Rat Model Of Hepatocellular Carcinoma Research Articles

Hepatic Artery Embolization Induces the Local Overexpression of Transforming Growth Factor β1 in a Rat Hepatoma Model

Introduction: The underlying mechanism involved in the recurrence of hepatoma after hepatic arterial embolization (HAE) is not adequately examined. An immunosuppressive cytokine, transforming growth factor β1 (TGF-β1), can lead to tumor progression and is affected by hypoxia in various cancers. The study aimed to assess the effect of HAE on the expression of TGF-β1 in a rat hepatoma model. Methods: Sprague-Dawley rats bearing N1S1 hepatoma cells underwent HAE (HAE group, n = 5) or sham treatment (sham group, n = 4). The animals were euthanized at 48 h, and liver tissues were harvested. Immunohistochemistry (IHC) and quantitative polymerase chain reaction (qPCR) were performed to compare the expression of TGF-β1 and hypoxia-inducible factor 1α (HIF-1α) between the HAE and sham groups. In vitro experiments with the N1S1 cell line were also performed under normoxic (21% O₂) or hypoxic (1% O₂) conditions for 48 h, and the expression of TGF-β1 and HIF-1α was assessed with western blotting and enzyme-linked immunosorbent assay. Statistical data comparisons were performed by Student t test. Results: IHC showed that both the TGF-β1-positive and HIF-1α-positive tumor peripheral areas were larger in the HAE group (6.59 ± 2.49 and 10.26 ± 4.14%; p < 0.001, respectively) than in the sham group (0.34 ± 0.41 and 0.40 ± 0.84% respectively). Similarly, qPCR showed that the mRNA expression levels of TGF-β1 and HIF-1α were higher (1.95 ± 0.38-fold and 1.62 ± 0.37-fold; p < 0.001 and p = 0.002, respectively) in the HAE group than those in the sham group. TGF-β1 expression was suppressed when HIF-1α inhibitors were added (p = 0.001), and HIF-1α expression was upregulated when exogenous TGF-β1 was added (p = 0.033) in N1S1 cells. Conclusion: HAE enhanced local TGF-β1 expression in a rat hepatoma model. In vitro experiments suggest that HAE-induced hypoxic stress may trigger the interdependent expression of TGF-β1 and HIF-1α.

Hepatic Arterial Bland Embolization Increases Th17 Cell Infiltration in a Syngeneic Rat Model of Hepatocellular Carcinoma.

To determine the tumor immune cell landscape after transcatheter arterial bland embolization (TAE) in a clinically relevant rat hepatocellular carcinoma (HCC) model. Buffalo rats (n = 21) bearing syngeneic McArdle RH-7777 rat hepatoma cells implanted into the left hepatic lobe underwent TAE using 70-150µm beads (n = 9) or hepatic artery saline infusion (n = 12). HCC nodules, peritumoral margin, adjacent non-cancerous liver, and splenic parenchyma were collected and disaggregated to generate single-cell suspensions for immunological characterization 14d after treatment. Changes in tumor-infiltrating immune subsets including CD4 T cells (Th17 and Treg), CD8 cytotoxic T cells (IFNγ), and neutrophils were evaluated by multiparameter flow cytometry. Migration and colony formation assays were performed to examine the effect of IL-17, a signature cytokine of Th17 cells, on McArdle RH-7777 hepatoma cells under conditions simulating post-embolization environment (i.e., hypoxia and nutrient privation). Statistical significance was determined by the Student unpaired t test or one-way ANOVA. TAE induces increased infiltration of Th17 cells in liver tumors when compared with controls 14d after treatment (0.29 ± 0.01 vs. 0.19 ± 0.02; p = 0.02). A similar pattern was observed in the spleen (1.41 ± 0.13 vs. 0.57 ± 0.08; p < 0.001), indicating both local and systemic effect. No significant differences in the percentage of FoxP3 + Tregs, IFNγ-producing CD4 T cells, and CD8 T cells were observed between groups (p > 0.05). In vitro post-embolization assays demonstrated that IL-17 reduces McA-RH7777 cell migration at 24-48h (p = 0.003 and p = 0.002, respectively). Transcatheter hepatic arterial bland embolization induces local and systemic increased infiltration of Th17 cells and expression of their signature cytokine IL-17. In a simulated post-embolization environment, IL-17 significantly reduced McA-RH7777 cell migration.

