Rat Model Of Hepatocellular Carcinoma Research Articles

Doxorubicin-loaded poly(lactic-co-glycolic acid) microspheres prepared using the solid-in-oil-in-water method for the transarterial chemoembolization of a liver tumor

Doxorubicin (DOX)-loaded poly(lactic-co-glycolic acid) (PLGA) microspheres (MSs) were fabricated using the solid-in-oil-in-water (S/O/W) emulsification method for transarterial chemoembolization (TACE) of a liver tumor. DOX-loaded PLGA MSs with a mean diameter of 26μm and a spherical shape were prepared. The biodegradation of PLGA MSs was observed in serum using a scanning electron microscope (SEM). Drug release from the PLGA MSs was accelerated at an acidic pH (pH 5.5) compared to a normal physiological pH (pH 7.4). According to the results of a pharmacokinetic study in rats, the area under the curve (AUC) value of a drug, which indicates the systemic exposure extent of the drug, of the PLGA MSs group was 29.9% of that of a hepatic arterial injection (HAI) group. The DOX concentration ratio for liver tumors compared to normal livers was significantly higher in the PLGA MSs group than that of the HAI group (p<0.05). After the TACE procedure was performed with DOX-PLGA MSs in a rat hepatoma model, the mean size increment of tumor in DOX-PLGA MSs group was found to be lower than that of the HAI group, and the viable portion of the DOX-PLGA MSs group was less than the other groups (p<0.05). All these findings suggested that the developed DOX-loaded PLGA MSs fabricated with the S/O/W method can be used as a promising drug delivery system in TACE for liver tumors.

Cell-type specific functions of epidermal growth factor receptor are involved in development of hepatocellular carcinoma.

Cell-type specific functions of epidermal growth factor receptor are involved in development of hepatocellular carcinoma.

Segmental Transarterial Embolization in a Translational Rat Model of Hepatocellular Carcinoma

PurposeTo develop a clinically relevant, minimally invasive technique for transarterial embolization in a translational rat model of hepatocellular carcinoma (HCC). Materials and MethodsOral diethylnitrosamine was administered to 53 male Wistar rats ad libitum for 12 weeks. Tumor induction was monitored using magnetic resonance imaging. Minimally invasive lobar or segmental transarterial embolization was performed through a left common carotid artery approach. Necropsy was performed to evaluate periprocedural mortality. Histologic analysis of tumors that received embolization was performed to assess percent tumor necrosis. ResultsSevere cirrhosis and autochthonous HCCs were characterized in a cohort of rats composed of two groups of rats identically treated with diethylnitrosamine with median survival times of 101 days and 105 days (n = 10/group). A second cohort was used to develop minimally invasive transarterial embolization of HCCs (n = 10). In a third cohort, lobar embolization was successfully performed in 9 of 10 rats and demonstrated a high rate of periprocedural mortality (n = 5). Necropsy performed for periprocedural mortality after lobar embolization demonstrated extensive tissue necrosis within the liver (n = 3) and lungs (n = 2), indicating nontarget embolization as the likely cause of mortality. In a fourth cohort of rats, a segmental embolization technique was successfully applied in 10 of 13 rats. Segmental embolization resulted in a reduction in periprocedural mortality (P = .06) relative to selective embolization and a 19% increase in average tumor necrosis (P = .04). ConclusionsMinimally invasive, segmental embolization mimicking the currently applied clinical approach is feasible in a translational rat model of HCC and offers the critical advantage of reduced nontarget embolization relative to lobar embolization.

