Liver Epithelial Cell Line Research Articles

Curcumin and Turmeric Modulate the Tumor-Promoting Effects of Iron In Vitro

ABSTRACTFree or loosely chelated iron has tumor-promoting properties in vitro. Curcumin, a polyphenol derived from the food spice turmeric (Curcuma longa), is a potent antioxidant that binds iron. The primary aim of this study was to investigate whether curcuminoids prevent tumor-promoting effects of iron in T51B cells, a non-neoplastic rat liver epithelial cell line. Purified curcuminoids (curcumin) or a standardized turmeric extract similarly reduced oxidative stress and cytotoxicity associated with iron overload (IC50 values near 10 μM, P < 0.05). Inhibition of iron-induced tumor promotion (seen upon treatment with 200 μM ferric ammonium citrate ± curcumin/turmeric for 16 wk in culture; subsequently assayed by soft agar colony formation) was nearly complete at 20 μM of total curcuminoids (P < 0.05), a concentration predicted to only partially chelate the added iron. Surprisingly, lower curcumin concentrations (10 μM) increased tumor promotion (P < 0.01). Curcuminoids delivered as a standardized turmeric extract were taken up better by cells, had a longer half-life, and appeared more effective in blocking tumor promotion (P < 0.01), suggesting enhanced curcuminoid delivery to cells in culture. The primary finding that curcuminoids can inhibit tumor promotion caused by iron in T51B cells is tempered by evidence for an underlying increase in neoplastic transformation at lower concentrations.

A protein kinase A-ezrin complex regulates connexin 43 gap junction communication in liver epithelial cells

Communication between adjacent cells can occur via gap junctions (GJ) composed of connexin (Cx) hexamers that allow passage of small molecules. One of the most widely and highly expressed Cxs in human tissues is Cx43, shown to be regulated through phosphorylation by several kinases including PKA. Ezrin is a membrane associated protein that can serve as an A-kinase anchoring protein (AKAP) and hold an anchored pool of PKA. Here, we used the liver epithelial cell line IAR20, which expresses Cx43 as the predominant GJ protein, to test the hypothesis that Ezrin may associate with Cx43 in cell types that form stable GJs and serve as an AKAP. Our biochemical and proteomics data indicate that Ezrin associates with Cx43 in epithelial cells. Analyses by confocal immunofluorescence microscopy and proximity ligation assays demonstrate that Ezrin and Cx43 co-localize, together with zonula occludens-1 (ZO-1) and PKA RIα and RIIα, at the cell membrane. Quantitative gap-FRAP experiments show increased GJ intercellular communication after cAMP stimulation. Moreover, loading of cells with the Ht31 peptide that displaces both PKA RIα and RIIα from the AKAP or a peptide that disrupts the Cx43-Ezrin interaction reverts the effect and reduces the level of communication, supporting the hypothesis that in IAR20 cells Ezrin associates with Cx43 (in complex with ZO-1) which places PKA in proximity to Cx43, enabling its phosphorylation and GJ opening.

Functional consequences of co-expressing connexin40 or connexin45 with connexin43 on intercellular electrical coupling

The functional characteristics of the co-expression of connexin43, connexin40, and connexin45 proteins in human myocardium are thought to play an important role in governing normal propagation of the cardiac electrical impulse and in generating the myocardial substrate for some arrhythmias and conduction disturbances.A rat liver epithelial cell line, that endogenously expresses connexin43, was used to induce also expression of connexin40 or connexin45 after stable transfection using an inducible ecdysone system. Electrical coupling was estimated from measurement of the input resistance of transfected cells using an intracellular microelectrode to inject current and record changes to membrane potential.However, varied expression of the transfected connexin40 or connexin45 did not change electrical coupling, although connexin43/40 co-expression led to better coupling than connexin43/45 co-expression. Quantification of endogenous connexin43 expression, at both mRNA and protein levels, showed that it was altered in a manner dependent on the transfected connexin isotype.The data using rat liver epithelial cells indicate an increased electrical coupling upon expression of connexin40 and connexin43 but decreased coupling with connexin45 and connexin43 co-expression.

Upregulation of long non coding RNA PCAT-1 contributes to cell proliferation, migration and apoptosis in hepatocellular carcinoma.

Long non-coding RNAs (lncRNAs) exert regulatory functions on various biological processes in cancer cells, including proliferation, apoptosis and mobility. Prostate cancer-associated transcript1 (PCAT-1) is a novel lncRNA that promotes cell proliferation in prostate cancer, however, the effect of PCAT‑1 in hepatocellular carcinoma (HCC) remains to be elucidated. The present study hypothesized that PCAT‑1 also exerts an important effect in HCC. The current study investigated PCAT-1 expression levels in HCC tissue samples and HepG2 and Bel‑7402 cell lines using the reverse transcription-quantitative polymerase chain reaction. The results demonstrated that PCAT-1 was upregulated in HCC tissue samples and cell lines compared with adjacent non‑cancerous tissues and the L02 normal liver epithelial cell line. PCAT‑1 suppression using PCAT‑1 small hairpin RNA in HepG2 and Bel‑7402 cells inhibited cell proliferation and migration, and induced apoptosis. Overexpression of PCAT‑1 induced synthetic plasmid vectors was demonstrated to increase cell proliferation and migration, and inhibit apoptosis. Results from the present study suggest that PCAT‑1 exerts an oncogenic effect in HCC and silencing PCAT-1 may be a potential novel therapeutic strategy for HCC.

