Hepatoblastoma Cell Line Research Articles

NMR-based metabolic profiling and in vitro antioxidant and hepatotoxic assessment of partially purified fractions from Golden germander (Teucrium polium L.) methanolic extract

Golden germander (Teucrium polium L.) is a Mediterranean shrub of the Labiatae family, used in traditional medicine for its diuretic, antipyretic, diaphoretic, antispasmodic, tonic, anti-inflammatory, antihypertensive, anorexic, analgesic, antibacterial and antidiabetic effects. Like other plants of the Teucrium genus, it was widely popular because of its hypoglycemic and hypolipidemic properties but various cases of T. polium-induced hepatitis have been reported. neo-Clerodane diterpenoids, considered chemotaxonomic markers for the Teucrium genus, are believed to be responsible for the observed hepatotoxicity. The plant also produces flavonoids and phenylethanoid glycosides to which the antioxidant and cytoprotective therapeutic properties of its preparations can be traced back. In order to establish a herbal formula that preserves the plant beneficial properties, T. polium leaf drug has been subjected to a bio-guided fractionation. The different phytocomplexes obtained were analyzed by means of an extensive antioxidant screening and hepatotoxicity evaluation against HepG2, a human hepatoblastoma cell line. The cytotoxicity of the fractions was also evaluated against HeLa and A549 cell lines. In order to identify the substances responsible for the bioactivities, NMR-based metabolic profiling techniques of all the phytocomplexes were performed. Data obtained highlighted the possibility of preparing strong antioxidant extracts, useful as food additives, such as MeOH-2, and MeOH-3, completely devoid of hepatotoxic components.

Observation of the absorption of the In‐DTPA‐hexa–lactoside (IDHL) and its distribution in vitro by ICP‐MS and TOF‐SIMS

The distribution of carbohydrate drug containing indiums ‘In‐DTPA‐hexa–lactoside (IDHL)’ in human hepatoblastoma cell line (HepG2) had been observed, and cell viability was investigated at the same time. The cell survival and mitochondrial metabolism of HepG2 after treated with 50 μΜ IDHL for 24 h were investigated by 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyltetra‐zolium bromide assay, and the cytotoxicity was evaluated by lactate dehydrogenase method (LDH assay). The intracellular indium ion concentration was quantitated by inductively coupled plasma mass spectrometer. Besides, the distribution of indium ion in HepG2 was also analyzed by time‐of‐flight secondary ion mass spectrometry for comparison. The results demonstrated that there were only small amount of indium ions distributed inside the cytoplasm and the cell membrane. This means that HepG2 had very limited IDHL absorption, and there was almost no attachment of IDHL onto the HepG2 cell membrane. In these experiments, a parsing technique was established, which can shed some light on the understanding toward the absorption and distribution of these isotope compounds in vitro. Copyright © 2012 John Wiley & Sons, Ltd.

Inflammatory stress exacerbates hepatic cholesterol accumulation via disrupting cellular cholesterol export

Both inflammation and cholesterol accumulation play important roles in the development of non-alcoholic fatty liver disease. This study was undertaken to investigate whether inflammation aggravated cholesterol accumulation via disrupting hepatic cholesterol export and we explored the underlying mechanisms. We used casein injection in C57BL/6J mice, and tumor necrosis factor alpha (TNF-α) stimulation in human hepatoblastoma cell line (HepG2) cells to induce inflammation. Intracellular cholesterol level was examined by Oil Red O staining and quantitative analysis. Bile acid level was quantified by colorimetric analysis. (3)[H] cholesterol assay by scintillation counting was performed to evaluate the cholesterol efflux. The mRNA and protein expression was examined by real-time polymerase chain reaction and Western blot. Inflammation increased cholesterol accumulation in livers of C57BL/6J mice and in HepG2 cells. High-fat diet in mice and low-density lipoprotein (LDL) loading in HepG2 cells increased bile acid synthesis and cholesterol efflux, enhanced the mRNA and protein expression of liver X receptor α (LXRα), peroxisome proliferator-activated receptors (PPARα, γ), cholesterol 7α-hydroxylase (CYP7A1) and ATP-binding cassette transporter A1 (ABCA1). However, inflammation reduced bile acid synthesis and cholesterol efflux even in high-fat-diet-fed mice and HepG2 cells in the presence of LDL loading. The enhanced effects of these genes and proteins expression due to high-fat diet and LDL loading were inhibited by inflammation both in vivo and in vitro. Inflammation disrupted PPAR-LXR-CYP7A1/ABCA1-mediated bile acid synthesis and cholesterol efflux resulting in exacerbated cholesterol accumulation in livers of C57BL/6J mice and HepG2 cells.

