Rat Hepatoma Research Articles

MRNA-miRNA networks identify metabolic pathways associated to the anti-tumorigenic effect of thyroid hormone on preneoplastic nodules and hepatocellular carcinoma.

BackgroundThyroid hormones (THs) inhibit hepatocellular carcinoma (HCC) through different mechanisms. However, whether microRNAs play a role in the antitumorigenic effect of THs remains unknown.MethodsBy next generation sequencing (NGS) we performed a comprehensive comparative miRNomic and transcriptomic analysis of rat hepatic preneoplastic lesions exposed or not to a short-term treatment with triiodothyronine (T3). The expression of the most deregulated miRs was also investigated in rat HCCs, and in human hepatoma cell lines, treated or not with T3.ResultsAmong miRs down-regulated in preneoplastic nodules following T3, co-expression networks revealed those targeting thyroid hormone receptor-β (Thrβ) and deiodinase1, and Oxidative Phosphorylation. On the other hand, miRs targeting members of the Nrf2 Oxidative Pathway, Glycolysis, Pentose Phosphate Pathway and Proline biosynthesis – all involved in the metabolic reprogramming displayed by preneoplastic lesions– were up-regulated. Notably, while the expression of most miRs deregulated in preneoplastic lesions was not altered in HCC or in hepatoma cells, miR-182, a miR known to target Dio1 and mitochondrial complexes, was down-deregulated by T3 treatment at all stages of hepatocarcinogenesis and in hepatocarcinoma cell lines. In support to the possible critical role of miR-182 in hepatocarcinogenesis, exogenous expression of this miR significantly impaired the inhibitory effect of T3 on the clonogenic growth capacity of human HCC cells.ConclusionsThis work identified several miRNAs, so far never associated to T3. In addition, the precise definition of the miRNA-mRNA networks elicited by T3 treatment gained in this study may provide a better understanding of the key regulatory events underlying the inhibitory effect of T3 on HCC development. In this context, T3-induced down-regulation of miR-182 appears as a promising tool.

Leukocyte cell-derived chemotaxin 2 regulates epithelial-mesenchymal transition and cancer stemness in hepatocellular carcinoma

Leukocyte cell-derived chemotaxin 2 (LECT2) acts as a tumor suppressor in hepatocellular carcinoma (HCC). However, the antineoplastic mechanism of LECT2, especially its influence on hepatic cancer stem cells (CSCs), remains largely unknown. In The Cancer Genome Atlas cohort, LECT2 mRNA expression was shown to be associated with stage, grade, recurrence, and overall survival in human HCC patients, and LECT2 expression was downregulated in hepatoma tissues compared with the adjacent nontumoral liver. Here, we show by immunofluorescence and immunoblot analyses that LECT2 was expressed at lower levels in tumors and in poorly differentiated HCC cell lines. Using functional assays, we also found LECT2 was capable of suppressing oncogenic behaviors such as cell proliferation, anchorage-independent growth, migration, invasiveness, and epithelial-mesenchymal transition in hepatoma cells. Moreover, we show exogenous LECT2 treatment inhibited CSC functions such as tumor sphere formation and drug efflux. Simultaneously, hepatic CSC marker expression was also downregulated, including expression of CD133 and CD44. This was supported by infection with adenovirus encoding LECT2 (Ad-LECT2) in HCC cells. Furthermore, in animal experiments, Ad-LECT2 gene therapy showed potent efficacy in treating HCC. We demonstrate LECT2 overexpression significantly promoted cell apoptosis and reduced neovascularization/CSC expansion in rat hepatoma tissues. Mechanistically, we showed using immunoblot and immunofluorescence analyses that LECT2 inhibited β-catenin signaling via the suppression of the hepatocyte growth factor/c-MET axis to diminish CSC properties in HCC cells. In summary, we reveal novel functions of LECT2 in the suppression of hepatic CSCs, suggesting a potential alternative strategy for HCC therapy.

Preclinical Therapeutic Evaluation of Lenvatinib-Eluting Microspheres for Transcatheter Arterial Chemoembolization of Hepatocellular Carcinoma.

