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Introduction Liver transplantation (LT) for hepatocellular carcinoma (HCC) is a satisfactory therapeutic choice in patients with “early HCC” selected according to Milan criteria. However, the risk of HCC recurrence after LT is about 7-20% at five years and molecular markers which can predict recurrence are still lacking. We investigated in HCC samples and LC surrounding tissues the significance of VEGFR-2, HSP90, and GRP78/BiP expression in patients with HCC who underwent LT in a western transplantation center and their possible role as molecular markers of recurrence. Methods 42 patients (35 M, 7 F) with early HCC who underwent LT between 2012 and 2013 were enrolled. The tumor recurrence rate was analyzed after a minimum follow-up of 36 months. Immunohistochemical expression of VEGFR-2, HSP90, and GRP78/BiP in HCC tissues and LC surrounding tissues, was correlated with clinicopathological variables and recurrence-free post-LT survival. At statistical analysis t Student’s or Mann Whitney U test were used where appropriate. To assess which variable measured at baseline was predictive of HCC recurrence, the univariate Cox proportional hazards model (Hr) was fitted to each variable. All variables with a P

FIBROSIS DIAGNOSTIC SCORES VALIDATION IN OBESE PATIENTS WITH NON-ALCOHOLIC LIVER DISEASE

Concept Gene therapy is a growing field of medicine with great potential for the treatment of several diseases and it is based on the delivery of nucleic acids (DNA, RNA, etc.,) to specific cells. To achieve their therapeutic effects, the nucleic acids need to cross several biological barriers and be protected from the degradation by nucleases, present in biological fluids and intracellular compartments, to successfully gain access to their intracellular targets. To overcome these hurdles, it is necessary to deliver the genetic material with biocompatible carriers able to facilitate its translocation across the cell membranes and protect it from being degraded while circulating in the bloodstream. At this purpose, viral and non-viral vectors have been used. Viral vectors usually have high transfection efficiencies, although there are several concerns about their use in human therapy such as an increased immune response, the possible recombination of oncogenes and the difficulty in scale-up. For these reasons, non-viral vectors have emerged as promising carriers due to their reduced immune response, low toxicity and their safety in comparison to viral vectors. Within this field, the use of solid lipid nanoparticles (SLN), made up of biocompatible and biodegradable lipids, solid at room and body temperatures, is of particular importance [1,2]. Motivations and Objectives In the present study, we aimed to develop cationic solid lipid nanoparticles able to efficiently bind, protect and deliver nucleic acids for the treatment of hepatocellular carcinoma. These nanosystems were designed in order to get features that made them suitable for parenteral administration (size, surface charge and hemocompatibility). We studied their potential as gene delivery systems carrying out biological assays (cytotoxicity, transfection, protection of the genetic material by DNasi degradation) on human hepatocellular carcinoma Hep3B cells. Results and Discussion Two different nanosystems were prepared, characterized in terms of size, polydipersity index and zeta potential, and complexed with siRNA and a DNA plasmid using different weight ratios. The physical binding between SLN and the nucleic acids was confirmed by Dynamic Light Scattering measurements and electrophoretic mobility studies. We also verified the acceptability of prepared formulations for parenteral administration performing hemocompatibility assays on human herytrocytes. Finally, in vitro biological characterization confirmed that these nanosystems are not toxic and able to protect and efficiently transfect genetic material to Hep3B cells.

Hepatocellular carcinoma (HCC) is the sixth most common cancer worldwide and the third leading cause of cancer-related deaths. In the United States, the incidence of HCC has almost tripled during the past two decades and HCC has become one of the fastest growing cancers. While surgical removal of tumor tissues is an effective approach to protect relatively healthy liver tissue, it is only applicable to a small subset of HCC patients with specific pathological conditions, such as confined tumor mass without portal hypertension. Therefore, there is an urgent need to develop novel therapeutic strategies to treat this deadly disease. Systemic tumor-targeted gene delivery is attracting increasing attention as a promising alternative to conventional therapeutic strategies. At this purpose a large number of viral and non-viral vectors have been studied and applied as systems of stable transfection with low toxicity. Although cationic polymers and liposome are promising systems, solid lipid nanoparticles (SLN) have been recently proved to be a really useful vehicle for gene therapy [1,2]. The aim of this work was to design and to obtain cationic SLNs capable of forming complexes with siRNA and DNA plasmid for the treatment of HCC. The physical binding between cSLN and nucleic acids was confirmed by the study of complexes’ zeta potential values that became more positive as higher was the amount of cSLN and via the electrophoretic mobility of the samples in agarose gel 0.8%. Transfection studies on different tumor cell line are in progress.