Abstract 4337: Effect of sorafenib in combination with cytostatic agents on hepatoblastoma in vitro and in vivo

Abstract Background: Hepatoblastoma (HB) represents the most common malignant liver tumour in childhood. For patients with high risk or recurrent HB treatment results remain still poor due to a developing multidrug resistance during chemotherapy. Therefore new therapy strategies have to be evaluated. The primary objective was to investigate the combined therapy of cytotoxic agents with sorafenib. Sorafenib has been shown to reduce tumour progression and angiogenesis. In order to investigate a treatment close to a clinical setting the effect of the combined administration of cytotoxic agents with sorafenib on HB cell lines and xenotransplanted HB tumours was determined. Methods: Cell viability of two HB cell lines (HUH6 and HepT1) was evaluated in using MTT assays after treatment with sorafenib and different cytostatic agents (cisplatin (CDDP), doxorubicin, irinotecan, topotecan). ERK signalling was investigated by Western blot analysis. NMRI mice (nu/nu) bearing subcutaneous HUH6 derived tumours were treated with Sorafenib orally (30 mg/kg, on days 4-13 and 17-24) alone or in combination with CDDP i.p. (3 mg/kg, on days 1-3 and 14-16). Tumor progression and viability were monitored by tumour volume and AFP levels. Apoptosis was assessed by TUNEL assay. Angiogenesis was determined by CD31 staining and mean vascular density (MVD) was calculated. Results: In vitro the combination of sorafenib with CDDP showed a remarkable decrease in cell viability compared to the other agents tested. However with increasing concentrations of CDDP this additive effect disappeared. CDDP alone showed an enhanced phosphorylation of ERK1/2, which is a common pathway for apoptosis induction through NOXA. CDDP in combination with sorafenib led to an overall reduced phosphorylation level of ERK1/2, which also resulted in a low HB cell viability. Since CDDP and sorafenib have an opposing effect on phosphorylation of ERK1/2, we avoid the concomitant administration of both drugs in vivo. In HB xenografts both sorafenib alone or in alternating combination with CDDP reduced tumour growth compared to the control untreated group (p < 0.05). The combination treatment showed a higher and earlier reduction of tumor volume than sorafenib alone. In addition AFP levels did not significantly change in the treatment groups. Apoptotic areas as determined by TUNEL analysis increased significantly after administration of sorafenib or sorafenib and CDDP (p < 0.08). Furthermore CD31 staining showed a significant inhibition of angiogenesis as revealed by determination of the MVD in both treatment groups (p < 0.02). Discussion: Taken together the combination therapy of Sorafenib and CDDP led to an enhanced decrease in cell viability as well as in tumour progression and angiogenesis than sorafenib alone. Therefore a combined therapy might be a promising option in the treatment of high risk or recurrent hepatoblastoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4337. doi:1538-7445.AM2012-4337

Abstract 2491: High-dose acetaminophen with N-acetylcysteine rescue enhances the efficacy of cisplatin chemotherapy

