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

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

Hyaluronated and PEGylated Liposomes as a Potential Drug-Delivery Strategy to Specifically Target Liver Cancer and Inflammatory Cells

Hepatocellular carcinoma (HCC) is the most frequent primary liver cancer and is characterized by poor clinical outcomes, with the majority of patients not being eligible for curative therapy and treatments only being applicable for early-stage tumors. CD44 is a receptor for hyaluronic acid (HA) and is involved in HCC progression. The aim of this work is to propose HA- and PEGylated-liposomes as promising approaches for the treatment of HCC. It has been found, in this work, that CD44 transcripts are up-regulated in HCC patients, as well as in a murine model of NAFLD/NASH-related hepatocarcinogenesis. Cell culture experiments indicate that HA-liposomes are more rapidly and significantly internalized by Huh7 cells that over-express CD44, compared with HepG2 cells that express low levels of the receptor, in which the uptake seems due to endocytic events. By contrast, human and murine macrophage cell lines (THP-1, RAW264.7) show improved and rapid uptake of PEG-modified liposomes without the involvement of the CD44. Moreover, the internalization of PEG-modified liposomes seems to induce polarization of THP1 towards the M1 phenotype. In conclusion, data reported in this study indicate that this strategy can be proposed as an alternative for drug delivery and one that dually and specifically targets liver cancer cells and infiltrating tumor macrophages in order to counteract two crucial aspect of HCC progression

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

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

Low P66shc with High SerpinB3 Levels Favors Necroptosis and Better Survival in Hepatocellular Carcinoma.

Cell proliferation and escape from apoptosis are important pathological features of hepatocellular carcinoma (HCC), one of the tumors with the highest mortality rate worldwide. The aim of the study was to evaluate the expression of the pro-apoptotic p66shc and the anti-apoptotic SerpinB3 in HCCs in relation to clinical outcome, cell fate and tumor growth. p66shc and SerpinB3 were evaluated in 67 HCC specimens and the results were correlated with overall survival. Proliferation and cell death markers were analyzed in hepatoma cells overexpressing SerpinB3, under different stress conditions. p66shc-/- mice and xenograft models were also used to assess the effects of p66shc and SerpinB3 on tumor growth. In patients with HCC, the best survival was observed in the subgroup with p66shc levels below median values and SerpinB3 levels above median values. Mice p66shc-/- showed high levels of SerpinB3, while in HepG2 cells overexpressing SerpinB3, p66shc expression was trivial. HepG2 overexpressing SerpinB3 cells were more prone to die after oxidizing treatments, such as diamide or high concentration H2O2. These cells injected in nude mice developed tumors five times smaller than those from control HepG2 cells. Tumors originating from HepG2 overexpressing SerpinB3 cells showed decreased activated Caspase-8, with concomitant increase of RIP3K and decreased levels of cleaved RIP3K, typical features of necroptosis. In conclusion, in patients affected by HCC, the pattern characterized by p66shc downregulation and elevated SerpinB3 levels was associated with markedly better survival. This pattern favored necroptosis in experimental high-stress conditions

Implicazioni biologiche di SerpinB3 nella fibrosi epatica e nell'epatocarcinoma

BACKGROUND SERPINB3 is a serine protease inhibitor belonging to the serpin family, characterized by pleyotropic functions. TGF-beta1 is the master cytokine in the pathogenesis of liver fibrosis. Inflammatory and mesenchymal cells, including hepatic stellate cells, have been identified as the principal source of this cytokine, however growing evidence also supports the ability of parenchymal hepatocytes to produce TGF-beta1. Recently, a direct correlation between TGF-beta1 and the ov-serpin SERPINB3 has been reported in idiopatic pulmonary fibrosis, whose pathological features recall cirrhosis pathogenesis. SERPINB3, a serine protease inhibitor up-regulated in cirrhosis, dysplastic nodules and hepatocellular carcinoma (HCC), can trigger epithelial to mesenchymal transition (EMT) in HepG2 cells. EMT is also triggered by hypoxia which is common in HCC and believed to favour selection of more invasive tumour cells and cancer progression. AIM Aim of the first study was to assess the potential correlation between SERPINB3 and TGF-beta1 in chronic liver diseases and to determine their relationship using in vitro models of cells transfected with SERPINB3. Aim of the second study was to investigated molecular mechanisms involved in SERPINB3-mediated EMT induction and the relationship between hypoxia and SERPINB3 expression in vitro, by employing normal and SERPINB3-transgenic HepG2 cells or in vivo by immunohistochemistry (IHC) on specimens from cirrhotic patients carrying HCC. METHODS In the first study SERPINB3 and TGF-beta1 expression in liver biopsies from 80 patients with chronic liver disease (59 chronic hepatitis and 21 liver cirrhosis) was evaluated by immunohistochemistry and by Real time PCR. To investigate whether SERPINB3 could affect TGF-beta1 expression, TGF-beta1 was determined in HepG2 cells transfected with SERPINB3 and in control HepG2 cells at transcription and protein level by Real time PCR and ELISA assay. In the second study SERPINB3 up-regulation, involvement of hypoxia inducible factors (HIFs) and signal transduction pathways, intracellular generation of reactive oxygen species (ROS), EMT and invasiveness were analysed by morphological, molecular and cell biology techniques in HepG2 cells, exposed to hypoxia and/or incubated with wild type and mutated SERPINB3 recombinant proteins. Moreover, liver specimens from either HCV cirrhotic patients carrying HCC or from transgenic mice over-expressing SerpinB3 were analyzed by immunohistochemistry. RESULTS Study I In chronic hepatitis and liver cirrhosis a strong positive correlation was observed between SERPINB3 and TGF-beta1 immunoreactivity. At transcription level, cDNA amplification profiles of SERPINB3 and TGFbeta1 were similar in the majority of the cases. A significant correlation was found between the degree of parenchymal TGF-beta1 1 and SERPINB3 expression and the severity of liver fibrosis. Neither of the two molecules were detectable in 20 normal livers. Time course analysis of TGF-beta1 1 over time in SERPINB3 transfected cells showed an increase of this cytokine, with a peak value at 48 hours for TGF-beta1 1 mRNA and at 72 h for protein production. Study II EMT and increased invasiveness triggered by wild-type and mutated recombinant SERPINB3 are independent of their anti-protease activity and involve intracellular generation of ROS and redox-dependent, phosphorylation of GSK3beta. SERPINB3 transcription, protein synthesis and release in the medium are up-regulated under hypoxia, almost abolished by specific silencing of HIF-2alpha (but not HIF-1alpha), significantly modulated by hypoxia-dependent intracellular generation of ROS and involve Ras/Erk and PI3K signalling pathways. In addition, liver specimens revealed co-localization of HIF-2alpha and SERPINB3 immunostaining in either HCC cells and in hepatocytes of surrounding cirrhotic liver. CONCLUSIONS Study I SERPINB3 is overexpressed in chronically damaged hepatocytes, where increased TGF-beta1 has also been documented. The positive correlation found between the expression of either TGF-beta1 and SERPINB3 and fibrosis grade supports the dynamic interplay of these molecules, suggesting that SERPINB3 could orchestrate the most peculiar aspects of chronic liver disease by inducing TGF-beta1 production, leading to enhanced collagen synthesis and by increasing the survival capacity of damaged hepatocytes. Study II Hypoxia, through a common redox sensitive signalling, can switch on both EMT program and SERPINB3 expression. Hypoxia may then sustain increased invasiveness in cancer cells by a release of HIF2-dependent SERPINB3 molecule. This serpin could elicit EMT and invasiveness even in normoxic surrounding cells, then enhancing its putative role in carcinogenesis.INTRODUZIONE SERPINB3 appartiene alla famiglia delle serpine, inibitori delle proteasi seriniche implicati in molte funzioni biologiche e nei processi di controllo dell'omeostasi cellulare. E' espressa normalmente negli epiteli squamosi, ma si trova iper-espressa nelle cellule neoplastiche di origine epiteliale e nell'epatocarcinoma ma anche in noduli cirrotici ad alto grado di displasia. TGF-beta1 una citochina multifunzionale, coinvolta nella regolazione della proliferazione, differenziamento e migrazione di molti tipi cellulari ed il più importante fattore implicato nel processo di fibrogenesi epatica, dove regola la sintesi di collagene nelle cellule stellate epatiche. Recentemente in un'altra patologia fibrosante progressiva, quale la fibrosi polmonare idiopatica, è stata documentata una correlazione diretta tra entità di espressione di TGF-beta1 e della serpina SERPINB3, con co-localizzazione delle due molecole a livello cellulare. Recenti studi sperimentali condotti nel nostro laboratorio suggeriscono il coinvolgimento in vitro di SERPINB3 in caratteristiche tipiche della transizione epitelio mesenchimale, (EMT) un fenomeno chiave anche nell'epatocarcinoma. Inoltre, ulteriori risultati descritti in letteratura identificano l'ipossia, una delle condizioni più comuni che si instaurano nel microambiente tumorale, come condizione in grado di indurre in modo indipendente EMT e lo stesso fenomeno di transizione epitelio mesechimale è indotto anche dalla citochina TGF-beta1 . SCOPO Studio I Lo scopo della prima parte della tesi è valutare la potenziale correlazione tra SERPINB3 e TGF-beta1 nel fegato in pazienti con danno epatico cronico e valutare la relazione tra queste due molecole utilizzando modelli in vitro di colture cellulari trasfettate con SERPINB3. Studio II Lo scopo della seconda parte della tesi è quello di caratterizzare gli stimoli regolatori e i meccanismi dell'induzione di SERPINB3 come fattore solubile in grado di indurre l'invasività e la transizione epitelio mesenchimale. Inoltre, ulteriore scopo è valutare la correlazione tra ipossia e SERPINB3 ed i relativi meccanismi di regolazione sia in modelli in vitro che in vivo. MATERIALI E METODI Per quanto concerne il primo studio sono stati studiati 80 pazienti, di cui 59 con epatite cronica e 21 con cirrosi epatica. L'espressione di SERPINB3 e TGF-beta1 stata analizzata su biopsie epatiche di pazienti con epatite cronica a livello proteico mediante immunoistochimica e a livello trascrizionale mediante PCR quantitativa. Il modello in vitro prevedeva la trasfezione transiente e stabile di cellule HepG2 con il gene SERPINB3 wild type e con differenti vettori caratterizzati da estensioni diverse di delezione del sito attivo della serpina, responsabile della sua attività  antiproteasica, con successiva analisi dell'espressione genica e proteica. Per quanto riguarda il secondo studio sono stati effettuate analisi di immunofluorescenza e invasività  in cellule HepG2 trattate con diverse forme di proteina SERPINB3 ricombinante per valutarne l'effetto di trasformazione epitelio-mesenchimale e la generazione intracellulare di specie reattive all'ossigeno (ROS). Inoltre le cellule sono state mantenute in condizioni di ipossia (3%O2 ) per studiare il comportamento di SERPINB3 ed è stato effettuato il silenziamento genico per i fattori inducibili in ipossia (HIF) al fine di determinare il loro coinvolgimento. L'espressione di SERPINB3 e dei fattori relativi all'EMT e all'ipossia sono stati valutati anche su biopsie epatiche in pazienti con epatocarcinoma. RISULTATI Studio I L'analisi immunoistochimica nel fegato ha documentato una significativa correlazione positiva con co-localizzazione di SERPINB3 e TGF-beta1 nelle cellule parenchimali del fegato. Anche a livello trascrizionale l'espressione di mRNA ha confermato un simile andamento, con livelli di espressione genica sostanzialmente sovrapponibili (r=0,7483, p=0,0070). Inoltre, l'espressione di entrambe le molecole è risultata e positivamente correlata con l'entità  della fibrosi. In cellule HepG2 trasfettate con il gene SERPINB3 è stata documentata un'induzione di TGF-beta1 sia dal punto di vista trascrizionale che della produzione proteica. I risultati ottenuti dalla trasfezione hanno dimostrato che è cruciale l'integrità  del sito attivo della serpina per l'induzione di TGF-beta1 , in quanto questa citochina è totalmente assente nei vettori con la delezione del sito antiproteasico di SERPINB3, mentre dove la delezione è minore, si riscontra una ridotta induzione, rispetto al controllo. Studio II SERPINB3 induce EMT e aumenta l'invasività  in cellule HepG2 mediante il coinvolgimento di ROS e la fosforilazione di GSK3b, indipendentemente dall'attività antiproteasica della serpina. In condizioni di ipossia si osserva un aumento della trascrizione e sintesi di SERPINB3, modulata dalle vie di segnale Ras/ERK e PI3K e completamente dipendente dal gene HIF-2alpha. Questi dati sono stati confermati nel topo transgenico per SERPINB3, dove è stata dimostrata un'iperespressione della molecola nelle zone ipossiche circostanti le vene centro globulari. Inoltre, in biopsie di pazienti con epatocarcinoma è stata confermata una colocalizzazione di SERPINB3 e HIF-2alpha CONCLUSIONI Studio I Nella malattia epatica cronica evolutiva esiste una correlazione diretta tra espressione di SERPINB3 e TGF-beta1. La correlazione di entrambe queste molecole con il grado di fibrosi epatica rafforza l'ipotesi di una loro potenziale interazione nel processo fibrogenetico epatico. In modelli in vitro SERPINB3 è in grado di indurre l'espressione di TGF-β e questo effetto dipende dalla sua attività  antiproteasica. Alla luce di queste considerazioni SERPINB3 potrebbe essere considerato un bersaglio terapeutico per contrastare il processo di fibrogenesi. Studio II L'ipossia può indurre SERPINB3 e conseguente trasformazione epitelio-mesenchimale mediante la produzione di radicali liberi dell'ossigeno. L'ipossia inoltre è il primo stimolo di rilevanza fisiopatologica descritto in letteratura capace di modulare la trascrizione e l'espressione di SERPINB3 attraverso un meccanismo dipendente dal gene HIF-2alpha