SerpinB3 as hepatic marker of post-resective shear stress

Post-resective liver failure is a frequent complication of liver surgery and it is due to portal hyperperfusion of the remnant liver and to arterial vasoconstriction, as buffer response of the hepatic artery. In this context, splenectomy allows a reduction of portal flow and increases the survival chance in preclinical models. SerpinB3 is over-expressed in the liver in oxidative stress conditions, as a mechanism of cell defense to provide survival by apoptosis inhibition and cell proliferation. In this study, the expression of SerpinB3 was assessed as predictor of liver damage in in vivo models of major hepatic resection with or without splenectomy. Wistar male rats were divided into 4 groups: group A received 30% hepatic resection, group B > 60% resection, group C > 60% resection with splenectomy and group D sham-operated. Before and after surgery liver function tests, echo Doppler ultrasound and gene expression were assessed. Transaminase values and ammonium were significantly higher in groups that underwent major hepatic resection. Echo Doppler ultrasound showed the highest portal flow and resistance of the hepatic artery in the group with > 60% hepatectomy without splenectomy, while the association of splenectomy determined no increase in portal flow and hepatic artery resistance. Only the group of rats without splenectomy showed higher shear-stress conditions, reflected by higher levels of HO-1, Nox1 and of Serpinb3, the latter associated with an increase of IL-6. In conclusion, splenectomy controls inflammation and oxidative damage, preventing the expression of Serpinb3. Therefore, SerpinB3 can be considered as a marker of post-resective shear stress

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

MiR-122 Targets SerpinB3 and Is Involved in Sorafenib Resistance in Hepatocellular Carcinoma

The only first-line treatment approved for advanced hepatocellular carcinoma (HCC) is sorafenib. Since many patients experience drug resistance, the discovery of more effective therapeutic strategies represents an unmet clinical need. MicroRNA (MiR)-122 is downregulated in most HCCs, while oncogenic SerpinB3 is upregulated. Here, we assessed the relationship between miR-122 and SerpinB3 and their influence on cell phenotype and sorafenib resistance in HCC. A bioinformatics analysis identified SerpinB3 among hypothetical miR-122 targets. In SerpinB3-overexpressing HepG2 cells, miR-122 transfection decreased SerpinB3 mRNA and protein levels, whereas miR-122 inhibition increased SerpinB3 expression. Luciferase assay demonstrated the interaction between miR-122 and SerpinB3 mRNA. In an HCC rat model, high miR-122 levels were associated with negative SerpinB3 expression, while low miR-122 levels correlated with SerpinB3 positivity. A negative correlation between miR-122 and SerpinB3 or stem cell markers was found in HCC patients. Anti-miR-122 transfection increased cell viability in sorafenib-treated Huh-7 cells, while miR-122 overexpression increased sorafenib sensitivity in treated cells, but not in those overexpressing SerpinB3. In conclusion, we demonstrated that miR-122 targets SerpinB3, and its low levels are associated with SerpinB3 positivity and a stem-like phenotype in HCC. MiR-122 replacement therapy in combination with sorafenib deserves attention as a possible therapeutic strategy in SerpinB3-negative HCCs

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

Hepatocyte-specific deletion of HIF2α prevents NASH-related liver carcinogenesis by decreasing cancer cell proliferation