PET Imaging of Oncolytic VSV Expressing the Mutant HSV-1 Thymidine Kinase Transgene in a Preclinical HCC Rat Model

Hepatocellular carcinoma (HCC) is the most predominant form of liver cancer and the third leading cause of cancer-related death worldwide. Due to the relative ineffectiveness of conventional HCC therapies, oncolytic viruses have emerged as novel alternative treatment agents. Our previous studies have demonstrated significant prolongation of survival in advanced HCC in rats after oncolytic vesicular stomatitis virus (VSV) treatment. In this study, we aimed to establish a reporter system to reliably and sensitively image VSV in a clinically relevant model of HCC for clinical translation. To this end, an orthotopic, unifocal HCC model in immune-competent Buffalo rats was employed to test a recombinant VSV vector encoding for an enhanced version of the herpes simplex virus 1 (HSV-1) thymidine kinase (sr39tk) reporter, which would allow the indirect detection of VSV via positron emission tomography (PET). The resulting data revealed specific tracer uptake in VSV-HSV1-sr39tk-treated tumors. Further characterization of the VSV-HSV1-sr39tk vector demonstrated its optimal detection time-point after application and its detection limit via PET. In conclusion, oncolytic VSV expressing the HSV1-sr39tk reporter gene allows for highly sensitive in vivo imaging via PET. Therefore, this imaging system may be directly translatable and beneficial in further clinical applications.

Synergistic curative effect of chicory extract and cisplatin against thioacetamide-induced hepatocellular carcinoma

Aim: Hepatocellular carcinoma (HCC) is the dominant form of primary liver cancer and is histologically and etiologically distinct from other forms of primary liver cancer. The objective of this study was to elucidate the synergistic effect and the role of chicory extract [inulin (IN)] as a chemo-sensitizer for cisplatin (CIS) treatment of HCC. Methods: Five groups of rats were treated for 4 months. These groups consisted of the control group, a group receiving thioacetamide (TAA) (200 mg/kg b.w) in drinking water, a group injected intraperitoneally with a single dose of CIS (7.5 mg/kg b.w) in addition to TAA for 4 months, a group receiving oral doses of IN (10 mg/kg b.w) in addition to TAA for 4 months, and a group injected intraperitoneally with a single dose of CIS (7.5 mg/kg b.w) and IN (10 mg/kg b.w) plus TAA for 4 months. Results: The current data exhibited increment of serum and liver enzyme (alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, creatine kinase, and lactate dehydrogenase) activity, serum lipid profile levels (total lipids, total cholesterol, triglycerides, low-density lipoprotein, and very low-density lipoprotein), and a significant increase in β-fetoprotein and bilirubin, accompanied with reduced total serum protein and albumin levels in a HCC rat model. Histopathologically, numerous alterations were detected in hepatic tissues of HCC rats, such as lymphocytic cell infiltration, damage of hepatocytes, dilated congested central vein with degenerated endothelial cells, and congested blood sinusoids in addition to Masson's trichrome staining blue collagen fibers in hepatocytes and central vein indicating hepatic fibrosis. Treatment of HCC rats with CIS or IN improved such deleterious effects, where IN is more effective than CIS, and the best effect can be observed in rats that received both CIS and IN. Conclusion: It could be concluded that IN in chicory extract acts as a chemo-sensitizer to CIS for treatment in an HCC rat model.

Characterization of Magnetic Viral Complexes for Targeted Delivery in Oncology

Oncolytic viruses are promising new agents in cancer therapy. Success of tumor lysis is often hampered by low intra-tumoral titers due to a strong anti-viral host immune response and insufficient tumor targeting. Previous work on the co-assembly of oncolytic virus particles (VPs) with magnetic nanoparticles (MNPs) was shown to provide shielding from inactivating immune response and improve targeting by external field gradients. In addition, MNPs are detected by magnet resonance imaging (MRI) enabling non-invasive therapy monitoring.In this study two selected core-shell type iron oxide MNPs were assembled with adenovirus (Ad) or vesicular stomatitis virus (VSV). The selected MNPs were characterized by high r2 and r2* relaxivities and thus could be quantified non-invasively by 1.5 and 3.0 tesla MRI with a detection limit below 0.001 mM iron in tissue-mimicking phantoms. Assembly and cell internalization of MNP-VP complexes resulted in 81 - 97 % reduction of r2 and 35 - 82 % increase of r2* compared to free MNPs. The relaxivity changes could be attributed to the clusterization of particles and complexes shown by transmission electron microscopy (TEM). In a proof-of-principle study the non-invasive detection of MNP-VPs by MRI was shown in vivo in an orthotopic rat hepatocellular carcinoma model.In conclusion, MNP assembly and compartmentalization have a major impact on relaxivities, therefore calibration measurements are required for the correct quantification in biodistribution studies. Furthermore, our study provides first evidence of the in vivo applicability of selected MNP-VPs in cancer therapy.