Assessment of the Antitumor Potentiality of Newly Designed Steroid Derivatives: Pre-Clinical Study.

Cancer is recognized as one of the most prevalent contributors to mortality in several nations and it remains one of the common health issues globally. In particular, hepatocellular carcinoma (HCC) has become a public health problem along with the increase of hepatitis B (HBV) and hepatitis C (HCV) virus infections. Based on this fact, our study goaled to synthesize newly hybrid drugs containing heterocyclic rings incorporated to steroid moiety and to examine the potential antitumor activity of the newly designed heterosteroid derivatives against HCC induced in animal model. Several heterocyclic steroids were synthesized 2-7 and confirmed via the analytical and spectral data (IR, 1H NMR13C NMR and Mass spectroscopy). Compounds 3, 4, and 5 were chosen to be investigated as anticancer agents in HCC rat model by means of validated biomarkers (alfa –fetoprotein, endoglin, lipocali-2 and heat shock protein-70). Following administration of compounds 3, 4 or 5, availability of the active tumor marker molecules was significantly dropped and a substantial decrease of the angiogenic and inflammatory mediators was also evident. These findings were supported by the histological examination of liver tissue. Taken together, this study indicates the potential anticancer activity of the newly synthesized heterosteroid derivatives against HCC in vivo. The antitumor activity of these compounds was likely attributable to modulating some signal transduction pathways involved in tumorigenesis, angiogenesis and inflammation.

Design And Characterisation Of Novel Sorafenib-Loaded Carbon Nanotubes With Distinct Tumour-Suppressive Activity In Hepatocellular Carcinoma.

PurposeOver the past 30 years, no consistent survival benefits have been recorded for anticancer agents of advanced hepatocellular carcinoma (HCC), except for the multikinase inhibitor sorafenib (Nexavar®), which clinically achieves only ~3 months overall survival benefit. This modest benefit is attributed to limited aqueous solubility, slow dissolution rate and, consequently, limited absorption from the gastrointestinal tract. Thus, novel formulation modalities are in demand to improve the bioavailability of the drug to attack HCC in a more efficient manner. In the current study, we aimed to design a novel sorafenib-loaded carbon nanotubes (CNTs) formula that is able to improve the therapeutic efficacy of carried cargo against HCC and subsequently investigate the antitumour activity of this formula.Materials and methodsSorafenib was loaded on functionalized CNTs through physical adsorption, and an alginate-based method was subsequently applied to microcapsulate the drug-loaded CNTs (CNTs-SFN). The therapeutic efficacy of the new formula was estimated and compared to that of conventional sorafenib, both in vitro (against HepG2 cells) and in vivo (in a DENA-induced HCC rat model).ResultsThe in vitro MTT anti-proliferative assay revealed that the drug-loaded CNTs formula was at least two-fold more cytotoxic towards HepG2 cells than was sorafenib itself. Moreover, the in vivo animal experiments proved that our innovative formula was superior to conventional sorafenib at all assessed end points. Circulating AFP-L3% was significantly decreased in the CNTs-SFN-MCs-treated group (14.0%) in comparison to that of the DENA (40.3%) and sorafenib (38.8%) groups. This superiority was further confirmed by Western blot analysis and immunofluorescence assessment of some HCC-relevant biomarkers.ConclusionOur results firmly suggest the distinctive cancer-suppressive nature of CNTs-SFN-MCs, both against HepG2 cells in vitro and in a DENA-induced HCC rat model in vivo, with a preferential superiority over conventional sorafenib.