Background: In hepatocellular carcinoma (HCC), different signaling pathways are de-regulated, and among them, the expression of the epidermal growth factor receptor (EGFR). Tyrphostin AG-1478 is a lipophilic low molecular weight inhibitor of EGFR, preferentially acting on liver tumor cells. In order to overcome its poor drug solubility and thus improving its anticancer activity, it was entrapped into nanostructured lipid carriers (NLC) by using safe ingredients for parenteral delivery. Results: Nanostructured lipid carriers (NLC) carrying tyrphostin AG-1478 were prepared by using the nanoprecipitation method and different matrix compositions. The best system in terms of mean size, PDI, zeta potential, drug loading and release profile was chosen to evaluate the anti-proliferative effect of drug-loaded NLC versus free drug on human hepatocellular carcinoma HA22T/VGH cells. Conclusions: Thanks to the entrapment into NLC systems, tyrphostin AG-1478 shows an enhanced in vitro anti-tumor activity compared to free drug. These finding raises hope of future drug delivery strategy of tyrphostin AG-1478 -loaded NLC targeted to the liver for the HCC treatment.

Hepatocellular carcinoma (HCC) is the sixth most prevalent cancer and the third most frequent cause of cancer-related death. Unlike most solid tumors the incidence and mortality of HCC have increased in the United States and Europe in the past decade. Most patients are diagnosed at advanced stages, so there is an urgent need for new systemic therapies [1,2]. To improve the delivery of therapeutic agents to tumor cells in vivo it’s necessary to overcome several problems, such as drug resistance at the tumor level due to physiological barriers (non cellular based mechanism), drug resistance at the cellular level (cellular mechanism), drug distribution, biotransformation and clearance of anticancer drugs in the body. At the purpose to increase selectivity of drugs towards cancer cells while reducing their toxicity towards normal tissues, a promising strategy could be to entrap antitumor drugs into colloidal nanoparticles. Nanosystems may act as drug vehicles able to target tumor tissues or cells, protecting it from premature inactivation during its transport. At the tumor level the accumulation mechanism of intravenously injected nanoparticles relies on a passive diffusion or convection across the leaky hyperpermeable tumor vasculature [3-5]. For the treatment of HCC several drugs are under development, but the only one with proven survival benefit is sorafenib. This agent is a multikinase inhibitor that blocks Raf signaling and VEGF, PDGF and c-Kit. It has antiproliferative and antiangiogenic activity and delays tumor progression [1,2]. In order to produce a new colloidal system able to deliver this drug in the tumor tissue and decrease its side effects, nanostructured lipid carriers (NLCs) were developed using tripalmitin, Miglyol 812 and sorafenib. The obtained nanosystems were characterized in terms of size, polidispersity index, zeta potential and morphology using Scanning Electron Microscopy. Moreover, technological-pharmaceutical characterization of NLC was made. Stability studies showed that the systems were stable up to 60 days, while particle aggregation occurred after a period of storage of 90 days. Afterwards, for this reason, the same systems were prepared with the addition of two cryoprotectors (threalose and glycerol) before freezing and lyophilization. Finally, in vitro biological studies were carried out by MTS assays using human hepatocellular carcinoma HepG2, Hep3B, Huh7 and PLC/PRF/5 cells. Results obtained demonstrate an improved efficacy of sorafenib-loaded NLC compared to the free drug in Huh7 cells, whereas in HepG2 and PLC/PRF/5 cells sorafenib-loaded NLCs maintain an antitumor activity comparable to free drug. Considering that solid tumours present much more favourable conditions for preferential accumulation of colloidal sized drug delivery systems, sorafenib-loaded NLC can be useful for application in liver cancer therapy. [1] A. Forner, J.M. Llovet and J. Bruix, Lancet, 379, 1245 (2012). [2] M. Cervello, J.A. McCubrey, A. Cusimano, N. Lampiasi, A. Azzolina and G. Montalto, Oncotarget, 3, 236 (2012). [3] I. Brigger, C. Dubernet and P. Couvreur, Adv. Drug Del. Rev., 54, 631 (2002). [4] M.L. Bondì, A. Azzolina, E.F. Craparo, G. Capuano, N. Lampiasi , G. Giammona and M. Cervello, Curr. Nanoscience, 5, 39 (2009). [5] M.L. Bondì, E.F. Craparo, G. Giammona, M. Cervello, A. Azzolina, P. Diana, A. Martorana and G. Cirrincione, Drug Deliv., 14, 61 (2007).

Hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide characterized by poor and often limited or no response to current drug therapies. The PI3K/Akt/mTOR pathway is a key regulator of cell proliferation and survival. Alterations in this pathway have been reported in many types of human cancer, including HCC. It has become evident that Akt inhibitors have great potential in cancer treatment. SC66 is a new allosteric Akt inhibitor that facilitates ubiquitination of Akt, favoring its degradation via the proteasome, thus inhibiting Akt signaling. In the present study, we investigated the anticancer activity of SC66 in HCC cell lines (HepG2, Huh7, Hep3B, PLC/PRF/5 and HA22T/VGH). Treatment with SC66 reduced cell viability in a dose- and time-dependent manner and inhibited colony formation. At the same time, SC66 induced anoikis, as demonstrated by alterations of cytoskeleton organization and reduction of E-cadherin, -catenin and Snail expression. SC66 induced apoptosis was revealed by flow cytometry analysis, cleavage of PARP, and decreased expression of the anti-apoptotic proteins survivin and Bcl2. In addition, after treatment with SC66 a dose-dependent ROS production was demonstrated by H2DCFDA-based fluorescence staining. Co-treatment with the ROS scavenger NAC prevented the SC66-induced cell growth inhibition and anoikis. In conclusion, our data indicated that the Akt inhibitor SC66 had antitumor effects on HCC cells. This was mediated by ROS production and induction of anoikis-mediated cell death. All together, our results provide a rational basis for the clinical use of SC66 in the treatment of liver cancer.

Introduction: TNF-a, COX-2 and Vascular Endothelial Growth Factor (VEGFA) are mediators of inflammation and angiogenesis, all of them are abundantly produced in liver cirrhosis (LC). It was proposed that there is an association between single nucleotide polymorphisms (SNPs) and hepatocellular carcinoma (HCC). These allelic variants influence the transcriptional activity of these genes, and therefore the proteins levels. The VEGF-A pathway is a potential therapeutic target in HCC, and several anti-angiogenic agents have entered clinical trials in HCC. Aims: 1) To evaluate thè frequency of SNPs of COX-2, TNF-a and VEGF-A genes in patients with HCC vs LC patients and a control group (C). 2) To verify thè correlation between thè allelic variations and the risk of developing HCC tumors. Methods: DNA extracted from whole blood of C, HCC and LC patients (all from Sicily, Italy), was used for the evaluation of SNPs by the RFLP-PCR method. The SNPs analyzed were at position -1195 G>A in the promoter regions, of COX-2 (A allele is associated with higher levels of thè enzyme), at position -308 G>A in thè promoter regions of TNF-a genes (G allele is associated with lower levels of the cytokine), and at position +936 C>T in the coding region of VEGFA gene (T allele is associated with high levels of the growth factor). X2, Fisher exact tests were used for statistical analysis. Results: We obtained a significant difference only for the SNP +936 C>7 of VEGF-A gene. Results obtained are shown in the following table.

The serine/threonine kinase glycogen synthase kinase-3 (GSK-3) was initially identified and studied in the regulation of glycogen synthesis. GSK-3 functions in a wide range of cellular processes. Aberrant activity of GSK-3 has been implicated in many human pathologies including: bipolar depression, Alzheimer's disease, Parkinson's disease, cancer, non-insulin-dependent diabetes mellitus (NIDDM) and others. In some cases, suppression of GSK-3 activity by phosphorylation by Akt and other kinases has been associated with cancer progression. In these cases, GSK-3 has tumor suppressor functions. In other cases, GSK-3 has been associated with tumor progression by stabilizing components of the beta-catenin complex. In these situations, GSK-3 has oncogenic properties. While many inhibitors to GSK-3 have been developed, their use remains controversial because of the ambiguous role of GSK-3 in cancer development. In this review, we will focus on the diverse roles that GSK-3 plays in various human cancers, in particular in solid tumors. Recently, GSK-3 has also been implicated in the generation of cancer stem cells in various cell types. We will also discuss how this pivotal kinase interacts with multiple signaling pathways such as: PI3K/PTEN/Akt/mTORC1, Ras/Raf/MEK/ERK, Wnt/beta-catenin, Hedgehog, Notch and others.

We tested the novel NF-kappaB inhibitor dehydroxymethylepoxyquinomicin (DHMEQ) in the hepatic cancer (HCC) HepG2, HA22T/VGH and HuH-6 cells. The sensitivity to the cell growth inhibitory and apoptotic effects of the agent increased along with the levels of constitutively activated NF-kappaB, which were low in HepG2 and higher in HA22T/VGH and HuH-6. In HA22T/VGH, DHMEQ exhibited synergy with cisplatin. In the same cells, DHMEQ exerted dose-dependent decreases in the nuclear levels of activated NF-kappaB and attenuated NF-kappaB activation by cisplatin. It down-regulated Bcl-XL mRNA in a dose-dependent manner and up-regulated that of Bcl-XS. It also decreased interleukin 6 (IL-6), NAIP and, after 16 h of exposure to the higher concentration tested (10 microg/ml), c-IAP-1 mRNA levels. At 10 microg/ml it caused significant increase in Bax, XIAP, cyclin D1 and beta-catenin mRNAs. The combination of DHMEQ with cisplatin produced unexpected significant decrease in c-IAP-2 and Bcl-XS mRNAs as well as additive decrease (IL-6, NAIP and, after 16 h, Bcl-XL) or increase (XIAP at 8 h) in gene expression. HA22T/VGH produce IL-6; in agreement with the results on mRNA, DHMEQ inhibited such a process. HA22T/VGH lack the IL-6 receptor alpha chain, ruling out that in these cells the antitumor effects of DHMEQ may be attributed to an interference with a growth stimulatory autocrine loop based on IL-6. However, the use of DHMEQ in HCC might be beneficial to contrast the adverse systemic effects of the released cytokine.