis were analyzed. SCCA-IgM was measured using the reference test (Hepa-IC, Xeptagen, Italy) that is based on rabbit oligoclonal anti-squamous cell carcinoma antigen (SCCA) and a dedicated ELISA with a mouse monoclonal anti-SCCA as the capture antibody. Results: SCCA-IgM concentrations measured with the reference assay (median value=87 AU/mL) were higher than those measured with the mouse monoclonal test (median value=78 AU/mL). However, the differences in the SCCA-IgM distribution were not statistically significant (p>0.05). When SCCA-IgM concentrations measured with both tests were compared, a linear correlation was found (r=0.77, p<0.05). Fractionation of the most reactive sera by gel-filtration chromatography showed that total recovery of SCCA-IgM reactivity was seen only in the fractions corresponding to components with a molecular weight higher than IgM and SCCA (>2000 kDa) with both tests. Conclusions: The equivalence of both SCCA-IgM assays and the absence of reactivity not related to immune complexes support the analytical specificity of SCCA-IgM measurements. The results validate the assessment of SCCA-IgM for prognostic purposes in patients with cirrhosis. Clin Chem Lab Med 2010;48:217–23.Peer Reviewe

Detection of high levels of Survivin-immunoglobulin M immune complex in sera from hepatitis C virus infected patients with cirrhosis

The identification and surveillance of patients with liver dysfunctions and the discovering of new disease biomarkers are needed in the clinical practice. The aim of this study was to investigate on Survivin-immunoglobulin (Ig)M immune complex (IC) as a potential biomarker of chronic liver diseases.Serum levels of Survivin-IgM were measured using an enzyme-linked immunoassay that had been standardized and validated in our laboratory in 262 individuals, including healthy subjects and patients with chronic viral hepatitis, cirrhosis and hepatocellular carcinoma (HCC).Survivin-IgM IC was lower in healthy subjects (median, 99.39 AU/mL) than in patients with chronic viral hepatitis (median, 148.03 AU/mL; P = 0.002) or with cirrhosis (median, 371.00 AU/mL; P  0.001). Among patients with cirrhosis, those with hepatitis C virus (HCV) infection showed the highest level of Survivin-IgM IC (median, 633.71 AU/mL; P  0.001). The receiver-operator curve analysis revealed that Survivin-IgM accurately distinguishes HCV correlated cirrhosis from chronic viral hepatitis (area under the curve [AUC], 0.738; sensitivity, 74.5%; specificity, 70.7%). A multivariate logistic regression model, including Survivin-IgM IC, aspartate aminotransferase (AST) and AST/alanine aminotransferase (ALT) ratio increased the prediction accuracy for the identification of the cirrhotic HCV patients (AUC, 0.818; sensitivity, 87.2%; specificity, 65.9%). Conversely, Survivin-IgM IC significantly decreased in HCC patients (median, 165.72 AU/mL; P = 0.022).Our results suggest that Survivin-IgM immune complex may be used as a potential biomarker for liver damage, particularly for the identification of the HCV-related cirrhotic population

Oncostatin M is overexpressed in NASH-related hepatocellular carcinoma and promotes cancer cell invasiveness and angiogenesis

: Oncostatin M (OSM) is a pleiotropic cytokine of the interleukin (IL)-6 family that contributes to the progression of chronic liver disease. Here we investigated the role of OSM in the development and progression of hepatocellular carcinoma (HCC) in NAFLD/NASH. The role of OSM was investigated in: a) selected cohorts of NAFLD/NASH HCC patients; b) liver cancer cells exposed to human recombinant OSM or stably transfected to overexpress human OSM; c) murine HCC xenografts; d) a murine NASH-related model of hepatic carcinogenesis. OSM was found to be selectively overexpressed in HCC cells of NAFLD/NASH patients, depending on tumor grade. OSM serum levels, barely detectable in patients with simple steatosis or NASH, were increased in patients with cirrhosis, and more evident in those carrying HCC. In this latter group, OSM serum levels were significantly higher in the subjects with intermediate/advanced HCCs and correlated with poor survival. Cell culture experiments indicated that OSM upregulation in hepatic cancer cells contributes to HCC progression by inducing epithelial-to-mesenchymal transition and increased invasiveness of cancer cells as well as by inducing angiogenesis, which is of critical relevance. In murine xenografts, OSM overexpression was associated with slower tumor growth, but an increased rate of lung metastases. Overexpression of OSM and its positive correlation with the angiogenic switch were also confirmed in a murine model of NAFLD/NASH-related hepatocarcinogenesis. Consistent with this, analysis of liver specimens from human NASH-related HCCs with vascular invasion showed that OSM was expressed by liver cancer cells invading hepatic vessels. In conclusion, OSM up-regulation appears to be a specific feature of HCC arising on a NAFLD/NASH background, and it correlates with clinical parameters and disease outcome. Our data highlight a novel pro-carcinogenic contribution for OSM in NAFLD/NASH, suggesting a role of this factor as a prognostic marker and a putative potential target for therapy. This article is protected by copyright. All rights reserved

The molecular signature of impaired diabetic wound healing identifies serpinB3 as a healing biomarker

Aims/hypothesis Chronic foot ulceration is a severe complication of diabetes, driving morbidity and mortality. The mechanisms underlying delaying wound healing in diabetes are incompletely understood and tools to identify such pathways are eagerly awaited. Methods Wound biopsies were obtained from 75 patients with diabetic foot ulcers. Matched subgroups of rapidly healing (RH, n = 17) and non-healing (NH, n = 11) patients were selected. Proteomic analysis was performed by labelling with isobaric tag for relative and absolute quantification and mass spectrometry. Differentially expressed proteins were analysed in NH vs RH for identification of pathogenic pathways. Individual sample gene/protein validation and in vivo validation of candidate pathways in mouse models were carried out. Results Pathway analyses were conducted on 92/286 proteins that were differentially expressed in NH vs RH. The following pathways were enriched in NH vs RH patients: apoptosis, protease inhibitors, epithelial differentiation, serine endopeptidase activity, coagulation and regulation of defence response. SerpinB3 was strongly upregulated in RH vs NH wounds, validated as protein and mRNA in individual samples. To test the relevance of serpinB3 in vivo, we used a transgenic mouse model with alpha 1-antitrypsin promoter-driven overexpression of human SERPINB3. In this model, wound healing was unaffected by SERPINB3 overexpression in non-diabetic or diabetic mice with or without hindlimb ischaemia. In an independent validation cohort of 47 patients, high serpinB3 protein content was confirmed as a biomarker of healing improvement. Conclusions/interpretation We provide a benchmark for the unbiased discovery of novel molecular targets and biomarkers of impaired diabetic wound healing. High serpinB3 protein content was found to be a biomarker of successful healing in diabetic patient

Purification of anticardiolipin and lupus anticoagulant activities by using cardiolipin immobilized on agarose beads.

none2We describe a novel method for the purification of aPL, in which pure CL is immobilized on octyl-sepharose beads by hydrophobic interaction. No lipid contamination was present in eluates, and the system could be reutilized three times without loosing extracting capacity. Four patients with antiphospholipid syndrome were studied. A marked decrease in aCL and LA activities was found in all patient plasmas after the passage through a CL-octyl-sepharose column. Both activities were recovered in eluates which contained beta 2-GP-I and IgGs. beta 2-GP-I was also present in normal plasma eluates, which showed no aCL and slight LA activity. This method represents an improvement in the purification of aPL, and could be useful in explaining the mechanism of action of antibodies that are obtained using pure phospholipid as extracting matrix.noneV. PENGO; BIASIOLO APengo, Vittorio; Biasiolo, Alessandr