Curcumin ameliorate DENA-induced HCC via modulating TGF-β, AKT, and caspase-3 expression in experimental rat model.

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. In laboratory animal models, diethylnitrosamine (DENA) is a well-known agent that has a potent hepatocarcinogenic effect that is used to induce HCC. As curcumin has a potent anti-inflammatory effect with strong therapeutic potential against a variety of cancers, our present study aims to investigate its curative effects and the possible mechanisms of action against DENA-induced HCC in male rats. Investigation of biochemical and molecular parameters of HCC animal model liver showed an overexpression of TGF-β and Akt proteins accompanied with a significant reduction of the proapoptotic marker caspase-3. DENA-induced hepatic cellular injury resulted also in a significant increase in liver function marker enzymes aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lipid peroxides in this group. Curcumin treatment partially reversed DENA-induced damage as it reduced the overexpression of the angiogenic and anti-apoptotic factors TGF-β and Akt and improved caspase-3 expression. Also, it could partially normalize the serum values of liver marker enzymes and lipid peroxidation and improve liver architecture. Curcumin shows a unique chemotherapeutic effect in reversing DENA-induced HCC in rat model. This effect is possibly mediated through its proapoptotic, antioxidant, anti-angiogenic, as well as antimitotic effects. It interferes and modulates cell signaling pathways and hence turns death signals and apoptosis on within tumor cells.

Curcumin: a unique antioxidant offers a multimechanistic approach for management of hepatocellular carcinoma in rat model.

This study was designed to investigate the role of curcumin against hepatocellular carcinoma (HCC) induced in rats. Forty rats were divided into five groups. Group (1) was negative control. Groups (2), (4), and (5) were orally administrated N-nitrosodiethylamine for HCC induction, then group (2) was left untreated, and group (4) was treated orally with curcumin, while group (5) was intraperitoneally injected with doxorubicin. Group (3) was served as curcumin control group. Serum alpha-fetoprotein, alpha L-fucosidase and vascular endothelial growth factor levels were analyzed. Gamma glutamyl transferase (GGT) and heat shock protein gp96 (HSPgp96) gene expressions were detected by RT-PCR. The immunohistochemical analysis of proliferating cell nuclear antigen (PCNA) and Ki-67 expressions was performed. Apoptosis was detected using DNA fragmentation assay. Also, histological investigation of liver tissue was achieved. Untreated HCC group showed significant elevation in the studied biochemical markers and significant upregulation in GGT and HSPgp96 gene expression as well as marked increase in PCNA and Ki-67 expression. Furthermore, this group revealed no DNA fragmentation. Histological investigation of liver tissue sections in HCC group revealed a typical anaplasia. On the other hand, the curcumin-treated group showed a significant depletion in the studied tumor markers and a significant downregulation in GGT and HSPgp96 gene expression. Also, this group displayed remarkable decrease in PCNA and Ki-67 expression. Moreover, this group revealed an obvious DNA fragmentation. Interestingly, treatment with curcumin showed remarkable improvement in the histological features of liver tissue. This study revealed the promising therapeutic role of curcumin against hepatocellular carcinoma owing to its antiangiogenic, antiproliferative, and apoptotic effects.