To evaluate the preclinical in vivo therapeutic response of Lenvatinib-eluting microspheres (LEN-EM) transcatheter arterial chemoembolization (LEN-TACE) in an hepatocellular carcinoma (HCC) rat model. Magnetic resonance imaging (MRI) visible LEN-EM was fabricated with poly(lactide-co-glycolide) and iron oxide nanoparticles by a double-emulsion method. The morphology, LEN loading/release kinetics, and MRI contrast effect of LEN-EM were evaluated. For in vivo study, N1S1 HCC rats were treated with LEN-TACE (LEN: 2.4mg/kg, n = 5) using LEN-EM, systemic LEN (LEN: 0.4mg/kg, oral gavage daily for 7days, n = 5), control (intra-arterial (IA) saline infusion, n = 5), and non-tumor control (n = 3). Tumor size changes were measured for 2weeks. Histology, comparative LEN plasma concentration, hematologic markers, liver profile, and serum chemistry among the groups were measured. LEN-EM with 33µm in average size was prepared in an optimized emulsion process. LEN loading efficiency was 58.7%. LEN was continuously released for 500h. LEN-TACE showed the delivered LEN-EM surrounding tumor tissue in MRI-T2* images. The LEN-TACE group demonstrated a statistically significant larger tumor volume reduction compared to the other groups at 2weeks post-procedure. Quantification data of Terminal deoxynucleotidyl transferase dUTP nick end labeling positive cells confirmed increased cancer cell death in the LEN-TACE group compared to control groups. Additional histology, hematologic markers, and liver profiles showed minimal side effects of LEN-TACE. LEN-TACE using IA delivery of LEN-EM demonstrated an effective therapeutic efficacy in an HCC rat animal model.

Elevated expression of ISY1, APOA-1, SYNE1, MTG1, and MMP10 at HCC initiation: HCC specific protein network involving interactions of key regulators of lipid metabolism, EGFR signaling, MAPK, and splicing pathways.

Identification of molecular regulators of hepatocellular carcinoma (HCC) initiation and progression is not well understood. We chemically induced HCC in male Wistar rats by administration of diethyl nitrosamine (DEN) and 2-acetylaminofluorene (2-AFF). Using 2D-electrophoresis and MALDI-TOF-MS/MS analyses, we characterized differentially expressed proteins in liver tissues at early stage of HCC progression. Using RT-PCR analysis, we quantified the mRNA expression of the characterized proteins and validated the transcript expression with tumor tissues of clinically confirmed HCC patients. Using bioinformatic tools, we analyzed a network among the introduced proteins that identified their interacting partners and analyzed the molecular mechanisms associated with signaling pathways during HCC progression. We characterized a protein, namely, pre-mRNA splicing factor 1 homolog (ISY1), which is upregulated at both transcriptome and proteome levels at HCC initiation, progression, and tumor stages. We analyzed the interacting partners of ISY1, namely, APOA-1, SYNE1, MMP10, and MTG1. Real-time PCR analysis confirmed elevated expression of APOA-1 mRNA at HCC initiation, progression, and tumor stages in animals undergoing tumorigenesis. The mRNA expression of the interacting partners was validated with tumor tissues of clinically confirmed liver cancer patients; the analysis revealed significant elevation in expression of transcripts. The transcriptome and proteome analyses complement each other and dysregulation in mRNA and protein expression of these regulators may play critical role in HCC initiation and progression.

Assessment Of Alpha-Fetoprotein Level After Treatment With Plectranthus Amboinicus Extract In Wister Albino Rats Induced By Carbon Tetra-Chloride

Plectranthus Amboinicus, plant extract has indispensible beneficial agents in traditional Indian medicine and Ayurveda. Incidence of several diseases, including Hepatocellular carcinoma (HCC) is increasing gradually to a new peak. The major goal of this investigation was to identify and estimate defensive capability of Plectranthus Amboinicus extract on enzymatic functions of liver and to investigate attenuating potential on HCC biomarker. The analyses include, effects of Plectranthus Amboinicus, in serum level of AST,ALT,ALP, Total bilirubin, and alpha fetoprotein (AFP) fluctuations in carbon tetrachloride (CCL4), induced HCC rats. Toxicity was stimulated by CCL4 with olive oil as vehicle in the ratio of (1:1 v/v);( olive oil 5mL/kg i.p). Sylimarin was used as a reference (standard) for intoxicated animal groups. Moreover, observation of histological studies favours the biochemical changes observed. Results showed P.Amboinicus, is highly potential in depleting the level of liver enzymes and AFP biomarker, based on dose regimen. These might have been due to the action of enriched phenolic content of P.Amboinicus, to get rid of free radicals. Study concluded that P.Amboinicus is a universal medicine and modulating biochemical changes in hepatotoxicity of liver cells induced HCC rat model.