Regulation of PKB/Akt-pathway in the chemopreventive effect of lactoferrin against diethylnitrosamine-induced hepatocarcinogenesis in rats.

Abnormal activation of protein kinase B (PKB) is associated with many cancers. This makes inhibition of PKB signaling pathway a promising strategy for cancer therapy. Lactoferrin (Lf) has been reported for its inhibition of tumor growth and metastasis, however, the mechanism is not completely understood. Its anti-hepatocarcinogenic activity has not taken the deserved recognition despite the additional advantages of Lf as an antiviral against hepatitis C virus, the main cause of hepatocellular carcinoma (HCC), and as a targeting ligand for delivering chemotherapeutics to hepatoma cells. This study evaluated the anti-hepatocarcinogenic effect of Lf, and the role of PKB in this effect using diethylnitrosamine (DENA)-induced HCC rat model, and a primary cell culture prepared from the induced hepatic lesions (DENA-HCC cell culture). Up-regulation of activated PKB in the hepatocytes of rats with DENA-induced HCC was observed, as measured biochemically in the liver homogenate, and localized immunohistochemically. This was accompanied by increment of hepatocytes proliferation, and expression of vascular endothelial growth factor and endothelial nitric oxide synthase. Involvement of PKB in DENA-induced HCC was confirmed by the observed decrease in cell proliferation in DENA-HCC cell culture that was treated with PKB inhibitor. In Lf-treated rats, a dose-dependent chemopreventive effect was observed, with decreased expression and activation of PKB, amelioration of the other DENA-induced alterations, and stimulation of apoptosis. In vitro, Lf blocked PKB activator-induced cell proliferation. These findings support the chemopreventive activity of Lf against HCC, and suggest regulation of PKB-pathway as a potential mechanism underlying this effect.

Zn(II)-curcumin solid dispersion impairs hepatocellular carcinoma growth and enhances chemotherapy by modulating gut microbiota-mediated zinc homeostasis

Zinc(II) complexes of curcumin display moderate cytotoxicity towards cancer cells at low micromolar concentrations. However, the clinical use of zinc(II) complexes is hampered by hydrolytic insolubility and poor bioavailability and their anticancer mechanisms remain unclear. Here, we investigated the efficacy and mechanism of action of a polyvinylpyrrolidone (PVP-k30)-based solid dispersion of Zn(II)-curcumin (ZnCM-SD) against hepatocellular carcinoma (HCC) in vitro and in vivo. In vitro assays revealed ZnCM-SD not only reduced the viability of HepG2 cells and SK-HEP1 cells in a dose-dependent manner, but also potently and synergistically enhanced cell growth inhibition and cell death in response to doxorubicin by regulating cellular zinc homeostasis. ZnCM-SD was internalized into the cells via non-specific endocytosis and degraded to release curcumin and Zn2+ ions within cells. The anticancer effects also occur in vivo in animals following the oral administration of ZnCM-SD, without significantly affecting the weight of the animals. Interestingly, ZnCM-SD did not reduce tumor growth or affect zinc homeostasis in HepG2-bearing mice after gut microbiome depletion. Moreover, administration of ZnCM-SD alone or in combination with doxorubicin significantly attenuated gut dysbiosis and zinc dyshomeostasis in a rat HCC model. Notably, fecal microbiota transplantation revealed the ability of ZnCM-SD to regulate zinc homeostasis and act as a chemosensitizer for doxorubicin were dependent on the gut microbiota. The crucial role of the gut microbiota in the chemosensitizing ability of ZnCM-SD was confirmed by broad-spectrum antibiotic treatment. Collectively, ZnCM-SD could represent a simple, well-tolerated, safe, effective therapy and function as a novel chemosensitizing agent for cancer.

Anticancer Effects of Gleditsia sinensis Extract in Rats Transplanted With Hepatocellular Carcinoma Cells.