Roles of transforming growth factor-β1 during bone mesenchymal stem cells transplantation for rat hepatic fibrogenesis and hepatoma model

Objective To investigate the effects of bone mesenehymal stem cells (BMSCs) transplantation on rat hepatic fibrogenesis/hepatoma via transforming growth factor-β1 (TGF-β1).Methods The hepatic fibrosis model and hepatoma model were induced by carbon tetrachloride (CCl4) and diethylnitrosamine respectively in rats (n =16 each).The BMSCs of the forth generation were respectively injected into the rats via the caudalis vena.Effects of TGF-β1 on the BMSCs transplantation on hepatic fibrogenesis and hepatoma in rats were investigated.Results In the hepatic fibrosis model,the mean value of Scheuer inflammation score,Schmid M fibrosis score and the activation score of HSC showed significant difference between control group and intervention group,with 3.58 and 0.42 (u =-4.22,P ＜ 0.01),5.17 and 1.58 (u =-3.65,P ＜0.01),2.25 and 0.58 (u =-2.93,P ＜0.01),respectively.There was also significant difference in the contents of TGF-β1 between two groups (F =11.024,P ＜ 0.05).In the hepatoma model,the cell cycle was restrained in intervention group and most cells were arrested in G1 phase,and in control group,most cells were arrested in S phase (G1 phase P ＜ 0.01,G2 phase P ＜ 0.01,S phase P ＜0.01).However,there was no significant difference in the incidence of liver cancer between two groups (P ＞ 0.05).Further more,the mean value of TGF-β1 showed no significant difference between two groups (P ＞ 0.05).Conclusion BMSCs transplantations can reduce hepatic stellate cells activation and thus slow down the process of liver fibrosis probably by inhibiting the secretion of TGF-β1.BMSCs can inhibit the cell cycle not through the TGF-β1 pathway. Key words: Bone mesenchymal stem cells; Hepatic fibrogenesis; Transforming growth factor-β1

Ginsenoside Rg3 attenuates hepatoma VEGF overexpression after hepatic artery embolization in an orthotopic transplantation hepatocellular carcinoma rat model.

BackgroundHypoxia-induced vascular endothelial growth factor (VEGF) upregulation and angiogenesis following treatment of hepatocellular carcinoma (HCC) with transarterial embolization (TAE) or transarterial chemoembolization (TACE) may be mediated by ginsenoside Rg3, an anti-angiogenic saponin extracted from ginseng.ObjectiveTo access the synergistic action of Rg3 and TAE treatment on HCC by VEGF and it’s receptor expressions decreasing in a rat model of HCC.MethodsAn orthotopic transplantation HCC model was established in Buffalo rats. HCC rats were treated with hepatic artery infusions of normal saline or iodized oil (0.1 mL) with or without Rg3 (1 mg/kg) (each n=15 in control, Rg3, TAE, and TAE + Rg3 groups). At 1, 2, 4, and 8 weeks, performance status (body weight), tumor progression (longest tumor diameter), metastasis rate, microvessel density (MVD), and overall survival rate were assessed. Additionally, cluster of differentiation 31 (CD31), VEGF, VEGF receptor 2 (VEGF-R2) and VEGF-R2 phosphorylation levels were assessed by immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), and Western blot.ResultsCombined Rg3 and TAE treatment reduced tumor progression, body weight loss, angiogenesis, and metastasis rate, and led to better overall survival in the HCC rat model. ELISA results showing VEGF expression in the control, Rg3, TAE, and TAE + Rg3 groups at 4 weeks following treatment were 132.6±2.38, 37.9±0.8, 87.4±0.7, and 45.3±0.4 pg/mL, respectively. Combined Rg3 and TAE reduced the protein expression of CD31 and VEGF-R2 phosphorylation, compared with those in the TAE group at 4 weeks of treatment.ConclusionCombined Rg3 and TAE treatment limited metastasis and promoted survival by downregulating VEGF overexpression in HCC tumors. Thus, this treatment may have potential clinical implications for HCC patients undergoing TAE or TACE.

Preparation and therapeutic evaluation of (188)Re-thermogelling emulsion in rat model of hepatocellular carcinoma.