Effective and cancer-specific siRNA delivery to human hepatocellular carcinoma cells mediated by poly(beta-amino ester) nanoparticles.

Event Abstract Back to Event Effective and cancer-specific siRNA delivery to human hepatocellular carcinoma cells mediated by poly(beta-amino ester) nanoparticles. Camila Zamboni1, 2, Kristen K. Kozielski1, Nicholas Radant1, Luke J. Higgins3, Martin G. Pomper4 and Jordan J. Green1, 5, 6 1 Johns Hopkins University School of Medicine, Biomedical Engineering, United States 2 Universidade de São Paulo, Instituto do Câncer do Estado de São Paulo, Brazil 3 Stanford, Radiology, United States 4 Johns Hopkins Medical Institutions, Russell H. Morgan Department of Radiology and Radiological Science, United States 5 Johns Hopkins University School of Medicine, Department of Material Science and Engineering, United States 6 Johns Hopkins University School of Medicine, Neurosurgery and Ophthalmology, United States Introduction: Liver cancer is the second leading cause of cancer mortality worldwide. Hepatocellular carcinoma (HCC) accounts for approximately 90%[1] of primary liver cancers and has a five year post-diagnosis survival rate of only 12%[2],[3]. Limited efficacy of the current treatment options is one of the most challenging problems existing in the field of liver oncology. Gene therapy offers promising advantages that might overcome these limitations. Unfortunately, the poor selectivity in vector targeting still represents a main obstacle in gene therapy. Here we demonstrate that bioreducible poly(beta-amino ester)s (PBAEs) nanoparticles suggest an effective and cancer-specific siRNA delivery strategy to target human HCC cells. Materials and Methods: A human liver epithelial cell line (THLE-3) and a human hepatocellular carcinoma cell line (HepG2) were separately cultured and treated with nanoparticles formed by PBAEs and either AllStars Hs Cell Death siRNA (death siRNA) or AllStars Negative Control siRNA (Qiagen, Netherlands). While the death siRNA consists of a blend of siRNAs that silence important genes related to cell survival, the negative control has no target within mammalian cells. Effective delivery of death siRNA leads to cell death and the negative control allows evaluation of non-specific cell toxicity. Two bioreducible PBAEs, that we synthesized and evaluated at five different concentrations, were used as vectors to carry either death siRNA or negative control siRNA. Three days post-treatment, DAPI/PI staining was applied to differentiate live from dead cells. Live cells were counted using the ImageJ software. Results and Discussion: Our bioreducible PBAE nanoparticles lead to effective siRNA delivery and concomitant siRNA-mediated cell death to the human HCC cell line. The bioreducible polymer R646 (Figure 1), at 270ng/ul was considered to form the favorite formulation. A siRNA-mediated cell death of 89.1 ± 5% for HepG2 and 30.2 ± 16% for THLE-3 cells were observed, with a statistically significant difference between the HCC cells when compared to the healthy hepatocytes (p<0.05). Non-specific toxicity to healthy hepatocytes remained below 26.1 ± 6%. Conclusion: Bioreducible poly(beta-amino ester)s nanoparticles not only seem to provide high siRNA-mediated death to HCC cells but also enable cancer-target in vitro. Even though further validation in vitro and in vivo is required, these findings suggest that biomaterial mediated siRNA delivery using bioreducible poly(beta-amino ester)s is a potentially viable strategy for liver cancer treatment.

MiR-214/199a/199a* cluster levels predict poor survival in hepatocellular carcinoma through interference with cell-cycle regulators.

To identify the clinical and functional association of miR-214/199a/199a* cluster in human hepatocellular carcinoma (HCC) and to clarify the mechanism of miR-214. Kaplan-Meier and Cox proportional regression analyses were used to determine the association of miR-214/199a/199a* cluster levels with the survival of HCC patients. The role of miR-214 in regulating HCC cell proliferation was studied with miR-214 mimics/inhibitor-treated cells. Furthermore, the inhibition effect of miR-214 on E2F2, cyclin-dependent kinase (CDK) 3 and CDK6 expression was assessed in HCC cell lines with miR-214 mimics/inhibitors to increase/decrease miR-214 expression. Direct binding of miR-214 to the 3'-untranslated regions of E2F2, CDK3, and CDK6 was verified by dual-luciferase reporter assay. In analyzing HCC clinical specimens and cell lines, we discovered a uniform decrease in miR-214/199a/199a* expression in comparison with noncancerous tissue or normal liver epithelial cell lines. Higher miR-214 levels were related with improved patient survival. Overexpression of miR-214 in HCC cells inhibited proliferation by inducing G1-S checkpoint arrest. Conversely, RNA interference-mediated silencing of miR-214 promoted cell-cycle progression and accelerated the proliferation of HCC cells. E2F2, CDK3 and CDK6 were each directly targeted for inhibition by miR-214, and restoring their expression reversed miR-214 inhibition of cell-cycle progression. The relationship between expression of miR-214 and its targets was confirmed in HCC tumor xenografts and clinical specimens. Our results demonstrate that miR-214 has tumor-suppressive activity in HCC through inhibition of E2F2, CDK3 and CDK6.