The thorns of Gleditsia sinensis have been historically used in Chinese medicine and are considered one of the fundamental therapeutic herbs. Its anticancer effects are currently being explored. Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and still requires the development of new drugs with higher efficiency. By using a rat HCC model implanted with cancerous Walker-256 cells, the therapeutic effects of G. sinensis extract (GSE) were assessed, as well as its regulatory effects on miRNAs. GSE significantly restored liver morphology and dramatically induced cell apoptosis in HCC rats. In addition, miR-21/181b/183 was upregulated in the HCC liver, and the elevation of these miRNAs could be alleviated by both GSE and sorafenib. PTEN/TIMP3/PDCD4 downregulation was consistent with the targets of miR-21/181b/183 in the HCC liver, and the alteration of these target genes was restored by both GSE and sorafenib. TIMP3 effects on MMP-2/9 expression were also determined. Our present findings indicate the potential of GSE in HCC treatment, and expand the understanding of miRNA-related mechanisms in the anticancer effects of GSE.

Folic acid conjugated polymeric drug delivery vehicle for targeted cancer detection in hepatocellular carcinoma.

Targeted therapies provide increased efficiency for the detection and treatment of cancer with reduced side effects. Folate receptor (alpha subunit) is overexpressed in multiple tumors including liver cancer. In this study, we evaluated the specificity and toxicity of a folic acid-containing drug delivery vehicle (DDV) in a hepatocellular carcinoma (HCC) model. The DDV was prepared with two units each of folic acid (FA) and fluorescein isothiocyanate (FITC) molecules and conjugated to a central poly (ethylene glycol) (PEG) core via a modified chemo-enzymatic synthetic process. Rat hepatoma (N1S1) and human monocytic (U937) cell lines were used for cell culture-based assays and tested for DDV uptake and toxicity. Folate receptor expressions in liver tissues and cell lines were verified using standard immunohistochemistry techniques. Rat HCC model was used for in vivo assessment. The DDV was injected via intra-arterial or intravenous methods and imaged with IVIS spectrum in vivo imaging system. Strong signals of FITC in the liver tumor region correlated to targeted DDV uptake. The use of PEG enhanced water-solubility and provided flexibility for the interaction of FA ligands with multiple cell surface folate receptors that resulted in increased specific uptake. Our study suggested that PEG incorporation and folate targeting via intra-arterial approach is an efficient strategy for targeted delivery in HCC therapy.

Abstract 3717: Animal model of cirrhosis with hepatocellular carcinoma: A reliable tool for testing new therapies