Abstract Acetaminophen (AAP) has been shown to enhance cisplatin (CDDP) cytotoxicity in hepatoblastoma cell lines. Further, one published case report demonstrated clinical efficacy of administering acetaminophen in conjunction with cisplatin and followed 6 hours later with n-acetylcysteine (NAC) rescue. In this study, atypical rhabdoid tumor (ATRT) and germinoma cells were treated with CDDP and AAP with or without NAC rescue. We then evaluated the cells with western blots for cleaved PARP and caspase 3 to demonstrate effects on apoptosis. Further, MTS assays were performed to evaluate cell viability of treated cells. To evaluate the effects of the various treatments on oxidative stress, dihydroethidium (DHE) and 2′,7′-dichlorofluorescin (DCFH) assays were performed to test for superoxide and hydrogen peroxide levels, respectively. It was demonstrated that AAP enhances CDDP cell kill and apoptosis in germinoma and ATRT cell lines. Further, both AAP and CDDP, independently and together, cause increased oxidative stress as measured by DHE and DCFE, but this oxidative stress is only reversed by NAC in the DCFE assay. This makes sense because NAC, a glutathione precursor, detoxifies peroxide but not superoxide. Further, our group has learned of five previously unpublished patients with recurrent liver tumors (hepatocellular carcinoma and hepatoblastoma) treated with AAP and CDDP with delayed NAC rescue. All patients tolerated the treatment well. Two of the five patients responded to the treatment, suggesting the potential of this approach. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2491. doi:1538-7445.AM2012-2491

45 HEPATO-SPECIFIC MICRORNA-122 UP-REGULATION CORRELATED WITH LIVER DEVELOPMENT AND HEPATIC STEM/PROGENITORS DIFFERENTIATION

S 20TH ANNUAL CONFERENCE OF INASL, MARCH 2–4, 2012 S26 © 2012, INASL A b tra ts 44 OVER-EXPRESSION OF MICRORNA-122 (MIR-122) INDUCES APOPTOSIS BY TARGETING CYCLING1 AND BCL-W GENES IN VITRO S Kumar1, YK Chawla2, S Ghosh1, A Chakraborti1 Departments of 1Experimental Medicine and Biotechnology, 2Hepatology, Postgraduate Institute of Medical Education and Research, Chandigarh Background: MicroRNA-122 (miR-122), liver-specific microRNA, plays an important role in many physiological and pathological conditions of liver, such as stress response, lipid metabolism and hepatocellular carcinoma (HCC). It has been reported that, miR-122 is frequently downregulated in HCC; in contrast, its target genes cyclinG1 and bcl-w is up-regulated. Aim: To study the pro-apoptotic and tumor suppressor function of miR-122. Method: Initially, HeLa (cervical epithelial carcinoma) and HepG2 (human hepatoblastoma) cell lines, which lack miR-122 expression, were transfected with pEGPmmu-mir-122 expression vector by lipofectamine. After transfection, miR-122 vector expression was confirmed by fluorescence microscopy, fluorescent assisted cell sorter (FACS) analysis and quantitatively by real-time PCR (qRTPCR). The cytopathic changes were analyzed at different time intervals (24 h, 48 h, 72 h, and 96 h post-transfection) by light microscopy, propidium iodide (PI) staining and ethidium bromide/acridine orange (EtBr/AO) staining. Subsequently, the expression of cyclinG1 and bcl-w genes was quantified by qRT-PCR. Result: The qRT-PCR analysis determined the threefold higher expression of miR-122 in transfected cells as compared to control. Light microscopy showed altered cell morphology, and a consistent increase in population of dead cells was visible after 96 hours post-transfection by PI staining. Also, the transfected cells showed the higher apoptotic index as compared to the control by EtBr/AO staining. Furthermore, the miR-122 target genes cyclinG1 and bclw showed approximately fivefold down-regulation during overexpression of miR-122. Conclusion: This study shed light on the functions of miR-122, which may act as an endogenous apoptosis regulator, and thus negatively regulates tumourigenesis. HEPATOCELLULAR CARCINOMA AND OTHER TUMORS

Novel Neo Glycolipid: Formulation into Pegylated Cationic Liposomes and Targeting of DNA Lipoplexes to the Hepatocyte-Derived Cell Line HepG2

Liver parenchymal cells are an important target for the treatment of several metabolic and viral disorders. Corrective gene delivery for this purpose is an avenue that is receiving increasing attention. In the present study, we report a novel neo glycolipid that may be formulated into cationic liposomes with or without poly(ethylene glycol) decoration. Lipoplexes formed with plasmid DNA are nuclease resistant and are targeted to the human hepatoblastoma cell line HepG2 by selective asialoglycoprotein receptor mediation. Transfection levels achieved by lipoplexes containing the targeting ligand cholesteryl-3β-N-(4-aminophenyl-β-D-galactopyranosyl) carbamate were sixfold greater than those obtained with similar but untargeted lipoplexes.