Radiolabeled Lipiodol® (Guerbet, Villepinte, France) is routinely used in hepatoma therapy. The temperature-sensitive hydrogel polyethylene glycol-b-poly-DL-lactic acid-co-glycolic acid-b-polyethylene glycol triblock copolymer is used as an embolic agent and sustained drug release system. This study attempted to combine the polyethylene glycol-b-poly-DL-lactic acid-co-glycolic acid-b-polyethylene glycol hydrogel and radio-labeled Lipiodol to form a new radio-thermogelling emulsion, rhenium-188–N,N’-1,2-ethanediylbis-L-cysteine diethyl-ester dihydrochloride–Lipiodol/hydrogel (188Re-ELH). The therapeutic potential of 188Re-ELH was evaluated in a rodent hepatoma model. Rhenium-188 chelated with N,N’-1,2-ethanediylbis-L-cysteine diethyl-ester dihydrochloride was extracted with Lipiodol to obtain rhenium-188–N,N’-1,2-ethanediylbis-L-cysteine diethyl-ester dihydrochloride–Lipiodol (188Re-EL), which was blended with the hydrogel in equal volumes to develop 188Re-ELH. The 188Re-ELH phase stability was evaluated at different temperatures. Biodistribution patterns and micro-single-photon emission computed tomography/computed tomography images in Sprague Dawley rats implanted with the rat hepatoma cell line N1-S1 were observed after in situ tumoral injection of ~3.7 MBq 188Re-ELH. The therapeutic potential of 188Re-EL (48.58±3.86 MBq/0.1 mL, n=12) was evaluated in a 2-month survival study using the same animal model. The therapeutic effects of 188Re-ELH (25.52±4.64 MBq/0.1 mL, n=12) were evaluated and compared with those of 188Re-EL. The responses were assessed by changes in tumor size and survival rates. The 188Re-ELH emulsion was stable in the gel form at 25°C–35°C for >52 hours. Biodistribution data and micro-single-photon emission computed tomography/computed tomography images of the 188Re-ELH group indicated that most activity was selectively observed in hepatomas. Long-term 188Re-ELH studies have demonstrated protracted reductions in tumor volumes and positive effects on the survival rates (75%) of N1-S1 hepatoma-bearing rats. Conversely, the 2-month survival rate was 13% in the control sham group. Therapeutic responses differed significantly between the two groups (P<0.005). Thus, the hydrogel enhanced the injection stability of 188Re-EL in an animal hepatoma model. Given the synergistic results, direct 188Re-ELH intratumoral injection is a potential therapeutic alternative for hepatoma treatment.

Establishment of animal models with orthotopic hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most serious health problems worldwide. Many researchers have investigated HCC at the level of genes, ribonucleic acid, proteins, cells, and animals. The resultant development of animal models and monitoring methods has improved the effectiveness of guidelines provided to researchers working with preclinical HCC models. HCC in animal models and clinical patients is monitored by various current imaging modalities such as ultrasound (US) imaging, computed tomography (CT), magnetic resonance imaging (MRI), single photon emission computed tomography (SPECT), positron emission tomography (PET) and bioluminescence imaging (BLI). These techniques are currently used for both preclinical and clinical assessment, and provide valuable diagnostic information. In this article, we have mainly reviewed the established animal models and the assessment of orthotopic HCC using imaging modalities. Additionally, we have introduced a method of orthotopic HCC rat model developed in our laboratory. We have furthermore evaluated the occurrence of tumor mass using molecular imaging techniques.