Histomorphological Progression of Diethylnitrosamine (DEN) Induced Hepatocellular Carcinoma Model in Rat

Hepatocellular carcinoma (HCC) commonly known as hepatoma is a frequently occurring liver cancer that occupies 75% of all types of liver cancers. It affects the hepatocellular cells which is the major cell of the liver. Some models can be developed for HCC in rats but it is time-consuming. Nitrosodiethylamine is a very popular carcinogenic drug, very useful to develop experimental animal models. The present study focuses on the establishment of an animal model through a simple approach of experimental protocol along with the duration of the experiment that leads to creating a scope for future studies associated with metastasis as well as anti-metastasis studies. For the present study 2 groups were formed where each group containing 16 rats. Phenobarbital was given for 7 days to each group after diluting with water at a concentration of 0.3g/L before administering the first DEN. The control group received the same number of vehicles for 8 weeks. 50mg/kg of DEN was received intraperitoneally by the animal of the 2nd group twice a week for eight weeks. Bodyweight, the weight of the liver, and all the blood parameters were analysed like alpha-fetoprotein, SGPT, SGOT, and ALP, and a comparison was made with the control group. After evaluating all biological and histological data it can be concluded that the outcome of the present study signifies a simple experimental protocol along with an appropriate experimental duration animal model which leads to help in the study of the mechanism of metastasis and anti-metastasis agent in near future. The current work also represents a straightforward experimental methodology that aids in the investigation of metastatic mechanisms and anti-metastasis agents, with phenobarbital serving as a good promoting agent.

Fabrication of a Tailored, Hybrid Extracellular Matrix Composite

The extracellular matrix (ECM) is a network of connective fibers that supports cells living in their surroundings. Native ECM, generated by the secretory products of each tissue's resident cells, has a unique architecture with different protein composition depending on the tissue. Therefore, it is very difficult to artificially design in vivo architecture in tissue engineering. In this study, a hybrid ECM scaffold from the basic structure of fibroblast-derived cellular ECMs is fabricated by adding major ECM components of fibronectin (FN) and collagen (COL I) externally. It is confirmed that while maintaining the basic structure of the native ECM, major protein components can be regulated. Then, decellularization is performed to prepare hybrid ECM scaffolds with various protein compositions and it is demonstrated that a liver-mimicking fibronectin (FN)-rich hybrid ECM promoted successful settling of H4IIE rat hepatoma cells. The authors believe that their method holds promise for the fabrication of scaffolds that provide a tailored cellular microenvironment for specific organs and serve as novel pathways for the replacement or regeneration of specific organ tissues.

Intravenous Immunoglobulin Inhibits Liver Cancer Progression by Promoting p38MAPK-Associated Apoptosis.

Objective The aim of this study is to explore the effect of intravenous immunoglobulin (IVIG) on the development of rat hepatocellular carcinoma and its possible molecular mechanism. Methods Sixty adult male Sprague-Dawley (SD) rats were randomly divided into three groups: control, diethylnitrosamine(DEN) + normal saline(NS), and DEN + IVIG groups, with 20 rats in each group. The rats in the DEN + NS group and DEN + IVIG group were given DEN 0.2 g/kg intraperitoneal injection once on day 1 and then 0.05% DEN aqueous solution in drinking water to establish a rat liver cancer model. Immunoglobulin (IgG) was injected intraperitoneally into the DEN + IVIG group twice a week at the dose of 100 mg/kg, and saline was administered intraperitoneally into the control group at a 50 mg/kg dosage. The body weight of each group of rats was recorded twice a week. All treatments were maintained continuously for 12 weeks. After the intervention, the liver function indexes of rats were measured by a fully automated biochemical analysis instrument. The liver histopathology was observed by hematoxylin-eosin(HE) staining. Immunohistochemistry was used to detect c-myc protein expression, and Western blotting was used to determine p38MAPK and p-p38MAPK protein expressions, as well as apoptosis-related proteins such as Bcl-2, Bax, and cleaved caspase-3. Results Compared with the rats in the DEN + NS group, rats in the DEN + IVIG group showed substantially higher body mass (P < 0.05), higher survival rate (P < 0.05), and lower liver function indexes (P < 0.05). Few focal necrosis of cancer cells and few nuclear division were observed in the rats in the DEN + IVIG group. The rats in the DEN + NS group showed lamellar necrosis of cancer foci, destruction of normal liver lobular structure, and hepatocellular carcinoma cells. Immunohistochemical analysis results revealed that the expression of c-myc was reduced in the DEN + IVIG group (P < 0.05), and Western blotting confirmed that the Bcl-2 expression was decreased (P < 0.05), while Bax, p38 MAPK, p-p38 MAPK, and cleaved caspase-3 protein expressions were increased (P < 0.05). Conclusion IVIG prophylactic injection can delay tumor development and induce apoptosis in primary hepatocellular carcinoma in rats. The mechanism is connected to the activation of the p38MAPK signaling pathway by upregulating the level of cleaved caspase-3 and Bax proteins while downregulating the level of Bcl-2 and c-myc proteins.

Regulation of Sirtuin-3 and ERK1/2/p38MAPK by the combination Ga nanoparticles/γ-radiation low dosage: an effective approach for treatment of hepatocellular carcinoma

BackgroundSynthesized gallium nanoparticles synthesized by grape seed extract were characterized with spherical shape and size range less than100 nm, possessing the functional groups of the biological material. The purpose of this study is to evaluate gallium nanoparticles synthesized by grape seed extract, as an antitumor agent with low dose of γ-radiation against hepatocellular carcinoma in rats. Aim of workThis work aimed to evaluate the antitumor effect of gallium nanoparticles synthesized (GaNPs) by grape seed extract and the co-binded treatment with low dose of γ-radiation on hepatocellular carcinoma in rats, through evaluating their effect on signaling pathways and tumor markers. ResultsCytotoxic activity of GaNPs synthesized by grape seed extract was estimated by mediated cytotoxicity assay on HepG2 cell line that recorded IC50 of 388.8 μg/ml. To achieve these goals, eighty Wistar male rats (120−150 g) will be divided into eight groups, each of 10 rats. The animals are administered with diethylnitrosamine to induce hepatocellular carcinoma and then orally administered with GaNPs synthesized by grape seed extract (38.5 mg/kg) in combination with the exposure of the total body to a low dose of γ-radiation (0.5 Gy). The treatment modulated plasma vascular endothelial growth factor and alpha-fetoprotein. In addition, the immunoblotting results of nuclear factor-kappa beta showed a marked downregulation of extracellular signal-regulated kinase, mitogen-activated protein kinase, and c-Jun NH2-terminal kinase alongside, significantly elevating the level of Sirtuin-3 and caspase-3. ConclusionsIt can be concluded that the combined treatment with GaNPs synthesized by grape seed extract and low dose γ-radiation may have antineoplastic activity against hepatocarcinogenesis by inhibiting signal pathways extracellular signal-regulated kinase/mitogen-activated protein kinase/c-Jun NH2-terminal kinase and stimulating apoptotic protein.

Studies On the Composition of The Low Molecular Weight Thiols Present in Tumour Cells– Is There "An Elephant in The Room”? An Autobiographical Review

This is an account of the author’s research investigations on the identification of the low molecular weight thiols (LMWT) present in a variety of eukaryote cells, particularly cancer cells. The work was carried out periodically over some 60 years in a number of university and private laboratories as, and when, resources permitted: as a result, this research can be roughly divided into 7 phases. The first phase arose from initial research into ways to improve cancer treatment by radiation treatment. The second was involved with studies of the thiols present in isolated nuclei and nucleoli. The third consisted of attempts to develop new techniques to isolate and identify the various nuclear protein thiol containing moieties of a rat hepatoma tumour. The fourth phase involved the successful isolation of the nuclear nonhistone proteins which contain the bulk of the nuclear thiol material, leading to study nuclear thiol changes during rat liver carcinogenesis. The fifth phase consisted of the discovery and isolation of “hidden” low molecular weight thiols in isolated nuclei from rat tumours and other tissues. After a break of some 14 years, when bulk preparations of whole human cells became available, it became possible to continue work again on the low molecular weight thiols. Phase six consisted of studies on the thiols found in the ASF of various tumour cell lines using the new analytical technologies which were developing at that time. The seventh phase evolved into studies on human prostate cancer cell lines. Here a method was developed which clearly demonstrated that, among the low molecular weight thiols present in these cells, there was “an elephant in the room”. This new thiol moiety, constituting 56.4% of the total cell thiols, was found to be concealed within the protein matrices of the cell, held in place, not covalently but possibly by ionic and hydrophobic bonding; deemed to be a “Conthiol”. It does not contain any of the constituents of known cellular thiols and has proved difficult to analyse due to its reactive nature.

Chemopreventive effects of Costus comosus Linn against diethylnitrosamine-induced hepatocellular carcinoma in rats

Introduction: Costus comosus is a potential medicinal plant used traditionally to treat various ailments. The present study aimed to evaluate antioxidant status, lipid peroxidation, and the ameliorative effect of its ethanolic leaf extract against diethylnitrosamine (DEN)-induced hepatocellular carcinoma (HCC) in rats. Methods: HCC was induced by 0.01 % v/v DEN through the drinking water for 16 weeks. The animals were treated with ethanolic leaf extract of C. comosus (EECC) at 200 and 400 mg/kg for 16 weeks. In this study, tumour incidence, tumour volume, tumour burden, lipid peroxidation, antioxidant activity, liver marker enzymes, and histological responses were measured in the animals. At the end of the study, rats were sacrificed, their livers were removed and the levels of antioxidant enzymes were measured in the liver homogenate. Results: In DEN-treated animals, there were 100% tumour occurrences probably due to an imbalance in carcinogen metabolizing enzymes and cellular redox state. The oral administration of ethanolic leaf extract of C. comosus therapy at a dose of 200 and 400 mg/kg reduced lipid peroxide levels and restored the increased activities of liver marker enzymes and antioxidant status to near normal. The biochemical findings corroborate histological findings, indicating that the leaf extract has a significant hepatoprotective impact in a dose-dependent manner. Conclusion: The results of the present study showed the promising anti-carcinogenic effects of ethanolic leaf extract of C. comosus against the DEN-induced HCC in rats.

1H-NMR-based serum metabolomic study to evaluate the effect of asarone and metformin on experimentally induced diabetic hepatocellular carcinoma in rats

BackgroundThe increased prevalence of hepatocellular carcinoma (HCC) in diabetic patients has focused on the need to characterize the role of altered metabolites in liver carcinogenesis. In this study, together with the serum biochemistry and histopathological observation, 1H nuclear magnetic resonance (1H-NMR)-based metabolomics was carried out to study the effect of asarone and metformin in diabetic HCC rats. Intraperitoneal administration of streptozotocin (STZ; 55 mg/kg b.w.) was used to induce diabetes in male Wistar rats. Further, 2 weeks later, after confirmation of diabetes, another group received diethylnitrosamine (DEN; 200 mg/kg b.w.) to simulate the diabetic HCC condition. The combined dose of α-and β-asarone (50 µg/kg b.w. in the ratio of 1:1) and metformin HCl (250 mg/kg b.w.) treatment was orally given to the diabetic HCC rats for 18 weeks. The serum samples were subjected to 1H-NMR-based metabolomics analysis to explore the metabolite changes at the end of the study.Results1H-NMR study quantitatively distinguished the metabolites, such as pyruvate, lactate, creatine, acetate, glutamine, valine, and alanine, which varied between the diabetic HCC and normal rats. Furthermore, compared to the diabetic HCC group, the administration of asarone and metformin resulted in a substantial change in metabolite levels. Histopathological examination indicated that treatment attenuates the magnitude of the toxic effect of STZ + DEN.ConclusionsThe aberrant glucose, lipid, and amino acid metabolisms were associated with developing hepatocarcinogenesis in rats during the diabetic condition. Treatment with asarone and metformin attenuated the metabolic changes due to STZ + DEN-induced diabetic HCC.

Selenium nanoparticles overcomes sorafenib resistance in thioacetamide induced hepatocellular carcinoma in rats by modulation of mTOR, NF-κB pathways and LncRNA-AF085935/GPC3 axis

AimsThe first-line treatment for advanced hepatocellular carcinoma (HCC) is the multikinase inhibitor sorafenib (SOR). Sofafenib resistance is linked to protein kinase B/ mammalian target of rapamycin (AKT/mTOR) and nuclear factor kappa B (NF-κB) activation, apoptosis inhibition and oxidative stress. This study investigated selenium nanoparticles (SeNps) to overcome SOR resistance in thioacetamide (TAA) induced HCC in rats. Materials and methodsTAA (200 mg/kg/twice weekly, i.p.) was administered for 16 weeks to induce HCC.s. Rats were treated with oral SOR (10 mg/Kg daily), selenium, and SeNps (5 mg/kg three times/week) alone or in combination, for two weeks. Apoptosis, proliferation, angiogenesis, metastasis and drug resistance were assessed. Cleaved caspase 3 (C. CASP3), mTOR, and NF-κB were determined by western blotting. Expression of p53 gene and long-noncoding RNA-AF085935 was determined by qRT-PCR. Expression of B- Cell Leukemia/Lymphoma 2 (Bcl2), Bcl associated X protein (Bax)and glypican 3 (GPC3) was determined by enzyme-linked immunosorbent assay. Liver functions, antioxidant capacity, histopathology and CD34 immunohistochemistry were performed. Key findingsSOR/SeNps reversed TAA-induced HCC in rats, through reduction of oxidative stress, activation of p53, Bax and CASP3, and inhibition of Bcl2. SOR/SeNps ameliorated the HCC-induced effect on cell proliferation and drug resistance by targeting mTOR and NF-κB pathways. SOR/SeNps decreased CD34 immunostaining indicating a decrease in angiogenesis and metastasis. SOR/SeNps regulated HCC epigenetically through the lncRNA-AF085935/GPC3 axis. SignificanceSOR/SeNps are a promising combination for tumor suppression and overcoming sorafenib resistance in HCC by modulating apoptosis, AKT/mTOR and NF-κB pathways, as well as CD34 and lncRNA-AF085935/GPC3 axis.

Effect of Acrylamide Treatment on Cyp2e1 Expression and Redox Status in Rat Hepatocytes.

Acrylamide (AA) toxicity is associated with oxidative stress. During detoxification, AA is either coupled to gluthatione or biotransformed to glycidamide by the enzyme cytochrome P450 2E1 (CYP2E1). The aim of our study was to examine the hepatotoxicity of AA in vivo and in vitro. Thirty male Wistar rats were treated with 25 or 50 mg/kg b.w. of AA for 3 weeks. Qualitative and quantitative immunohistochemical evaluation of inducible nitric oxide synthase (iNOS), CYP2E1, catalase (CAT), superoxide dismutase 1 (SOD1), and SOD2 expression in liver was carried out. Bearing in mind that the liver is consisted mainly of hepatocytes, in a parallel study, we used the rat hepatoma cell line H4IIE to investigate the effects of AA at IC20 and IC50 concentrations on the redox status and the activity of CAT, SOD, and glutathione-S-transferase (GST), their gene expression, and CYP2E1 and iNOS expression. Immunohistochemically stained liver sections showed that treatment with AA25mg induced a significant decrease of CYP2E1 protein expression (p < 0.05), while treatment with AA50mg led to a significant increase of iNOS protein expression (p < 0.05). AA treatment dose-dependently elevated SOD2 protein expression (p < 0.05), while SOD1 protein expression was significantly increased only at AA50mg (p < 0.05). CAT protein expression was not significantly affected by AA treatments (p > 0.05). In AA-treated H4IIE cells, a concentration-dependent significant increase in lipid peroxidation and nitrite levels was observed (p < 0.05), while GSH content and SOD activity significantly decreased in a concentration-dependent manner (p < 0.05). AA IC50 significantly enhanced GST activity (p < 0.05). The level of mRNA significantly increased in a concentration-dependent manner for iNOS, SOD2, and CAT in AA-treated H4IIE cells (p < 0.05). AA IC50 significantly increased the transcription of SOD1, GSTA2, and GSTP1 genes (p < 0.05), while AA IC20 significantly decreased mRNA for CYP2E1 in H4IIE cells (p < 0.05). Obtained results indicate that AA treatments, both in vivo and in vitro, change hepatocytes; drug-metabolizing potential and disturb its redox status.

Abstract No. 359 Embolization induced tumor-associated macrophage polarization in tumor immune microenvironment can be reprogrammed by lenvatinib in a rat hepatoma model

Abstract No. 359 Embolization induced tumor-associated macrophage polarization in tumor immune microenvironment can be reprogrammed by lenvatinib in a rat hepatoma model

Neutralization and receptor use of infectious culture-derived rat hepacivirus as a model for HCV.

Lack of tractable immunocompetent animal models amenable to robust experimental challenge impedes vaccine efforts for HCV. Infection with rodent hepacivirus from Rattus norvegicus (RHV-rn1) in rats shares HCV-defining characteristics, including liver tropism, chronicity, and pathology. RHV in vitro cultivation would facilitate genetic studies on particle production, host factor interactions, and evaluation of antibody neutralization guiding HCV vaccine approaches. We report an infectious reverse genetic cell culture system for RHV-rn1 using highly permissive rat hepatoma cells and adaptive mutations in the E2, NS4B, and NS5A viral proteins. Cell culture-derived RHV-rn1 particles (RHVcc) share hallmark biophysical characteristics of HCV and are infectious in mice and rats. Culture adaptive mutations attenuated RHVcc in immunocompetent rats, and the mutations reverted following prolonged infection, but not in severe combined immunodeficiency (SCID) mice, suggesting that adaptive immune pressure is a primary driver of reversion. Accordingly, sera from RHVcc-infected SCID mice or the early acute phase of immunocompetent mice and rats were infectious in culture. We further established an in vitro RHVcc neutralization assay, and observed neutralizing activity of rat sera specifically from the chronic phase of infection. Finally, we found that scavenger receptor class B type I promoted RHV-rn1 entry in vitro and in vivo. The RHV-rn1 infectious cell culture system enables studies of humoral immune responses against hepacivirus infection. Moreover, recapitulation of the entire RHV-rn1 infectious cycle in cell culture will facilitate reverse genetic studies and the exploration of tropism and virus-host interactions.

The Intracellular Cholesterol Pool in Steroidogenic Cells Plays a Role in Autophagy, Basal Steroidogenesis and Mitochondrial Dynamics

In steroidogenesis, cholesterol‐ the precursor substrate molecule for all steroid hormones is central and the framework of steroidogenesis across steroidogenic cells is constructed around it— including its cellular uptake, storage in intracellular lipid droplets, mobilization upon steroidogenic stimulation, and finally, its transport to the mitochondria where cholesterol is converted to pregnenolone by the P450 side chain cleavage enzyme. Thus, cholesterol and the mitochondria are highly interconnected in all steroidogenic cells. Moreover, accruing evidence suggests that autophagy and mitochondrial dynamics are important cellular events in the regulation of trophic hormone‐induced cholesterol homeostasis and steroidogenesis. However, a potential role of cholesterol in the regulation of steroidogenic events and factors remain unexplored. We tested the hypothesis that cholesterol plays a role in the regulation of cell‐intrinsic factors and events involving steroidogenesis. We used an intermediary approach to investigate the role of cholesterol in the regulation of steroidogenesis in different steroidogenic cell types such as MA‐10 cells (a murine Leydig cell line), BeWo cells (placental cell line) producing different steroid hormones. Steroidogenic cells were cultured in a lipoprotein‐depleted medium that leads to the depletion of the intracellular cholesterol pool and lipid droplets. Furthermore, cholesterol deprivation induced steroidogenic events. To get a snapshot of cholesterol deprived (CD)‐induced changes in lipid droplets, autophagy, and mitochondrial dynamics at an ultrastructural level we analyzed steroidogenic cells using transmission electron microscopy. As expected, cholesterol deprivation resulted in the depletion or loss of intracellular lipid droplets and the cholesterol pool across steroidogenic cell lines. Moreover, a difference in autophagy‐related structural changes was observed between cells cultured under the normal and CD conditions. In general, the structural characteristics of autophagy were more apparent in the CD group when compared with the normal control group across different steroidogenic cell types. Moreover, a change in mitochondrial shape was observed between the cells from two groups. For instance, the elongated or tubular mitochondria showing signs of mitochondrial dynamics (fusion or fission) were more common in the CD experimental group. Importantly, an increase in lysosome size and numbers were consistently found in cells under CD conditions which inversely correlated with the depletion of lipid droplets and the cholesterol pool, suggesting a compensatory increase in cholesterol mobilization. Taken together, this data suggests that cholesterol indeed plays a role in the regulation of autophagy and mitochondrial dynamics across steroidogenic cells in a context‐dependent manner. Such effects of cholesterol deprivation on autophagy and mitochondrial dynamics were not observed in the non‐steroidogenic cells such as HepG2 (human hepatoma cells) and H4IIE (rat hepatoma cells), indicating that cholesterol insufficiency‐induced changes in steroidogenic cells are specific to steroidogenesis. Thus, we unraveled this previously unknown role of cholesterol in steroidogenesis beyond being a mere substrate for steroid hormones. The implications of our findings are broad and offer new insights into trophic hormone‐dependent and independent steroidogenesis during development, as well as in health and disease.

Suppressive effects of thymoquinone on the initiation stage of diethylnitrosamine hepatocarcinogenesis in rats.

Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related death globally. Chemoprevention is the most effective technique for reducing HCC incidence. Thymoquinone (TQ), the main bioactive constituent of Nigella sativa, exhibits anti-inflammatory and antineoplastic activities against various cancers. Therefore, TQ was tested as an inhibitor of the initial phase of diethylnitrosamine (DEN)-induced HCC in rats.Twenty-four male Wistar albino rats were randomly placed into four equal groups. Group 1 received saline and acted as the negative control; Group 2 received TQ; Group 3 received DEN; and Group 4 received TQ for 7 days and DEN on the 8th day. After 24 h of fasting, blood samples were taken from the slaughtered rats. Additionally, each rat's liver was dissected and separated into two halves for histological and biochemical investigation.DEN-induced hepatotoxicity was detected by elevated hepatic enzymes and HCC biomarkers reduced antioxidant and proapoptotic statuses. DEN administration caused a significant increase in the levels of glutathione, superoxide dismutase, malondialdehyde, caspase-3, alpha-fetoprotein (AFP), AFPL3, glypican 3, and the expression of BAX. However, DEN significantly decreased glutathione peroxidase, catalase, and CYP2E1 and the expression of BCl-2. Furthermore, it caused histological changes and showed a strong positive GSH S-transferase P expression in the hepatic parenchyma. Pretreatment with TQ prevented the histopathological and most of the biochemical changes and improved the antioxidant status.TQ supplementation appears to suppress the development of DEN-initiated liver cancer by reducing oxidative stress, activating the intrinsic mitotic apoptosis pathway, and retaining the antioxidant enzymes.

Aflatoxin B1 DNA-Adducts in Hepatocellular Carcinoma from a Low Exposure Area.

Aflatoxin B1 (AFB1) is a class 1 carcinogen with an ascertained role in the development of hepatocellular carcinoma (HCC) in high exposure areas. Instead, this study aimed to assay whether chronic/intermittent, low-dose AFB1 consumption might occur in low-exposure geographical areas, ultimately accumulating in the liver and possibly contributing to liver cancer. AFB1-DNA adducts were assayed by immunostaining in liver tissues from three Italian series of twenty cirrhosis without HCC, 131 HCC, and 45 cholangiocarcinoma, and in an AFB1-induced HCC rat model. CD68, TP53 immunostaining, and TP53 RFLP analysis of R249S transversion were used to characterize cell populations displaying AFB1-DNA adducts. Twenty-five HCCs displayed AFB1-adducts both in neoplastic hepatocytes and in cells infiltrating the tumor and non-tumor tissues. Nuclear immunostaining was observed in a few cases, while most cases showed cytoplasmic immunostaining, especially in CD68-positive tumor-infiltrating cells, suggestive for phagocytosis of dead hepatocytes. Similar patterns were observed in AFB1-induced rat HCC, though with higher intensity. Cholangiocarcinoma and cirrhosis without HCC did not displayAFB1-adducts, except for one case. Despite not providing a causal relationship with HCC, these findings still suggest paying attention to detection and control measures for aflatoxins to ensure food safety in low exposure areas.

Cinnamaldehyde Relieves Induced Hepatocellular Carcinoma in Rat Model via Targeting Wnt/β-Catenin Pathway

Cinnamaldehyde (CA) is a natural compound that has promising biological activity. The current study investigates the antitumor activity of CA in thioacetamide induced hepatocellular carcinoma (HCC) in rats through targeting the Wnt/β-catenin pathway and evaluates the capability of CA to relieve hepatocytes oxidative stress in the HCC-rat model. After 16 weeks of HCC induction by thioacetamide (TAA), rats were treated for 7 consecutive weeks with CA daily; i.p. injection, Alpha-fetoprotein (AFP) level, necroinflammatory score and fibrosis percentage were measured to assess HCC development. The Wnt/β-catenin pathway was evaluated by measuring the hepatic protein level of Wnt-3a, β-catenin, cyclin D, matrix metalloproteinase-9 (MMP-9), and vascular endothelial growth factor (VEGF). Furthermore, hepatocytes’ oxidative stress was assessed by measuring hepatic GSH and MDA contents. Results showed that CA was significantly inhibiting the Wnt/β-catenin pathway through the downregulation of hepatic Wnt-3a, β-catenin, cyclin D, MMP-9, and VEGF. Moreover, CA ameliorates hepatocytes’ oxidative stress via lowering hepatic MDA content and rising hepatic GSH content. Thus, in conclusion, CA is a promising treatment for HCC. It not only has an effective antitumor activity but also ameliorates hepatocytes’ oxidative stress.