A Strategy in The Design of Micellar Shape for Cancer Therapy

For cancer therapy, optimization of carrier features is necessary to effectively deliver the targeting agents to tumor sites. Biodegradable poly(ether-anhydrides) micelles with filamentous, rod-like, and spherical shapes are fabricated. Their size and morphology are characterized by AFM and TEM. The encapsulation of doxorubicin hydrochloride (DOX) into the micelles does not impact their shape. The effect of micellar shape on the drug loading capacity and encapsulation efficiency, as well as in vitro drug release, is investigated. The cellular uptakes are evaluated using fluorescence microscopy, confocal laser scanning microscopy and flow cytometry on co-cultures of human hepatoblastoma cell line (HepG2), lung epithelial cancer cell line (A549), and human nasopharyngeal epidermoid carcinoma cells (KB) and fibroblast normal cells mixed with the different shapes of DOX-loaded micelles. The results show that the spherical DOX-loaded micelles are more readily taken up by all types of cells. The impact of micellar shape on in vivo antitumor function is also assessed from changes of tumor volume, body weight loss, and survival rate of 4T1-bearing mice and the immunostaining of tumor sections for analysis of tumor cell proliferation. The results reveal that the filamentous DOX-loaded micelles possess the highest safety to body and the best therapeutic effects to artificial solid tumors. Therefore, the filamentous shape is deemed the most suitable morphology for design and engineering of drug vehicles for cancer therapy.

Transcription Factor GATA‐4 Is Abundantly Expressed in Childhood but Not in Adult Liver Tumors

Transcription factor GATA-4 is expressed in early fetal liver and essential for organogenesis. It is also implicated in carcinogenesis in several endoderm-derived organs. Hepatoblastoma (HB), the most common malignant pediatric liver tumor, has features of fetal liver including extramedullary hematopoiesis. We investigated the expression of GATA-4 and its purported target gene erythropoietin (Epo) in liver tumors and the role of GATA-4 in HB pathogenesis. Immunohistochemistry, Western blotting, and reverse transcription-polymerase chain reaction were used for liver samples from patients with HB or hepatocellular carcinoma. To further investigate the role of GATA-4 in pediatric liver tumors, we used adenoviral transfections of wild-type or dominant negative GATA-4 constructs in the human HB cell line, HUH6. We found abundant GATA-4 expression in both types of liver tumors in children, whereas it was absent in adult hepatocellular carcinoma. A close family member GATA-6 was expressed in a minority of childhood but not adult liver tumors. Epo, present in the fetal liver, was also expressed in childhood liver tumors. Moreover, cell line HUH6 was GATA-4 positive and produced Epo. We found that altering the amount of functional GATA-4 in HUH6 cells did not significantly affect either proliferation or apoptosis. GATA-4 is abundant in pediatric liver tumors, but unraveling its exact role in these neoplasms requires further investigation.

Treatment effects of the multikinase inhibitor sorafenib on hepatoblastoma cell lines and xenografts in NMRI‐Foxn1nu mice

Multidrug resistance is a major reason for poor treatment results in advanced hepatoblastoma (HB). Several alternative treatment options are currently under investigation to improve the prognosis of affected patients This study aimed to analyse the impact of sorafenib on the viability of HB cells and xenotransplanted HB tumours. Cell viability and apoptosis were evaluated in two HB cell lines (HUH6 and HepT1) after treatment with sorafenib using MTT and Caspase 3 activation assay. Extracellular signal-regulated kinase (ERK) phosphorylation was investigated using Western blot. In addition, sorafenib (30 mg/kg) was administered orally to NMRI mice bearing subcutaneous HUH6 derived tumours. Tumour progression and viability were monitored by tumour volume and α-fetoprotein (AFP) levels, and apoptosis was assessed using TUNEL assay. Tumour angiogenesis and mean vascular density (MVD) was determined using CD31 staining, ERK phosphorylation was detected using indirect immunofluorescence. Treatment with sorafenib led to decreased ERK phosphorylation, reduced cell viability and induction of apoptosis in HepT1 and HUH6 cells. In HB xenografts, sorafenib significantly reduced tumour growth compared with control (P < 0.05). AFP levels were lower in the sorafenib group (P = 0.07). Relative apoptotic areas detected using TUNEL assay were increased (P = 0.003). CD31 staining revealed inhibition of angiogenesis, and mean vascular density was lower in the sorafenib group (P = 0.02). ERK phosphorylation was reduced in tumours tissues after sorafenib treatment. Treatment with sorafenib led to a potent inhibition of cell viability, tumour progression and angiogenesis. Sorafenib might therefore also be a promising treatment option for high risk or recurrent HB.

Target-Specific Cellular Uptake of Folate-Decorated Biodegradable Polymer Micelles

For cancer therapy, folate (FA) and β-cyclodextrin (β-CD) decorated micelles based on the biodegradable pluronic F127-b-poly(ε-caprolactone) copolymer were fabricated. These micelles were measured by dynamic light scattering measurements and atomic force microscopy. The in vitro release of doxorubicin hydrochloride (DOX·HCl) from the biodegradable polymer micelles was performed in a phosphate-buffered saline solution at pH 7.4 and acetate buffer solution at pH 5.0 at the temperatures of 4, 25, and 37 °C, and the results show that the release was obviously influenced by the pH and temperature. The material cytotoxicity and the tumor cell growth inhibition assays of DOX·HCl-loaded micelles were studied with the human hepatoblastoma cell line (HepG2), the lung epithelial cancer cell line (A549), and human nasopharyngeal epidermoid carcinoma cells (KB) and fibroblast normal cells using fluorescence microscopy as well as confocal laser scanning microscopy. The cellular uptake was quantitatively analyzed to further evaluate the active targeting behaviors of the micelles by flow cytometry. These quantitative and qualitative results of cellular uptake of the micelles provide evidence for the different targeting efficiencies of FA decoration for HepG2, KB, and A549 tumor cells as well as fibroblast normal cells. It also suggested that FA- and β-CD-decorated doxorubicin-loaded micelles may have great potential as nanocarriers for targeted drug delivery.

Nuclear receptor mRNA expression by HBV in human hepatoblastoma cell lines

Recent studies have implicated nuclear receptors (NRs) in the development of hepatocarcinogenesis. We assumed that hepatitis B virus (HBV) alters the expression of NRs and coregulators, and compared the gene expression profiling for 84 NRs and related genes between HpeG2.2.15, which secretes complete HBV virion, and HepG2 by real-time RT-PCR with SyBr green. Forty (47.6%) genes were upregulated 2-fold or greater, and only 5 (5.9%) were downregulated 2-fold or more, in HepG2.2.15 compared to HepG2. These results suggest that HBV affects NRs and their related signal transduction, and that they play important roles in viral replication and HBV-related hepatocarcinogenesis.

Human liver fatty acid binding protein (hFABP1) gene is regulated by liver-enriched transcription factors HNF3β and C/EBPα

The human liver fatty acid binding protein (hFABP1) participates in cellular long-chain fatty acid trafficking and regulation of lipid metabolism and changes in hFABP1 are associated with an increased risk for type 2 diabetes, cardiovascular disease (CVD), and metabolic syndromes. Gene regulation of hFABP1 is not fully understood. Therefore, in the present study, the full length hFABP1 promoter (nucleotides −2125 to +51) and a series of truncated promoter regions were cloned. A luciferase reporter assay revealed that nucleotides −255 to +50 in the promoter region contained full of maximum hFABP1 promoter activity compared with the full length promoter. Furthermore high activity was shown when the plasmid was transfected into liver-derived cells such as the human hepatoblastoma cell line HepG2 and the hepatoma cell line Huh7. TFSEARCH and TESS programs were used to predict potential transcription factor binding sites. Two putative binding sites for the liver-enriched transcription factors hepatocyte nuclear factor 3β (HNF3β) and CCAAT/enhancer binding protein α (C/EBPα) were identified in the −255 nt to −155 nt hFABP1 promoter region. Site-directed mutagenesis of these two sites reduced dramatically hFABP1 promoter activity. In addition, the electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation assay (ChIP) revealed that these binding sites were recognized by HNF3β and C/EBPα respectively. Overexpression of HNF3β and C/EBPα enhanced the transcription of hFABP1 and consequently improved the protein level of hFABP1 in HepG2 cells, while knockdown of HNF3β and C/EBPα showed the inverse effects. Taken together, the hFABP1 gene is highly transcribed in liver-derived cells, and regulated predominantly by liver-enriched transcription factors HNF3β and C/EBPα.

Porous and single-skinned polyethersulfone membranes support the growth of HepG2 cells: A potential biomaterial for bioartificial liver systems

In this study, we evaluated a porous and single-layer skin polyethersulfone (PES) membrane as a material for use in hybrid bioartificial liver support systems. The PES membrane has been characterized as a single-layer skin structure, with a rough porous surface. Specifically, we studied the ability of the human hepatoblastoma cell lines (HepG2) to adhere, grow, and spread on the PES membrane. Furthermore, we examined albumin secretion, low-density lipoprotein uptake, and CYP450 activity of HepG2 cells that grew on the membrane. HepG2 cells readily adhered onto the outer surfaces of PES membranes. Over time, HepG2 cells proliferated actively, and confluent monolayer of cells covered the available surface area of the membrane, eventually forming cell clusters and three-dimensional aggregates. Furthermore, HepG2 cells grown on PES membranes maintained highly specific functions, including uptake capability, biosynthesis and biotransformation. These results indicate that PES membranes are potential substrates for the growth of human liver cells and may be useful in the construction of hollow fiber bioreactors. Porous and single-layer skin PES membranes and HepG2 cells may be potential biomaterials for the development of biohybrid liver devices.

Abstract LB-372: Expression and functional role of erbB2 in human hepatoblastoma

Abstract The receptor tyrosine kinase erbB2 is known to play a role in the development of hepatic progenitor cells and is often found to be expressed and mutated in different solid tumors. Activated erbB2 is a marker of poor prognosis in several malignancies and to date, successful strategies have been developed to target erbb2 receptor. The aim of this study was to determine the expression of erbb2 in human hepatoblastoma, the most common primary hepatic malignancy in early childhood, and its functional role in hepatoblastoma cell biology. Expression of erbB2 on the protein level was analyzed by Western Blot, immunofluorescence and RT-PCR of lysates of primary tumors and cell lines HuH6, HepT1, HepT3 and HepG2. Immunohistochemical staining was performed to determine phosphorylated erbB2 and subsequent kinases. Single strand conformation polymorphism (SSCP) analysis was used to scan known hotspots for mutations (exons 6, 8, 18–23 of erbB2) in a panel of 56 tumor samples. Cells were stimulated with rhHRG1-β. Activation of subsequent kinases was determined via Western Blot and MTT assay was used to determine proliferation after stimulation and overexpression of erbb2 via transient transfection. The influence of rhHRG1-β on the activity of the Wnt/ß-catenin pathway was measured by transfection with the TOP-/FOP-Flash reporter assay. The effects of erbB2 kinase knockdown by transient transfection with siRNA against erbB2 on apoptosis and proliferation were also evaluated. Expression of erbB2 was detected in all four cell lines and most of the primary tumors. Immunohistochemistry indicated expression of phosphorylated (activated) Erbb2 proteins in 90% and activation of subsequent kinases, which are downstream targets of erbb2. Mutations were not present in the erbB2 gene. Stimulation with HRG1-β induced phosphorylation of Akt, Erk1/2 and p70S6K and resulted in increased cell proliferation. This effect was not mediated by an increase of activity of the Wnt/ß-catenin signaling pathway because TOP-Flash reporter activity was not further increased in the presence of rhHRG1-β. Overexpression of erbB2 resulted in enhanced proliferation after stimulation with HRG1-β in all four cell lines, whereas knockdown of erbB2 decreased the proliferative effect of HRG1-β on hepatoblastoma cell lines. Knockdown of erbB2 did not induce apoptosis in hepatoblastoma cell lines. These results indicate that erbB2 might play an important functional role in hepatoblastoma cell biology. Further experiments are needed to evaluate the clinical significance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-372. doi:10.1158/1538-7445.AM2011-LB-372

Inflammatory stress exacerbates hepatic cholesterol accumulation via increasing cholesterol uptake and de novo synthesis

Cholesterol accumulation plays an important role in the progression of non-alcoholic fatty liver disease. We have demonstrated that inflammation aggravated cholesterol accumulation, causing tissue injury in the vessel and kidney. This study was undertaken to investigate whether inflammatory stress exacerbates hepatic cholesterol accumulation and we explored the underlying mechanisms. We used casein injection in C57BL/6J mice, interleukin-1β and interleukin-6 stimulation in human hepatoblastoma cell line (HepG2) cells to induce inflammatory stress. Oil Red O staining and intracellular cholesterol assay were used to quantify cellular cholesterol levels. Real-time reverse transcription polymerase chain reaction and Western blot were used to measure messenger RNA (mRNA) and protein expression of target genes. HMGCoA reductase (HMGCoA-r) enzymatic activity and cellular cholesterol synthesis were measured by radioactive methods. We demonstrated that inflammatory stress increased hepatic cholesterol accumulation and enhanced sterol regulatory element binding protein 2 (SREBP2), low-density lipoprotein receptor (LDLr) and HMGCoA-r mRNA and protein expression in livers of C57BL/6J mice and in HepG2 cells. A high-fat diet in mice or LDL loading in HepG2 cells inhibited mRNA and protein expression of these genes. However, the suppressive effect was overridden by inflammatory stress both in vivo and in vitro. Inflammatory stress increased HMGCoA-r enzymatic activity and cellular cholesterol synthesis in HepG2 cells in the absence or presence of LDL loading. Inflammatory stress disrupted hepatic SREBP2-mediated low-density lipoprotein receptor and HMGCoA-r feedback regulation resulting in exacerbated cholesterol accumulation in livers of C57BL/6J mice and HepG2 cells.

Effects of PCB mixtures on recognized targets of endocrine disrupters exposure: search for early biomarkers

Polychlorinated Biphenyls (PCBs) are Endocrine Disrupters (EDs) relevant to human dietary exposure. Since liver is the main organ involved in metabolism, a human hepatoblastoma cell line (HuH6) was used as in vitro model. A panel of nuclear receptors (NRs) was selected as early markers of PCBs exposure and analysed by qPCR. PCB congeners, grouped in three mixtures according to similarities in the modes of action, were used at the concentrations derived from previous data on human internal exposure. Preliminary results indicated that PCB mixtures exert different NRs modulations in HuH6.

Hepatitis B virus and hepatocellular carcinoma at the miRNA level

To study Hepatitis B virus (HBV) infection and its association with hepatocellular carcinoma (HCC) at the miRNA level. Three cellular models were used to investigate miRNA expression changes during HBV infection: human HepG2 hepatoblastoma cell line as a model without HBV infection; HepG2 cell line transfected with a 1.3-fold full-length HBV genome as an acute infection model; and HepG2.2.15 cell line, which is derived from HepG2 and stably transfected with a complete HBV genome, as a chronic infection model. The miRNA levels were examined using microarray technology. To explore the relationship between HBV infection and HCC genesis at the miRNA level, we downloaded from national center for biotechnology information Gene Expression Omnibus an miRNA expression dataset derived from HCC patients, most of whom are HBV carriers. We compared the miRNA expression alterations during HBV infection with those in HCC patients, by analyzing miRNA expression change profiles statistically. Seventy-seven and 48 miRNAs were differentially expressed during acute and chronic HBV infection, respectively. Among these miRNAs, 25 were in common, the intersection of which was significant under the hypergeometric test (P = 1.3 × 10⁻¹¹). Fourteen miRNAs were observed to change coherently in the acute and chronic infections, with one upregulated and 13 downregulated. Eleven showed inverse changes during the two phases of infection; downregulated in the acute infection and upregulated in the chronic infection. The results imply that common and specific mechanisms exist at the miRNA level during acute and chronic HBV infection. Besides, comparative analysis of the miRNA expression changes during HBV infection with those in HCC indicates that, although miRNA expression changes during HBV infection are distinct from those in HCC patients (P < 2.2 × 10⁻¹⁶), they exhibited significant correlations (P = 0.0229 for acute infection; P = 0.0084 for chronic infection). Perturbation of miRNA expression during chronic HBV infection was closer to that in HCC patients than that during acute HBV infection. This observation implies the contribution of miRNAs to HCC genesis from HBV infection. According to their patterns of differential expression in acute and chronic HBV infection, as well as in HCC, miRNAs of potential research interest could be identified, such as miR-18a/miR-18b, miR-106a, miR-221 and miR-101. For instance, the gradient expression alteration of miR-221 in the above three phases, which is downregulated in acute HBV infection, normally expressed in chronic HBV infection, and upregulated in HCC, indicates that it may be a key effector for progression of the disease. Our analysis provides insights into HBV infection and related HCC in relation to miRNAs, and reveals some candidate miRNAs for future studies.