Therapeutic effects of mesenchymal stem cells on hepatocellular carcinma: tracking of cells using iron oxide nanoparticles (87.3)

Recently, a significant increase in the incidence of hepatocellular carcinoma (HCC) has been reported. However, early detection of the disease can help in selecting from various available therapies. Unfortunately, in advanced liver cancer cases, treatment options are very limited. In the present study, we point to the need to identify a new effective, less aggressive treatment approach. Advances in stem cell research, led us to consider cell‐based therapy for treating liver cancer.It was previously reported that bone marrow derived mesenchymal stem cells (MSCs) have the tumor suppressive effects in an experimental HCC model in rats. In this work, we investigated the possible role of Wnt signaling in hepatic carcinogenesis and how it is influenced by by MSCs labeled with iron oxide nanoparticles.Forty rats were used and were divided equally into four groups: a normal control group and 3 groups that received diethylnitroseamine and CCl4 to induce HCC. Then after induction, one group was treated with MSCs only, the second group with PBS (vehicle) only, and the third group with labeled MSCs with iron‐oxide nanoparticles. Gene expression of Wnt signaling target genes by reverse transcription‐polymerase chain reaction (RT‐PCR), in rat liver tissue, was measured. In addition, serum levels of liver function parameters and alpha fetoprotein were performed in all groups. Histopathological examination of the liver and organ samples from all groups was performed. Magnetic resonance imaging (MRI) was used to visualize MSCs loaded with iron oxide nanoparticles in the affected liver. We detected a significant tumor‐mass reduction in the group which received MSCs compared to the control groups.The results of this work confirm the previous finding of a possible therapeutic effect of MSCs on HCC. In addition, the use of iron oxide nanoparticles may prove to be successful in tracking and localizing MSCs to the site of the lesion, which may provide a documentation of their therapeutic effect.Grant Funding Source: Supported by grant from the Deanship of Scientific Research, King Abdulaziz University Jeddah, KSA

8:18 AM; Abstract No. 148 - Superselective trans-arterial embolization in a translational rat model of hepatocellular carcinoma

Recent advances in the molecular biology of hepatocellular carcinoma (HCC) have identified new therapeutic targets that may potentiate the effects of transarterial embolization (TAE). Development of these new therapies requires a pre-clinical model that recapitulates the human disease and allows minimally invasive TAE that mimics the protocol used in patients. We describe a minimally invasive, superselective TAE in a translational rat model of HCC.

Everolimus is a potent inhibitor of activated hepatic stellate cell functions in vitro and in vivo, while demonstrating anti-angiogenic activities

Progression of liver fibrosis to HCC (hepatocellular carcinoma) is a very complex process which involves several pathological phenomena, including hepatic stellate cell activation, inflammation, fibrosis and angiogenesis. Therefore inhibiting multiple pathological processes using a single drug can be an effective choice to curb the progression of HCC. In the present study, we used the mTOR inhibitor everolimus to observe its effect on the in vitro activation of hepatic stellate cells and angiogenesis. The results of the present study demonstrated that everolimus treatment blocked the functions of the immortalized human activated hepatic stellate cell line LX-2 without affecting the viability and migration of primary human stellate cells. We also observed that treatment with everolimus (20 nM) inhibited collagen production by activated stellate cells, as well as cell contraction. Everolimus treatment was also able to attenuate the activation of primary stellate cells to their activated form. Angiogenesis studies showed that everolimus blocked angiogenesis in a rat aortic ring assay and inhibited the tube formation and migration of liver sinusoidal endothelial cells. Finally, everolimus treatment reduced the load of tumoral myofibroblasts in a rat model of HCC. These data suggest that everolimus targets multiple mechanisms, making it a potent blocker of the progression of HCC from liver fibrosis.

Seven-Tesla Magnetic Resonance Imaging Accurately Quantifies Intratumoral Uptake of Therapeutic Nanoparticles in the McA Rat Model of Hepatocellular Carcinoma

After inducing McA tumors in Sprague-Dawley rats (McA-SD), the following hypotheses were tested: first, that hypervascular McA tumors grown in Sprague-Dawley rats provide a suitable platform to investigate drug delivery; and second, that high-field MRI can be used to measure intratumoral uptake of DOX-SPIOs. McA cells were implanted into the livers of 18 Sprague-Dawley rats. In successfully inoculated animals, 220-μL DOX-SPIOs were delivered to tumors via the intravenous or intra-arterial route. Pretreatment and posttreatment T2*-weighted images were obtained using 7-T MRI, and change in R2* value (ΔR2*) was obtained from mean signal intensities of tumors in these images. Tumor iron concentration ([Fe]), an indicator of DOX-SPIO uptake, was measured using mass spectroscopy. The primary outcome variable was the Pearson correlation between ΔR2* and [Fe]. Tumors grew successfully in 13 of the 18 animals (72%). Mean (SD) maximum tumor diameter was 0.83 (0.25) cm. The results of phantom studies revealed a strong positive correlation between ΔR2* and [Fe], with r = 0.98 (P < 0.01). The results of in vivo drug uptake studies demonstrated a positive correlation between ΔR2* and [Fe], with r = 0.72 (P = 0.0004). The McA tumors grown in the Sprague-Dawley rats demonstrated uptake of nanoparticle-based therapeutic agents. Magnetic resonance imaging quantification of intratumoral uptake strongly correlated with iron concentrations in pathological specimens, suggesting that MRI may be used to quantify uptake of iron-oxide nanotherapeutics.

Increasing matrix stiffness upregulates vascular endothelial growth factor expression in hepatocellular carcinoma cells mediated by integrin β1

Matrix stiffness as a novel regulation factor involves in modulating the pathogenesis of hepatocellular carcinoma (HCC) invasion or metastasis. However, the mechanism by which matrix stiffness modulates HCC angiogenesis remains unknown. Here, using buffalo rat HCC models with different liver matrix stiffness backgrounds and an in vitro cell culture system of mechanically tunable Collagen1 (COL1)-coated polyacrylamide gel, we investigated the effects of different matrix stiffness levels on vascular endothelial growth factor (VEGF) expression in HCC cells and explored its regulatory mechanism for controlling HCC angiogenesis. Tissue microarray analysis showed that the expression levels of VEGF and CD31 were gradually upregulated in tumor tissues with increasing COL1 and lysyl oxidase (LOX) expression, indicating a positive correlation between tumor angiogenesis and matrix rigidity. The expression of VEGF and the phosphorylation levels of PI3K and Akt were all upregulated in HCC cells on high-stiffness gel than on low-stiffness gel. Meanwhile, alteration of intergrin β1 expression was found to be the most distinctive, implying that it might mediate the response of HCC cells to matrix stiffness simulation. After integrin β1 was blocked in HCC cells using specific monoclonal antibody, the expression of VEGF and the phosphorylation levels of PI3K and Akt at different culture times were accordingly suppressed and downregulated in the treatment group as compared with those in the control group. All data suggested that the extracellular matrix stiffness stimulation signal was transduced into HCC cells via integrin β1, and this signal activated the PI3K/Akt pathway and upregulated VEGF expression. This study unveils a new paradigm in which matrix stiffness as initiators to modulate HCC angiogenesis.

Branched-Chain Amino Acids Ameliorate Fibrosis and Suppress Tumor Growth in a Rat Model of Hepatocellular Carcinoma with Liver Cirrhosis