URG4/URGCP enhances the angiogenic capacity of human hepatocellular carcinoma cells in vitro via activation of the NF-κB signaling pathway.

BackgroundAngiogenesis is essential for tumor growth. Hepatocellular carcinoma (HCC) is characterized by hypervascularity; high levels of angiogenesis are associated with poor prognosis and a highly invasive phenotype in HCC. Up-regulated gene-4 (URG4), also known as upregulator of cell proliferation (URGCP), is overexpressed in multiple tumor types and has been suggested to act as an oncogene. This study aimed to elucidate the effect of URG4/URGCP on the angiogenic capacity of HCC cells in vitro.MethodsExpression of URG4/URGCP in HCC cell lines and normal liver epithelial cell lines was examined by Western blotting and quantitative real-time PCR. URG4/URGCP was stably overexpressed or transiently knocked down using a shRNA in two HCC cell lines. The human umbilical vein endothelial cell (HUVEC) tubule formation and Transwell migration assays and chicken chorioallantoic membrane (CAM) assay were used to examine the angiogenic capacity of conditioned media from URG4/URGCP-overexpressing and knockdown cells. A luciferase reporter assay was used to examine the transcriptional activity of nuclear factor kappa – light – chain - enhancer of activated B cells (NF-κB). NF-κB was inhibited by overexpressing degradation-resistant mutant inhibitor of κB (IκB)-α. Expression of vascular endothelial growth factor C (VEGFC), tumor necrosis factor-α (TNFα), interleukin (IL)-6, IL-8 and v-myc avian myelocytomatosis viral oncogene homolog (MYC) were examined by quantitative real-time PCR; VEGFC protein expression was analyzed using an ELISA.ResultsURG4/URGCP protein and mRNA expression were significantly upregulated in HCC cell lines. Overexpressing URG4/URGCP enhanced - while silencing URG4/URGCP decreased - the capacity of HCC cell conditioned media to induce HUVEC tubule formation and migration and neovascularization in the CAM assay. Furthermore, overexpressing URG4/URGCP increased - whereas knockdown of URG4/URGCP decreased - VEGFC expression, NF-κB transcriptional activity, the levels of phosphorylated (but not total) IκB kinase (IKK) and IκB-α, and expression of TNFα, IL-6, IL-8 and MYC in HCC cells. Additionally, inhibition of NF-κB activity in HCC cells abrogated URG4/URGCP-induced NF-κB activation and angiogenic capacity.ConclusionsThis study suggests that URG4/URGCP plays an important pro-angiogenic role in HCC via a mechanism linked to activation of the NF-κB pathway; URG4/URGCP may represent a potential target for anti-angiogenic therapy in HCC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1378-7) contains supplementary material, which is available to authorized users.

Zataria multiflora: chemical and biological diversity in the essential oil

The essential oils from six chemo-types of Zataria multiflora (ZMO) was obtained and examined for chemical composition and antioxidant, antimicrobial and cytotoxic activities. Chemical analysis of ZMO was done by using gas chromatography and mass spectrometry (GC/MS). The antioxidant capacity of ZMO was examined through measuring ABTS, H2O2, nitrite and malondialdehyde (MDA) scavenging effects. The antibacterial activity toward Salmonella typhimurium, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, Aspergillus niger and Candida albicans were tested by minimum inhibitory concentration (MIC) determination. The cytotoxic effect of ZMO was tested on the human nasopharyngeal cancer cell line (KB), liver hepatocellular carcinoma cell line (HepG-2) and epithelial breast cancer cell line (MCF-7). The main components of ZMO were carvacrol (4–57%), thymol (1–45%), p-cymene (4–20%), γ-terpinene (1–38%) and linalool (1–33%). ZMO exhibited good antioxidant activity against ABTS, H2O2, nitrite and MDA at concentrations less than 10 μg/mL. ZMO showed good cytotoxic activity against tested bacteria and fungi, and tumor cell line at concentrations less than 10 and 20 μg/mL, respectively. Thymol-rich ZMO exhibited stronger antioxidant activity, while carvacrol-rich ZMO exhibited stronger antibacterial, antifungal and antitumor activities.

Multiple compound-related adverse properties contribute to liver injury caused by endothelin receptor antagonists.

Drug-induced liver injury has been observed in patients treated with the endothelin receptor antagonists sitaxentan and bosentan, but not following treatment with ambrisentan. The aim of our studies was to assess the possible role of multiple contributory mechanisms in this clinically relevant toxicity. Inhibition of the bile salt export pump (BSEP) and multidrug resistance-associated protein 2 was quantified using membrane vesicle assays. Inhibition of mitochondrial respiration in human liver-derived HuH-7 cells was determined using a Seahorse XF(e96) analyzer. Cytochrome P450 (P450)-independent and P450-mediated cell toxicity was assessed using transfected SV40-T-antigen-immortalized human liver epithelial (THLE) cell lines. Exposure-adjusted assay ratios were calculated by dividing the maximum human drug plasma concentrations by the IC50 or EC50 values obtained in vitro. Covalent binding (CVB) of radiolabeled drugs to human hepatocytes was quantified, and CVB body burdens were calculated by adjusting CVB values for fractional drug turnover in vitro and daily therapeutic dose. Sitaxentan exhibited positive exposure-adjusted signals in all five in vitro assays and a high CVB body burden. Bosentan exhibited a positive exposure-adjusted signal in one assay (BSEP inhibition) and a moderate CVB body burden. Ambrisentan exhibited no positive exposure-adjusted assay signals and a low CVB body burden. These data indicate that multiple mechanisms contribute to the rare, but potentially severe liver injury caused by sitaxentan in humans; provide a plausible rationale for the markedly lower propensity of bosentan to cause liver injury; and highlight the relative safety of ambrisentan.

Gap Junctional Intercellular Communication Increases Cytotoxicity and Reduces Resistance to Hydroxyurea

Background: Gap junctions enable small molecules to diffuse between adjacent cells and have been associated with greater cytotoxicity of radiation and anti-cancer drugs. We investigated whether this gap junctional intercellular communication (GJIC) affected the cytotoxicity of the classic ribonucleotide reductase (RR) inhibitor and anti-cancer agent, hydroxyurea (HU). Materials and Methods: We used GJIC-proficient and deficient, connexin 43-expressing WB rat liver epithelial cell lines. We compared HU toxicity by crystal violet assay, effects of the drug on deoxynucleotide pools by HPLC, and ability of GJIC to increase toxicity of HU-resistant cells through a bystander effect in co-culture experiments. Results: GJIC-proficient cells were three- to five-fold more sensitive (IC50 0.1 mM) to HU than GJIC-deficient derivatives (IC50 0.3 - 0.5 mM). This sensitivity depended upon GJIC because treatment of GJIC-proficient cells with the GJIC blocker oleamide decreased HU toxicity by approximately 60% - 80% and restoration of GJIC in GJIC-deficient cells by stable transduction of connexin 32-encoding Gjb1 increased HU toxicity (IC500.1 mM). The effects were not due to connexin expression per se or its localization since all cell lines expressed comparable quantities of connexin 43 that was localized to the plasma membrane. Also HU sensitivity was not related to differential effects on nucleotide metabolism in the cells. Thymidine triphosphate levels increased and deoxyadenosine triphosphate levels decreased similarly (15% - 20%) in GJIC-proficient and deficient cells over 24 h of HU treatment. More importantly, when HU-resistant cells were co-cultured with sensitive cells, the resistant cells were killed only when GJIC was present. Conclusion: The data suggest that GJIC enhances cytotoxicity and decreases resistance to HU. These results may be important clinically if GJIC can be enhanced in drug-resistant cells.

2-Methoxyestradiol binding of GPR30 down-regulates angiotensin AT1 receptor

Controlling angiotensin AT1 receptor function has been shown to be protective for many pathophysiological disorders. Although estrogen metabolite, 2-methoxyestradiol (2ME2) can down-regulate angiotensin AT1 receptor expression independently of nuclear receptors, no specific cellular targets have been identified. This study was focused on identification and validation of a cellular target responsible for 2ME2-mediated angiotensin AT1 receptor down-regulation in a continuously passaged rat liver epithelial cell line. Cell membranes were isolated and used to determine 2ME2 specific binding. Cell membranes exposed to [3H]2ME2 showed specific saturable binding, which was found to be pertussis toxin (PTx) sensitive. Under similar conditions, G-protein coupled receptor 30 (GPR30) agonist (G1) and antagonist (G15) inhibited 2ME2 specific binding. In these cells GPR30 was found localized to endoplasmic reticulum (ER) membranes. In intact cells, G1 down-regulated angiotensin AT1 receptor expression and this effect was reversed by G15. Furthermore, 2ME2 mediated activation of epidermal growth factor receptor (EGFR) followed by ERK1/2 phosphorylation, an essential signaling step in angiotensin AT1 receptor down-regulation, was abrogated by G15, suggesting that this signal is GPR30 dependent. Additionally, EGF was found to independently down-regulate angiotensin AT1 receptor in an ERK1/2-dependent manner. In summary, our results demonstrate for the first time that 2ME2 down-regulation of angiotensin AT1 receptor is dependent on ER membrane-associated GRP30. Moreover, this effect is facilitated by GPR30 dependent transactivation of EGFR and ERK1/2 phosphorylation. This study provides further understanding of the physiological significance of 2ME2 and its role in modulating angiotensin AT1 receptor expression.

Protective action of n-3 fatty acids on benzo[a]pyrene-induced apoptosis through the plasma membrane remodeling-dependent NHE1 pathway

Plasma membrane is an early target of polycyclic aromatic hydrocarbons (PAH). We previously showed that the PAH prototype, benzo[a]pyrene (B[a]P), triggers apoptosis via DNA damage-induced p53 activation (genotoxic pathway) and via remodeling of the membrane cholesterol-rich microdomains called lipid rafts, leading to changes in pH homeostasis (non-genotoxic pathway). As omega-3 (n-3) fatty acids can affect membrane composition and function or hamper in vivo PAH genotoxicity, we hypothesized that addition of physiologically relevant levels of polyunsaturated n-3 fatty acids (PUFAs) might interfere with B[a]P-induced toxicity. The effects of two major PUFAs, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), were tested on B[a]P cytotoxicity in the liver epithelial cell line F258. Both PUFAs reduced B[a]P-induced apoptosis. Surprisingly, pre-treatment with DHA increased the formation of reactive B[a]P metabolites, resulting in higher levels of B[a]P-DNA adducts. EPA had no apparent effect on B[a]P metabolism or related DNA damage. EPA and DHA prevented B[a]P-induced apoptotic alkalinization by affecting Na+/H+ exchanger 1 activity. Thus, the inhibitory effects of omega-3 fatty acids on B[a]P-induced apoptosis involve a non-genotoxic pathway associated with plasma membrane remodeling. Our results suggest that dietary omega-3 fatty acids may have marked effects on the biological consequences of PAH exposure.

Cortactin is a sensitive biomarker relative to the poor prognosis of human hepatocellular carcinoma

BackgroundCortactin is an important regulator involved in invasion and migration of hepatocellular carcinoma (HCC). The aim of this study was to elucidate the forecasting role of cortactin in resectable HCCs.MethodsWe compared the invasiveness and motility among liver epithelial cell line and HCC cell lines by using Transwell assay and wound healing assay. We further investigated the CTTN mRNA expression by real-time PCR. Next, 91 HCC and 20 normal liver tissue samples were detected by IHC and real-time PCR. Finally, we analyzed the clinicopathologic features and survival time of the HCC cases.ResultsWe identified that HepG2, LM3, and SK-Hep-1 had more invasiveness and motility (P <0.05). Compared with liver epithelial cell line, CTTN expression was higher in LM3, HepG2, and MHCC97-L (P <0.01) and lower in SK-Hep-1 (P <0.05). IHC examination showed cortactin expression was closely relative to TNM stage (AJCC/UICC), cancer embolus, and metastasis (P <0.01). Cortactin overexpression indicated a longer survival time of 52 ± 8.62 months and low expression of a shorter survival time of 20 ± 4.95 months (P <0.01). Cortactin examination has more predictive power in patients with Child-Pugh grade A and BCLC stage 0-B.ConclusionsOverexpression of cortactin is closely associated with poor human HCCs prognosis that caused by cancer embolus and metastasis. Cortactin and CTTN should be used for differentiating varieties of survival for patients after HCC resection.

Molecular risk assessment of methyl isocyanate-induced liver carcinogenesis

Introduction: The increased incidence of hepatocellular carcinoma (HCC) over the last 50-60 years has been largely attributed to three factors: lifestyle, age and diffusion of environmental carcinogens. Molecular implications of isocyanates, ubiquitous chemicals with wide range of industrial applications, on liver carcinogenesis still remain elusive.Aims: To study the role of isocyanate in liver carcinogenesis.Methods: Experiments were performed on the cultured human liver epithelial cell line to ascertain the possible molecular risks associated with isocyanate exposure using N-succinimidyl N-methylcarbamate, a chemical entity that mimics the effects of methyl isocyanatein vitro. Qualitative and quantitative assessment of phosphorylation states of ATM, H2AX, ATR and p53, put forth the kinetics of DNA damage. Analyses of apoptosis in treated cells and inflammatory cytokines secreted in culture supernatant were done through annexin-V FITC/PI assay and cytometric bead array respectively. Cytogenetic studies along with inter simple sequence repeat PCR were done in control and treated cells to check for the possible precariousness at the chromosomal and microsatellite levels of the genome.Results: An increasing trend of all the DNA damage responsive parameters along with the apoptotic index were displayed in treated cells. Isocyanate exposed cells secreted an elevated levels of inflammatory cytokines in the culture supernatant. Cytogenetic analysis and inter simple sequence repeat PCR revealed varied chromosomal anomalies with greater instability at the microsatellite level.Conclusion: To conclude, these findings demonstrate that human liver epithelial milieu could confront with molecular risks due to isocyanate exposure attributing to defective DNA repair pathway along with de-regulated cell cycle and genomic instability which may accelerate the development of HCC. Introduction: The increased incidence of hepatocellular carcinoma (HCC) over the last 50-60 years has been largely attributed to three factors: lifestyle, age and diffusion of environmental carcinogens. Molecular implications of isocyanates, ubiquitous chemicals with wide range of industrial applications, on liver carcinogenesis still remain elusive. Aims: To study the role of isocyanate in liver carcinogenesis. Methods: Experiments were performed on the cultured human liver epithelial cell line to ascertain the possible molecular risks associated with isocyanate exposure using N-succinimidyl N-methylcarbamate, a chemical entity that mimics the effects of methyl isocyanatein vitro. Qualitative and quantitative assessment of phosphorylation states of ATM, H2AX, ATR and p53, put forth the kinetics of DNA damage. Analyses of apoptosis in treated cells and inflammatory cytokines secreted in culture supernatant were done through annexin-V FITC/PI assay and cytometric bead array respectively. Cytogenetic studies along with inter simple sequence repeat PCR were done in control and treated cells to check for the possible precariousness at the chromosomal and microsatellite levels of the genome. Results: An increasing trend of all the DNA damage responsive parameters along with the apoptotic index were displayed in treated cells. Isocyanate exposed cells secreted an elevated levels of inflammatory cytokines in the culture supernatant. Cytogenetic analysis and inter simple sequence repeat PCR revealed varied chromosomal anomalies with greater instability at the microsatellite level. Conclusion: To conclude, these findings demonstrate that human liver epithelial milieu could confront with molecular risks due to isocyanate exposure attributing to defective DNA repair pathway along with de-regulated cell cycle and genomic instability which may accelerate the development of HCC.

A case of severe jaundice caused by thiamazole therapy

S 21 ANNUAL CONFERENCE—2013 D LI industrial applications, on liver carcinogenesis still remain elusive. Aims: To study the role of isocyanate in liver carcinogenesis. Methods: Experiments were performed on the cultured human liver epithelial cell line to ascertain the possible molecular risks associated with isocyanate exposure using Nsuccinimidyl N-methylcarbamate, a chemical entity that mimics the effects of methyl isocyanatein vitro. Qualitative and quantitative assessment of phosphorylation states of ATM, H2AX, ATR and p53, put forth the kinetics of DNA damage. Analyses of apoptosis in treated cells and inflammatory cytokines secreted in culture supernatant were done through annexin-V FITC/PI assay and cytometric bead array respectively. Cytogenetic studies along with inter simple sequence repeat PCR were done in control and treated cells to check for the possible precariousness at the chromosomal and microsatellite levels of the genome. Results: An increasing trend of all the DNA damage responsive parameters along with the apoptotic index were displayed in treated cells. Isocyanate exposed cells secreted an elevated levels of inflammatory cytokines in the culture supernatant. Cytogenetic analysis and inter simple sequence repeat PCR revealed varied chromosomal anomalies with greater instability at the microsatellite level. Conclusion: To conclude, these findings demonstrate that human liver epithelial milieu could confront with molecular risks due to isocyanate exposure attributing to defective DNA repair pathway along with de-regulated cell cycle and genomic instability which may accelerate the development of HCC. Corresponding author. Kewal Krishan Maudar. E-mail: maudarji@yahoo.com A CASE OF SEVERE JAUNDICE CAUSED BY THIAMAZOLE THERAPY Maria Sergeevna Zharkova, Marina Trofimovich Maevskaya, Elena Nikolaevna Shirokova, Tatjana Petrovna Nekrasova, Marina Victorovna Ivashkin Hepatology Department, University Clinical Hospital N 2, First Moscow State Medical University, Moscow, Russia Objective:Hepatotoxicity is a rare serious adverse reaction to antithyroid agents. It occurs with a frequency of 0.1– 0.2% of treated patients. Here we describe a case of severe jaundice with expected poor prognosis in a Thiamazoletreated patient with thyrotoxicosis. Case report: A 48-year-old woman with thyrotoxicosis developed jaundice after two weeks of Thiamazole therapy (40 mg/day). Viral hepatitis and mechanical biliary obstruction were excluded. Drug-induced liver injury had S38 been suspected. Despite drug discontinuation, weakness, skin itching, weight loss intensified. She was hospitalized to our clinic six weeks after disease onset. There were significant jaundice and visible traces after scratching; tachycardia. Liver and spleen were not palpable. Laboratory analyses showed elevation of ALT> AST (5-6 times higher ULN), total bilirubin (36 times higher ULN) due to both fractions, hypoalbuminemia (2.6 g/dl). Tests for autoantibodies were negative. Serum thyroid-stimulating hormone level was less than 0.01 uIU/ml (N: 0.4–4), free thyroxine two times higher ULN. Liver biopsy revealed parenchymatous and canalicularbilirubinostasis, necrosis of some hepatocytes. We diagnosed: Thiamazole-induced hepatitis. Hepatic failure: jaundice, hypoalbuminemia, thyrotoxicosis. In spite of drug withdrawal and administration of Ademethionine, UDCA, plazmapheresis, we observed a very slow regression of jaundice. Prednisolone prescription led to a rapid full patient's recovery with normalization of all biochemical markers during a month. The patient passed later radioactive iodine ablation of the thyroid gland. Discussion: Here we observed a mixed (dose-dependent and idiosyncratic) liver injury. There was an untypical form of hepatotoxicity hepatocellular damage with jaundice, without increase in laboratory markers of cholestasis, what makes the uniqueness of this clinical case. Conclusion: Severe jaundice is a rare adverse effect of antithyroid drugs’ therapy. The liver injury is usually reversible after drug withdrawal but in some cases can require Prednizolone administration. Physicians should keep in mind such a rare adverse reaction of antithyroid drugs. Corresponding Author. Maria Sergeevna Zharkova. E-mail: zharkovamaria@mail.ru HCV INFECTION IN THE HIV PATIENT IN ORAN ALGERIA Nadjet Allab Mouffok, Fatima Bensadoun, Ahmed Kouiad Belkadi

Distinct gene expression signatures during development of distant metastasis

Using cDNA microarrays, we have conducted a systematic characterization of global gene expression in v-raf or v-raf/v-myc transformed rat liver epithelial (RLE) cell lines exhibiting both non-metastatic and metastatic phenotypes. Seven transformed cell lines were compared with the non-transformed parental RLE cell. The hierarchical clustering analysis of gene expression profiles revealed two groups reflecting the in vivo metastatic potential of the cells. Surprisingly, one non-metastatic cell line T1 was co-clustered with metastatic cell lines, suggesting that T1 underwent significant genetic changes. The T1 cell line was further compared against all the metastatic cell lines in order to reveal the critical genes required for metastatic conversion but not expressed in the T1. These data demonstrated that expression of genes involved in apoptosis and immune cell homing were altered in all metastatic cell lines. Survival of both intravasated cells in circulation systems and extravasated cells in a new tissue environment might be critical for the final step in the metastatic process. Our study provides gene expression signatures consistent with two critical events in the metastatic process, namely, the acquisition of early homing capacity and increased survival potential of the tumor cells.

Characterization of THLE-Cytochrome P450 (P450) Cell Lines: Gene Expression Background and Relationship to P450-Enzyme Activity

The hepatic SV40 large T-antigen immortalized human liver epithelial (THLE) cell line and sublines transfected with cytochromes P450 (P450s) are increasingly being used for evaluation of potential drug-induced liver injury. So far, the available information on transporter and enzyme expression in these transfected cell systems is scattered. The purpose of this study was to characterize THLE cell lines with respect to transporter and enzyme expression. The mRNA expression of 96 typical drug absorption, distribution, metabolism and excretion genes, which encode a selection of transporters, phase I and II drug-metabolizing enzymes, and nuclear hormone receptors, was investigated in five THLE cell lines transfected with individual human P450s and in mock-transfected THLE-null cells using real-time polymerase chain reaction. The majority of the analyzed genes was either absent or expressed at low levels in the THLE-null and THLE-P450 cells, apart from housekeeping genes and the individual transfected P450s. Enzyme activity measurements provided confirmatory functional data for CYP2C9 and CYP3A4. Comparison with gene expression in human liver revealed an overall much lower gene expression in the THLE cell lines. The low levels of expression of a broad range of P450 genes in the THLE cell lines highlight the value of studies undertaken with P450-expressing cell lines for investigation of mechanisms of P450 metabolite-mediated hepatotoxicity. However, when attempting to translate between data obtained in THLE cell lines in vitro and functional consequences in vivo, it is important to take account of their limited expression of genes encoding many other drug-metabolizing enzymes and hepatic transporters.

Tumor promoting effects of cyanobacterial extracts are potentiated by anthropogenic contaminants – Evidence from in vitro study