Functional characterisation of the H365Y mutation of the 21-hydroxylase gene in congenital adrenal hyperplasia

The study subject was a 13 day-old boy admitted to hospital, with weight loss since birth. He presented with the vomiting and hypotension that are classical features of congenital adrenal hyperplasia (CAH). The most common type of CAH is an autosomal recessive disorder caused by mutations in the 21-hydroxylase (CYP21A2) gene. To examine the CYP21A2 gene, gene-specific PCR was carried out, followed by sequencing. The baby was shown to be a compound heterozygote H365Y/R356W for two CYP21A2 gene mutations each inherited from a different parent. One of the mutations has not previously been functionally characterised. The mutations were reconstructed in an expression plasmid and characterised in vitro after transient transfection into human embryonic kidney (HEK293T) and hepatoblastoma (C3A) cell lines followed by measurement of enzyme activity. The CYP21A2 H365Y mutant exhibited minimal 21-hydroxylase activity to convert 17-hydroxyprogesterone to 11-deoxycortisol or progesterone to 11-deoxycorticosterone. Western immunoblotting indicated that the H365Y enzyme was produced in more variable amounts than wild type; in particular, the H365Y mutant protein may be unstable and/or subject to a more rapid degradation by the human proteosome as well as catalytically inefficient. The double mutant genotype with a severe mutation on each allele is compatible with the clinical presentation.

Development of a drug resistance model for hepatoblastoma

Multidrug resistance (MDR) is a major reason for poor treatment results in hepatoblastoma (HB). The objective of this study was to establish a drug resistance model for HB to analyse alternative treatment options in vitro. Both HB cell lines HUH6 and HepT1 were xenotransplanted in NMRI mice (nu/nu) and 2 cycles of cisplatin (CDDP) treatment were administered. Thereafter, xenotransplants were excised and viable tumour cells were re-cultured. 3D cultures of HUH6 and HepT1 cells were generated on a low binding culture surface. Cell viability in response to CDDP/DOXO (doxorubicin) and apoptosis was assessed by MTT-assay and caspase 3 activity, respectively. Efflux of doxorubicin was measured by flow cytometry. Cellular levels of ABC-transporters (MDR1, MRP1, cMOAT and BRCP) were determined by real time rt-PCR. Only HepT1 cells isolated from HB xenografts showed resistance to CDDP, but did not survive repeated passages. Culturing HUH6 and HepT1 cells as spheroids was successful and 3D cultures showed an IC50-drift to higher drug concentrations for CDDP and DOXO compared to 2D cultures. Treatment with CDDP and DOXO led to homogeneous apoptosis in spheroids. Increased doxorubicin efflux in HUH6 spheroids was not influenced by the P-glycoprotein inhibitor tariquidar. Expression levels of MDR1, MRP1, cMOAT and BRCP in 3D cultures were similar to those in 2D cultures and were higher in HepT1 than in HUH6 cells. In conclusion, a 3D cell culture model for multidrug resistance was established for hepatoblastoma. The underlying mechanism involves altered accessibility of the cells for drugs rather than up-regulation of ABC-transporters.