Background & aims: Hypoxia and hypoxia-inducible factors (HIFs) are involved in chronic liver disease progression. We previously showed that hepatocyte HIF-2\u3b1 activation contributed significantly to nonalcoholic fatty liver disease progression in experimental animals and human patients. In this study, using an appropriate genetic murine model, we mechanistically investigated the involvement of hepatocyte HIF-2\u3b1 in experimental nonalcoholic steatohepatitis (NASH)-related carcinogenesis. Methods: The role of HIF-2\u3b1 was investigated by morphologic, cellular, and molecular biology approaches in the following: (1) mice carrying hepatocyte-specific deletion of HIF-2\u3b1 (HIF-2\u3b1-/- mice) undergoing a NASH-related protocol of hepatocarcinogenesis; (2) HepG2 cells stably transfected to overexpress HIF-2\u3b1; and (3) liver specimens from NASH patients with hepatocellular carcinoma. Results: Mice carrying hepatocyte-specific deletion of HIF-2\u3b1 (hHIF-2\u3b1-/-) showed a significant decrease in the volume and number of liver tumors compared with wild-type littermates. These effects did not involve HIF-1\u3b1 changes and were associated with a decrease of cell proliferation markers proliferating cell nuclear antigen and Ki67. In both human and rodent nonalcoholic fatty liver disease-related tumors, HIF-2\u3b1 levels were strictly associated with hepatocyte production of SerpinB3, a mediator previously shown to stimulate liver cancer cell proliferation through the Hippo/Yes-associated protein (YAP)/c-Myc pathway. Consistently, we observed positive correlations between the transcripts of HIF-2\u3b1, YAP, and c-Myc in individual hepatocellular carcinoma tumor masses, while HIF-2\u3b1 deletion down-modulated c-Myc and YAP expression without affecting extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase, and AKT-dependent signaling. In\ua0vitro data confirmed that HIF-2\u3b1 overexpression induced HepG2 cell proliferation through YAP-mediated mechanisms. Conclusions: These results indicate that the activation of HIF-2\u3b1 in hepatocytes has a critical role in liver carcinogenesis during NASH progression, suggesting that HIF-2\u3b1-blocking agents may serve as novel putative therapeutic tools

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

Caratterizzazione di un modello di topo transgenico per SERPINB3 umana

the serpins are a family of serine protease inhibitors involved in several biological functions and in the maintenance of cell homeostasis. SERPINB3 (known as SCCA1), a member of the ovalbumin family, is normally expressed in squamous epithelium, and it is over-expressed in tumours of epithelial origin and in hepatocellular carcinoma. The SERPINB3 involvement in the control of proteolytic processes has important implications in neoplastic transformation, whereas the balance between proteases and their inhibitors can affect cell motility, invasiveness, proliferation and apoptosis. Considering the potential role of SERPINB3 and the lack of human biological tissues for clinical research, the possibility to use a transgenic animal model to explore in vivo the role of this serpin in several cell processes could be of great value. The aim of the present study was to assess the SERPINB3 involvement in several pathologies by a transgenic mouse model for human SERPINB3, expressed in different organs as brain, lung and liver.Le serpine sono una famiglia di inibitori delle proteasi seriniche implicata in molte funzioni biologiche e nei processi di controllo dell’omeostasi cellulare. SERPINB3 (chiamata anche SCCA1), appartenente alle ov-serpine, è espressa normalmente negli epiteli squamosi, ma si trova iper-espressa nelle cellule neoplastiche di origine epiteliale e nell’epatocarcinoma. Il coinvolgimento di SERPINB3 nella regolazione dei processi proteolitici ha importanti implicazioni a livello dei processi neoplastici, dal momento che l’equilibrio tra le proteasi ed i loro inibitori, può influenzare la mobilità, l’invasività, la proliferazione e la morte cellulare stessa. Dato il potenziale ruolo di SERPINB3 ed il limite determinato dalla disponibilità di materiale biologico per la ricerca clinica sull'uomo, risulta importante poter utilizzare un modello animale che permetta di sperimentare in vivo il ruolo che la proteina può rivestire in molteplici processi cellulari. Lo scopo dello studio è quello di sperimentare il coinvolgimento della serpina in diverse patologie utilizzando un modello animale, costituito da un topo transgenico per SERPINB3 umana, caratterizzato dall’espressione della serpina in diversi organi quali il cervello, i polmoni ed il fegato

SERPINB3 protects from oxidative damage by chemotherapeutics through inhibition of mitochondrial respiratory complex I

none7noneFrancesco Ciscato; Marco Sciacovelli; Gianmarco Villano; Cristian Turato; Paolo Bernardi; Andrea Rasola; Patrizia PontissoFrancesco, Ciscato; Marco, Sciacovelli; Villano, Gianmarco; Turato, Cristian; Bernardi, Paolo; Rasola, Andrea; Patrizia, Pontiss