PurposeRecent studies have revealed that branched-chain amino acids (BCAA) reduce the development of hepatocellular carcinoma (HCC) in patients with obesity and hepatitis C virus infection by improving insulin resistance (IR). The aim of this study was to examine the anti-cancer and anti-fibrotic effects of BCAA on the development of diethylnitrosamine (DEN)-induced HCC and liver cirrhosis in a rat model.MethodsMale SD rats received weekly intraperitoneal injections of DEN (50 mg/kg of body weight) for 16 weeks to induce HCC. They were fed a diet containing 3% casein, 3% or 6% BCAA for 13 weeks beginning 6 weeks after DEN administration. DEN was used to induce HCC through stepwise development from cirrhosis to HCC. The effect of BCAA was evaluated in tumor tissues by histopathologic analyses, reverse transcription-polymerase chain reaction, and Western blotting.ResultsThe mean area and number of dysplastic nodules (DNs) and tumors in the casein group tended to be larger than those in the BCAA group 16 weeks after DEN administration. The mean fibrotic area in the BCAA group was smaller than that in the casein group. The BCAA group showed decreased mRNA levels for markers of fibrosis, angiogenesis, and apoptosis inhibition. Compared with the casein group, the BCAA group had lower levels of α-smooth muscle actin, vascular endothelial growth factor, p-β-catenin, p-p38 mitogen-activated protein kinase, proliferating cell nuclear antigen, and caspase-3 protein expression, as well as a higher level of cleaved caspase-3 protein expression.ConclusionsBCAA supplementation of the diet ameliorated liver fibrosis and HCC development in a DEN-induced rat model of HCC with liver cirrhosis, but not in the IR model. These results provide a rationale for anti-fibrosis and chemoprevention using BCAA treatment for HCC with liver cirrhosis, as well as decreasing the ammonia level.

Radiofrequency ablation suppresses distant tumour growth in a novel rat model of multifocal hepatocellular carcinoma

RFA (radiofrequency ablation) is an established therapy for HCC (hepatocellular carcinoma). The multikinase inhibitor sorafenib prolongs survival in advanced HCC. We examined the effects of RFA alone and in combination with sorafenib on a bystanding tumour in a two-tumour rat model of HCC. A total of 80 rats were implanted with two liver tumours and randomized to four treatment groups: vehicle and sham operation (control), sorafenib and sham operation (Sora/Sham), vehicle and RFA (Vh/RFA), and sorafenib and RFA (Sora/RFA) (n=10/group per time point). RFA or sham-operation was performed on the left lobe tumour on day 15. Animals were killed at day 18 and day 30. Non-RFA-targeted right lobe tumours were analysed for angiogenesis, growth factors [HGF (hepatocyte growth factor), EGF (epidermal growth factor) and VEGF (vascular endothelial growth factor)] and infiltrating immune cells (CD3 and CD68). At day 30, the non-RFA-targeted tumours were significantly smaller in all three treatment groups compared with control (Sora/Sham P≤0.0001, Vh/RFA P=0.005 and Sora/RFA P≤0.0001). The smallest tumours were observed in animals treated with a combination of sorafenib and RFA, whereas the size reduction seen in the RFA-only group indicated an RFA-mediated distant suppression of tumour growth. Growth factor measurement revealed transiently decreased EGF levels after RFA (P=0.008), whereas sorafenib treatment decreased HGF levels (P=0.001). MVD (microvessel density) was reduced by sorafenib (P=0.002) despite increased VEGF levels (P≤0.0001). The immune parameters revealed augmented T-cells and IL-10 (interleukin 10) levels in all three treatment groups; sorafenib additionally increased macrophage numbers (P≤0.0001). RFA and sorafenib alone resulted in significant volume reduction of the non-RFA-targeted tumour; this effect was enhanced when both modalities were combined.

Inhibitory effect of dsRNA TLR3 agonist in a rat hepatocellular carcinoma model

Hepatocellular carcinoma (HCC) is one of the most common types of malignant tumor. Studies have demonstrated that the toll‑like receptor 3 (TLR3)/interferon pathway is inhibitory in cancer cell proliferation, suggesting that the activation of this pathway may have therapeutic potential. In the present study, the inhibitory effects of BM‑06, a double‑stranded (ds)RNA TLR3 agonist, against HCC were studied in vivo. Using a 2‑acetylaminofluorene-induced HCC rat model, histological examination and analysis of corresponding biomarkers following treatment with BM-06, showed a decrease in tumor growth and cell proliferation, and an increase in apoptosis compared with that in a phosphate‑buffered saline control group. In addition, the observed antitumor effect of BM‑06 in the HCC rat model was demonstrated to be superior to the known TLR3 agonist, polyinosinic-polycytidylic acid.