Abstract Background and aim: Liver cancer is now the second most common cause of cancer related death worldwide, with hepatocellular carcinoma (HCC) accounting for the majority of these cases . 90% of HCC are associated with liver fibrosis or cirrhosis developed from chronic liver injuries.Small animal models represent essential tools in cancer research. As fibrosis/cirrhosis modifies liver vascularization, extracellular matrix composition, and drugs metabolism, it is essential to use a cirrhotic animal model to test HCC drugs for their efficiency against tumour initiation and/or progression. Current mouse model failed to reproduce all fibrosis stages, especially cirrhosis. One of the rodent models that most faithfully reproduce human cirrhosis is diethyl nitrosamine-injured rats (DEN rats). The aim of our project is to deeply characterize DEN-induced HCC rat model during cirrhosis progression and HCC development with special focus on liver inflammatory micro-environment. Method: 6-weeks-old Fischer 344 male rats were treated weekly with intra-peritoneal injections of 50 mg/kg DEN. 9 rats are sacrificed before starting DEN-injections at 0 week (control group), after 8 weeks of injections (8 weeks), after 14 weeks of injections (14 weeks) and at 20 weeks after start of DEN-injections (20 weeks). Histopathological analysis, immunohistochemical and FACS analysis were performed. Results were analysed in a double blind manner. Results: Chronic DEN treatment induces tumour development that starts at 14 weeks. Tumour size significantly increases between 14 and 20 weeks but not tumour number. DEN injection was associated with a significant increase of hepatocyte proliferation at 8 weeks, 14 weeks, and 20 weeks when compared to 0 weeks. Similarly, CD34 positive cells were increased, suggesting enhanced angiogenesis at 14 &amp; 20 weeks when compared to 0 weeks. DEN-induced liver fibrosis was significantly and gradually enhanced as suggested by upregulation of fibrosis deposition at 8 weeks, 14 weeks, 20 weeks compared to 0 weeks. We also observed significant decrease in CD4+ and increase in CD8+ T lymphocytes in the blood and non-tumour liver tissue at each time points. In parallel, CD8+ T lymphocytes were significantly decreasing in hepatic tumour tissue. CD152 expression was significantly increasing in blood samples and non- tumour liver tissues at 8, 14 and 20 weeks compared to 0 weeks. Conclusion: DEN-induced HCC rat model displays tumour initiation, development and different stages of liver fibrosis, up to cirrhosis and also helps to understand related modulation of immune micro environment. Indeed we demonstrated CTLA-4 expression is upregulated in this model. In this context, DEN-induced cirrhotic HCC rat model might be a relevant tool as pre-clinical models to evaluate new HCC treatment efficacy and tolerance in liver cirrhotic background. Citation Format: Keerthi Kurma, Zuzana Macek Jilkova, Patrice N. Marche, Nathalie Sturm, Hervé Lerat, Thomas Decaens. Animal model of cirrhosis with hepatocellular carcinoma: A reliable tool for testing new therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3717.

Histological Study of Emerging Role of Exosomes Based Therapeutic in Management of Hepatocellular Carcinoma Rat Model

Histological Study of Emerging Role of Exosomes Based Therapeutic in Management of Hepatocellular Carcinoma Rat Model

Histological Study of Emerging Role of Exosomes Based Therapeutic in Management of Hepatocellular Carcinoma Rat Model

Background: hepatocellular carcinoma (HCC) is the most common type of primary liver cancers with high incidence and mortality rates. The major risk factor for HCC is cirrhosis, which occurs usually due to HBV and HCV infection. Exosomes thought that have a major role on the promotion and spread of HCC, which can be uased as therapeutic and diagnostic marker. Objectives: we aimed to use evaluate their role in HCC assessment and their use as potential selected diagnostic biomarkers, and to characterize the efficacy of Hesperidin on modulating exosomal production and exosomal non-coding RNA related HCC expression using animal models. Materials and Methods: the study included 30 Rats devised to 5 groups 6 on each, controle and DiethyleNitros amine (DEN) and Acetamidofluorene (2-AAF) to iduce HCC group and Hispiridine treated groups with three different dosos 50 mg, 100 mg and 200 mg respectively for three groups. infants with neonatal cholestasis in three groups. Results: improvement of all of them, regarding liver histopathology wich the HCC focus regressed , regarding exosomal abundance in the serumand tissues while the evaluation of exosomal RAB11A-mRNA in rats' samples was performed by real time PCR, then the results were statistically analyzed by the SPSS software. Finally regarding to liver function test which the ALT, bilirubin and AFP reduced in the treated groups. Conclusion: exosomal RAB11A-mRNA as biomarkers for HCC inducedrats and target these exosomal markers using Hesperidin in animal model and hence we can uses as thrapuetic bases in HCC in the future.