Inhibition of gap junctional intercellular communication (GJIC) is affiliated with tumor promotion process and it has been employed as an in vitro biomarker for evaluation of tumor promoting effects of chemicals. In the present study we investigated combined effects of anthropogenic environmental contaminants 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB 153) and fluoranthene, cyanotoxins microcystin-LR and cylindrospermopsin, and extracts of laboratory cultures of cyanobacteria Aphanizomenon gracile and Cylindrospermopsis raciborskii, on GJIC in the rat liver epithelial cell line WB-F344. Binary mixtures of PCB 153 with fluoranthene and the mixtures of the two cyanobacterial strains elicited simple additive effects on GJIC after 30min exposure, whereas microcystin-LR and cylindrospermopsin neither inhibited GJIC nor altered effects of PCB 153 or fluoranthene. However, synergistic effects were observed in the cells exposed to binary mixtures of anthropogenic contaminants (PCB 153 or fluoranthene) and cyanobacterial extracts. The synergistic effects were especially pronounced after prolonged (6–24h) co-exposure to fluoranthene and A. gracile extract, when mixture caused nearly complete GJIC inhibition, while none of the individual components caused any downregulation of GJIC at the same concentration and exposure time. The effects of cyanobacterial extracts were independent of microcystin-LR or cylindrospermopsin, which were not detected in cyanobacterial biomass. It provides further evidence on the presence of unknown tumor promoting metabolites in cyanobacteria. Clear potentiation of the GJIC inhibition observed in the mixtures of two anthropogenic contaminants and cyanobacteria highlight the importance of combined toxic effects of chemicals in complex environmental mixtures.

Abstract 1323: Liver-specific knockout mice and liver-derived cell lines provide insights into potential roles for the GP73/GOLM1 HCC serum biomarker - association with sustained cell proliferation

Abstract Golgi Protein-73kDa (GP73/GOLM1) is a resident cis Golgi Type II membrane protein that is nearly undetectable in normal liver, but whose expression is upregulated in hepatocellular carcinoma (HCC) and whose circulating levels increase in the plasma of patients with chronic liver disease and HCC. As such, it is the subject of ongoing clinical trials investigating its use as a serum biomarker for HCC. GP73/GOLM1 is cleaved by a proprotein convertase and secreted by proliferating cells, an activity possibly elevated in cancer, yet its biological role in the liver and in the pathogenesis of HCC remains unclear. In order to investigate possible biological roles for GP73/GOLM1, liver-specific knockout mice (C57BL/6) were generated using the Cre-loxP system, characterized by a “floxed” GOLM1 gene and Cre recombinase driven by the albumin promoter. GP73/GOLM1 Cre+/−genotypes were confirmed by PCR analysis. GP73/GOLM1Fl/Fl/Cre(+) animals showed no obvious biological phenotype compared to their Cre(-) littermates, and exhibited normal growth, behavior, and successful mating, suggesting that hepatic GP73/GOLM1 is not vital for normal physiological function, as might be hypothesized from its normally low expression in the liver. Previous studies indicated that GP73/GOLM1 expression was not upregulated in post-hepatectomy liver regeneration, suggesting that its activated expression in the liver is linked to inflammatory or carcinogenic processes rather than transiently increased mitosis. Examination of GP73/GOLM1 expression in 14 human HCC cell lines revealed similar expression levels in epithelial and mesenchymal cell lines, and in an SV40-transformed nontumorigenic human liver epithelial cell line. Rodent in vitro models of hepatocellular transformation revealed equal GP73/GOLM1 expression levels in nontumorigenic parent cell lines and their transformed tumorigenic derivatives. Collectively, the data from the in vivo and in vitro models thus far do not support a direct association of GP73/GOLM1 expression with liver mitogenesis or differentiation, but rather suggest that its expression may be linked to a sustained commitment to cell proliferation - such as occurs in cell lines, liver disease and cancerous transformation, promoted by an inflammatory microenvironment. Possible mechanistic roles for GP73/GOLM1 in HCC development and progression are currently being tested in carcinogenesis models with liver-specific knockout mice. A website on GP73/GOLM1 has also been established as a resource for the research community interested in the study of this protein. 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 1323. doi:1538-7445.AM2012-1323

Side Population Cells From an Immortalized Human Liver Epithelial Cell Line Exhibit Hepatic Stem-Like Cell Properties.

The existence of hepatic stem cells in human livers is controversial. We investigated whether the side population (SP) cells derived from an immortalized human liver epithelial cell line THLE-5b possess the properties of hepatic stem-like cells. SP cells derived from THLE-5b were isolated using flow cytometry and were assayed for the expression of phenotypic markers by reverse transcription polymerase chain reaction and immunostaining. THLE-5b SP cells retained the capacity to generate both SP and non-SP cells, showed a capacity for self-renewal, and were more efficient in colony formation than non-SP cells. Neither the SP nor the non-SP cells formed tumors when transplanted into athymic nude mice or severe combined immunodeficient mice. The expression level of stem cell-associated markers such as an ATP-binding cassette membrane transporter, epithelial cell adhesion molecule, c-kit, Thy-1, and octomer binding transcription factor 4 was higher in SP cells than in non-SP cells. When cultivated as rotation-mediated aggregates, the expression of liver-specific genes including tryptophan oxygenase and CYP3A4 was up-regulated in SP cells, suggesting that THLE-5b SP cells have the ability to differentiate into a hepatocyte phenotype. One of the clonal cell lines derived from the SP cells expressed stem cell-associated markers. These results indicate that SP cells derived from THLE-5b possess hepatic stem-like cell properties and suggest that THLE-5b can be used as a model of normal human liver progenitor or stem cell line.