Abstract 3856: MTLCEBPA, a drug candidate for hepatocellular-carcinoma enhances efficacy of Sorafenib

Abstract MTLCEBPA, a non-toxic therapeutic oligonucleotide formulated inside SMARTICLES (liposomal nanoparticles) is currently under a Phase I study for patients with advanced hepatocellular carcinoma (HCC). We have demonstrated in multiple in vivo liver disease models that MTLCEBPA can improve liver function and inhibit HCC tumor growth. Here we show that upregulation of hepatic CEBPA expression by MTLCEBPA enhances the effects of Sorafenib in a (Diethylnitrosamine) DEN induced cirrhotic HCC rat model by significantly reducing liver tumor volume after only two weeks of combination treatment when compared to untreated control or single agent treatment only. In this study, we established cirrhosis and HCC by exposing male Wistar rats to DEN for 6 weeks. Animals were randomized into 5 groups comprising of: 1. PBS control; 2. MTL-CEBPA treatment (TIW at 3mg/kg) for one week; 3. MTL-CEBPA treatment (TIW at 3mg/kg) for two weeks; 4. Sorafenib TIW at 10mg/kg for two weeks; and 5. MTLCEBPA at 3mg/kg at week one combined with Sorafenib 10mg/kg at week 2 group. The results here indicate that Sorafenib combined with MTLCEBPA significantly inhibits tumor growth when compared to single agent treatment. Tumor sizes averaged at 644.7mm3 (± 88.57) in group 1 compared to 326mm3 (± 53.05) in group 2; 199.7mm3 (± 54.73) in group 3 and 299.5mm3 (± 77.1) in group 4. In combination treatment (group 5) the tumor sizes averaged at 101.3mm3 (± 37.48). Serum AFP change (posttreatment values - pre-treatment) was more prominent in the MTLCEBPA/Sorafenib combination treatment (group 5) where there was a 4-fold reduction in values after treatment when compared to control groups 1-4. Serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) and total bilirubin remained unchanged across all the treatment groups suggesting no deterioration in liver function during the study period. These results suggest that MTLCEBPA improved the efficacy of Sorafenib in this DEN induced HCC model, indicating a promising therapeutic option for advanced HCC patients who can no longer be treated with potentially more effective local therapies. Citation Format: Nagy A. Habib, Kai-Wen Huang, Vikash Reebye, Mikael Sodergren, John Rossi. MTLCEBPA, a drug candidate for hepatocellular-carcinoma enhances efficacy of Sorafenib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3856.

Lipopolysaccharide induces the differentiation of hepatic progenitor cells into myofibroblasts constitutes the hepatocarcinogenesis-associated microenvironment.

Hepatocellular carcinoma (HCC) generally occurs in the presence of chronic liver injury, often as a sequela of liver fibrosis. Hepatic progenitor cells (HPCs) are known to be capable of forming both hepatocytes and cholangiocytes in chronic liver injury, which are also considered a source of myofibroblasts and tumor-initiating cells, under carcinogenic circumstances. However, the underlying mechanisms that activate HPCs to give rise to HCC are still unclear. In current study, the correlation between HPCs activation and liver fibrosis and carcinogenesis was investigated in rats and human specimens. We analyzed the role of HPCs in tumorigenesis, by transplanting exogenous HPCs in a diethylnitrosamine-induced rat HCC model. Our data indicated that HPC activation correlated with hepatic fibrosis and hepatocarcinogenesis. We further found that exogenous HPC infusion promoted liver fibrosis and hepatocarcinogenesis, while lipopolysaccharides (LPS) played an important role in this process. However, results of our study indicated that LPS did not induce HPCs to form tumor in nude mice directly. Rather, LPS induced myofibroblast-like morphology in HPCs, which enhanced the tumorigenic potential of HPCs. Further experiments showed that LPS/Toll-like receptor 4 (TLR4) signaling mediated the differentiation of HPCs into myofibroblasts and enhanced the production of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), which led to the aberrant expression of Ras and p53 signaling pathways in HPCs, and finally, promoted the proliferation and malignant transformation of HPCs, by long non-coding RNA regulation. Besides, examination of HCC clinical samples demonstrated that IL-6 and TNF-α production correlated with HPC activation, hepatic fibrosis, and HCC recurrence. Our study indicates that both myofibroblasts and tumor cells are derived from HPCs. HPC-derived myofibroblasts create tumor microenvironment and contribute to the proliferation and malignant transformation of HPCs. Furthermore, LPS present in the chronic liver inflammation microenvironment might play an important role in hepatocarcinogenesis, by regulating the plastic potential of HPCs.

Melatonin maximizes the therapeutic potential of non-preconditioned MSCs in a DEN-induced rat model of HCC

Pretreatment of mesenchymal stem cells (MSCs) with melatonin (Mel) improves their potential therapeutic effect on chronic diseases and cancers. However, this preconditioning strategy may direct the effect of Mel toward MSCs alone and deprive cancer cells of the oncostatic effect of Mel. Herein, we hypothesized that Mel given before transplantation of non-preconditioned MSCs may maximize the therapeutic outcome via the oncostatic effect of Mel by preparing a suitable tumor microenvironment for MSCs. Female rats (n = 60) were equally divided into 6 groups; normal control, diethylnitrosamine (DEN), DEN + Mel, DEN + MSCs, DEN + MSCs preconditioned with Mel, and DEN + MSCs + Mel. The obtained data revealed that administration of Mel before MSCs treatment without preconditioning yielded a better ameliorative effect against DEN-induced hepatocellular carcinoma (HCC) as evidenced by: 1) reduced serum levels of alpha fetoprotein and gamma-glutamyl transferase; 2) decreased number and area of glutathione S-transferase placental positive foci; 3) induced apoptosis (as indicated by increased cleaved caspase-3 activity, upregulated expression of proapoptotic genes Bax and caspase 3 and downregulated expression of anti-apoptotic genes Bcl2, survivin); 4) decreased malondialdehyde level and increased activities of superoxide dismutase, catalase, and glutathione peroxidase enzymes; and 5) reduced inflammation, angiogenesis and metastasis as indicated by downregulated expression of interleukin 1 beta, nuclear factor kappa B, vascular endothelial growth factor, and matrix metallopeptidase 9 genes and upregulated expression of metalloproteinase inhibitor 1 gene. Thus, administration of Mel before MSCs (without preconditioning) fostered the survival and therapeutic potential of MSCs in HCC, possibly through induction of apoptosis and inhibition of inflammation and oxidative stress. This new strategy showed better therapeutic outcomes and may improve MSC-based therapies for HCC.

Abstract No. 498 Modulating tumoral perfusion using antivascular ultrasound (AVUS) in a rat model of hepatocellular carcinoma

This study evaluates changes in tumor perfusion following low- and high-dose antivascular ultrasound (AVUS) therapy in a translational rat model of hepatocellular carcinoma (HCC). HCC was induced in 35 Wistar rats via ingested diethylnitrosamine (DEN). Rats received AVUS treatments at low, high, and sham doses. Low-dose (n=8): 1 W/cm2 ultrasound (US), 3 min, 0.2 mL perflutren lipid microbubbles (uB) IV. High-dose (n=21): 2 W/cm2, 6 min, 0.7 mL uB IV. Sham: no US, 0.7 mL normal saline IV. Perfusion was assessed by contrast-enhanced ultrasound imaging with nonlinear contrast (NLC) and power Doppler (PD) modes pre and post therapy. Delta projection (DP) of NLC created a maximum intensity projection within tumor vessels. From each mode, peak enhancement (PE) and perfusion index (PI) were measured. H&E of tumor sections was compared with perfusion metrics. Perfusion measures decreased following high-dose AVUS therapy and increased following low-dose AVUS. NLC was most sensitive to these changes, with mean peak enhancement increasing from 2.5 to 4 (p= 0.03) in low-dose and decreasing from 6.2 to 3.2 (p= 0.001) in high-dose therapy. NLC maximal intensity projection (delta projection) and power Doppler showed a similar trend without reaching significance level in all parameters. Histologic analysis showed 5.0% (2.6) area necrosis in low-dose treated tumors and 25.0% (7.1) necrosis in the high-dose group (p = 0.028). High-dose AVUS decreased tumoral perfusion and caused increased necrosis, while low-dose AVUS increased perfusion. Nonlinear contrast-enhanced ultrasound proved the most sensitive method of detecting perfusional changes due to therapy.

Abstract No. 620 Which rat hepatocellular carcinoma model is the best in translational researches of interventional oncology?

Abstract No. 620 Which rat hepatocellular carcinoma model is the best in translational researches of interventional oncology?

Brivanib in combination with Notch3 silencing shows potent activity in tumour models

BackgroundSorafenib is the first targeted agent proven to improve survival of patients with advanced hepatocellular carcinoma (HCC) and it has been used in first line treatments with heterogeneous response across patients. Most of the promising agents evaluated in first-line or second-line phase III trials for HCC failed to improve patient survival. The absence of molecular characterisation, including the identification of pathways driving resistance might be responsible for these disappointing results.Methods2D DIGE and MS analyses were used to reveal proteomic signatures resulting from Notch3 inhibition in HepG2 cells, combined with brivanib treatment. The therapeutic potential of Notch3 inhibition combined with brivanib treatment was also demonstrated in a rat model of HCC and in cell lines derived from different human cancers.ResultsUsing a proteomic approach, we have shown that Notch3 is strongly involved in brivanib resistance through a p53-dependent regulation of enzymes of the tricarboxylic acid (TCA), both in vitro and in vivo.ConclusionWe have demonstrated that regulation of the TCA cycle is a common mechanism in different human cancers, suggesting that Notch3 inhibitors combined with brivanib treatment may represent a strong formulation for the treatment of HCC as well as Notch3-driven cancers.

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.

Preclinical feasibility study of combination therapy of sorafenib and theragnostic ultrasound contrast microbubble for rat model of hepatocellular carcinoma

Preclinical feasibility study of combination therapy of sorafenib and theragnostic ultrasound contrast microbubble for rat model of hepatocellular carcinoma

Antiproliferative and antiangiogenic effects of zerumbone from Zingiber zerumbet L. Smith in sprague dawley rat model of hepatocellular carcinoma

Context: Zerumbone (ZER) is known to exhibit anticancer properties on various cancer cells both in vitro and in vivo. However, the anti-angiogenesis effect of ZER on liver cancers in vivo is not yet addressed clearly. Aims: This study aimed to investigate the in vivo antiproliferative and antiangiogenesis effects of ZER using rats with diethylnitrosamine-induced hepatocellular carcinoma (HCC). Materials and Methods: The antiproliferative and anti-angiogenesis effects of ZER were determined using the hepatosomatic index, vascular endothelial growth factor (VEGF) immunoassay, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, histopathology, and immunohistochemistry analysis. Results: Nontreated rats with HCC had higher median liver weight than those treated with ZER or suramin. The expression of VEGF, matrix metalloprotease, and Ki-67 that were high in nontreated HCC rats was down-regulated with ZER or suramin treatments. Statistical Analysis Used: Statistical analyses were performed using the Statistical Package for Social Science version 21.0 (SPSS Inc, IBM, Maryland, USA). The data were expressed as the mean ± standard deviation and analyzed using a one-way analysis of variance. P Abbreviations used: ZER: Zerumbone; HCC: Hepatocellular carcinoma; MMP-9: Matrix metalloproteinase-9; VEGF: Vascular endothelial growth factor; DEN: Diethylnitrosamine; PBS: Phosphate-buffered saline; ELISA: Enzyme-Linked Immunosorbent Assay; TUNEL: Terminal deoxynucleotidyl transferase dUTP nick end labeling; FFPE: Formalin-fixed and paraffin-embedded; H and E: Hematoxylin and Eosin; DAB: Dako Envision®+Dual Link System; HRP: Horseradish peroxidase; H2O2: Hydrogen peroxide; PBST: PBS in Tween 20; SPSS: Statistical Package for Social Science; RUGS: Research University Grant Scheme. Correspondence: