Rat Hepatocellular Carcinoma Cell Lines Research Articles

A Comparison of Primary Human Hepatocytes and Hepatoma Cell Lines to Model the Effects of Fatty Acids, Fructose and Glucose on Liver Cell Lipid Accumulation.

Non-alcoholic fatty liver disease (NAFLD) begins with lipid accumulation within hepatocytes, but the relative contributions of different macronutrients is still unclear. We investigated the impact of fatty acids, glucose and fructose on lipid accumulation in primary human hepatocytes (PHH) and three different cell lines: HepG2 (human hepatoblastoma-derived cell line), Huh7 (human hepatocellular carcinoma cell line) and McA-RH7777 (McA, rat hepatocellular carcinoma cell line). Cells were treated for 48 h with fatty acids (0 or 200 μM), glucose (5 mM or 11 mM) and fructose (0 mM, 2 mM or 8 mM). Lipid accumulation was measured via Nile Red staining. All cell types accumulated lipid in response to fatty acids (p < 0.001). PHH and McA, but not HepG2 or Huh7 cells, accumulated more lipid with 11 mM glucose plus fatty acids (p = 0.004, fatty acid × glucose interaction, for both), but only PHH increased lipid accumulation in response to fructose (p < 0.001). Considerable variation was observed between PHH cells from different individuals. Lipid accumulation in PHH was increased by insulin (p = 0.003) with inter-individual variability. Similarly, insulin increased lipid accumulation in both HepG2 and McA cells, with a bigger response in McA in the presence of fatty acids (p < 0.001 for fatty acid × insulin). McA were more insulin sensitive than either HepG2 or Huh7 cells in terms of AKT phosphorylation (p < 0.001 insulin × cell type interaction). Hence, glucose and fructose can contribute to the accumulation of lipid in PHH with considerable inter-individual variation, but hepatoma cell lines are not good models of PHH.

Establishment of an Immunocompetent Metastasis Rat Model with Hepatocyte Cancer Stem Cells.

Simple SummaryCancer stem cells (CSCs) are considered responsible for the maintenance, metastasis, and recurrence of various tumors. Here, we isolated a novel CSCs, TW-1, which can self-renew, differentiate and resistant to sorafenib. We also established novel orthotopic and metastasis models in immunocompetent rats with TW-1. These immunocompetent animal model can provide a normal physiological condition for anti-cancer drug screening. Using machine learning, we predicted HCC (AUCs > 0.9) with eight biomarkers including 6 highly expressed in TW-1/HTC and 2 well-known biomarkers from recent HCC studies. In conclusion, our results showed that TW-1 was a novel rat CSC line, and the animal model established with TW-1, is not only useful for the study of metastasis of HCC but also preclinical cancer drug screening.Hepatocellular carcinoma (HCC) is one of the leading causes of cancer mortality. Cancer stem cells (CSCs) are responsible for the maintenance, metastasis, and relapse of various tumors. The effects of CSCs on the tumorigenesis of HCC are still not fully understood, however. We have recently established two new rat HCC cell lines HTC and TW-1, which we isolated from diethylnitrosamine-induced rat liver cancer. Results showed that TW-1 expressed the genetic markers of CSCs, including CD133, GSTP1, CD44, CD90, and EpCAM. Moreover, TW-1 showed higher tolerance to sorafenib than HTC did. In addition, tumorigenesis and metastasis were observed in nude mice and wild-type rats with TW-1 xenografts. Finally, we combined highly expressed genes in TW-1/HTC with well-known biomarkers from recent HCC studies to predict HCC-related biomarkers and able to identify HCC with AUCs > 0.9 after machine learning. These results indicated that TW-1 was a novel rat CSC line, and the mice or rat models we established with TW-1 has great potential on HCC studies in the future.

Abstract No. 428 Parthenolide induces thiol oxidation-mediated cytotoxicity in human and rat hepatocellular carcinoma cell lines

Parthenolide (PTL) is a naturally occurring sesquiterpene lactone that has anti-tumor activity. The redox environment of hepatocellular carcinoma (HCC) becomes an important target due to the differences between cancer and normal tissue metabolism. We hypothesize that in vitro PTL will induce HCC cell death through increased thiol oxidation. Half maximal inhibitory concentrations (IC50) response to PTL was determined in human (HepG2, SNU-423) and rat (McARH7777) hepatoma cell lines using MTT assays for 24 h. Cellular respiration response to PTL was measured over 24 h using Seahorse Flux Analyzer. Intracellular glutathione (GSH) and oxidized glutathione (GSSG) levels were measured using spectrophotometry at 2 h, 4 h and 24 h. The overall oxidation status of cell lines was also determined using thiol redox-sensitive ratiometric sensor roGFP. PTL had over 90% cytotoxicity at high concentrations 20 μM for SNU-423, 54 μM for HepG2, and 40 μM for rat HCC after 24 h. IC50 concentrations of 6.6 μM for SNU-423, 18 μM for HepG2 and 13 μM for rat HCC were determined after 24 h. Cytotoxic doses of PTL induced a decline in cellular respiration of the HCC cell lines after 2 h. Furthermore, 2 h PTL treatment at IC50 or high concentration caused cyto-roGFP sensor to be oxidized 2.5- to 4-fold in human and rat HCC. PTL at IC50 doses resulted in increased percent GSSG as early as 2 h in human and rat cells. This increase was compensated by upregulation of total GSH levels to minimize cell toxicity in HepG2 cells, which appeared to be offset at high doses of PTL. In HepG2 cells increased percent GSSG levels also demonstrated a similar trend in both IC50 and high PTL doses. In vitro PTL decreased cellular respiration, increased overall oxidation status and increased glutathione oxidation in HCC as early as 2 h after treatment. In HepG2 cells, IC50 dose caused a compensatory upregulation of GSH levels to ostensibly overcome thiol oxidation induced cytotoxicity. However, higher concentrations of PTL overwhelmed this mechanism and caused significant cytotoxicity in all 3 cells lines suggesting PTL can potentially be delivered at high concentrations as locoregional therapy for HCC in vivo.

03:18 PM Abstract No. 220 Retinoic acid enhances doxorubicin-induced cytotoxicity in rat hepatocellular carcinoma cell lines

03:18 PM Abstract No. 220 Retinoic acid enhances doxorubicin-induced cytotoxicity in rat hepatocellular carcinoma cell lines

Biotransformation and oxidative stress responses in rat hepatic cell-line (H4IIE) exposed to racemic ketoprofen (RS-KP) and its enantiomer, dexketoprofen (S(+)-KP)

Pharmaceuticals such as racemate ketoprofen (RS-KP) and its enantiomer, dexketoprofen (S(+)-KP) are highly detectable non-steroidal anti-inflammatory drugs (NSAIDs) in the aquatic environment and therefore are designated as one of the most emerging groups of pollutants that can affect environmental and human health. The potential impact of these pharmaceuticals was assessed for the first time in vitro using a rat hepatocellular carcinoma cell line (H4IIE). Cells were exposed to low and high concentrations of these drugs. Cytotoxicity was determined by MTT reduction assay; CYP1A1 transcriptional and enzymatic levels together with canonical oxidative stress responsive markers (GPx, GR, GST and CAT) were also investigated. Cells exposed to RS-KP and S(+)-KP did not show cytotoxicity effect at the concentrations tested. However, this study highlighted differences between RS-KP and S(+)-KP in most of the evaluated markers, showing compound-, concentration- and time-specific effect patterns which suggest a potential stereo-selective toxicity of these drugs.

Preliminary evaluation of indigenous 90 Y-labelled microspheres for therapy of hepatocellular carcinoma.

Background & objectives:Yttrium-90 (90Y)-based radioembolization has been employed to treat hepatocellular carcinoma (HCC) as commercial radioactive glass and polymeric resin microspheres. However, in India and other Asian countries, these preparations must be imported and are expensive, validating the need for development of indigenous alternatives. This work was aimed to develop an economically and logistically favourable indigenous alternative to imported radioembolizing agents for HCC therapy.Methods:The preparation of 90Y-labelled Biorex 70 microspheres was optimized and in vitro stability was assessed. Hepatic tumour model was generated in Sprague-Dawley rats by orthotopic implantation of N1S1 rat HCC cell line. In vivo localization and retention of the 90Y-labelled Biorex 70 microspheres was assessed for seven days, and impact on N1S1 tumour growth was studied by histological examination and biochemical assays.Results:Under optimal conditions, >95% 90Y-labelling yield of Biorex70 resin microspheres was obtained, and these showed excellent in vitro stability of labelling (>95%) at seven days. In animal studies, 90Y-labelled Biorex 70 microspheres were retained (87.72±1.56% retained in liver at 7 days). Rats administered with 90Y-labelled Biorex 70 microspheres exhibited lower tumour to liver weight ratio, reduced serum alpha-foetoprotein level and greater damage to tumour tissue as compared to controls.Interpretation & conclusions:90Y-labelled Biorex 70 microspheres showed stable retention in the liver and therapeutic effect on tumour tissue, indicating the potential for further study towards clinical use.

Connexin 32 and luteolin play protective roles in non-alcoholic steatohepatitis development and its related hepatocarcinogenesis in rats.

Non-alcoholic steatohepatitis (NASH) has the potential to lead to the development of cirrhosis and hepatocellular carcinoma (HCC). Connexin (Cx) 32, a hepatocyte gap-junction protein, plays a preventive role in hepatocarcinogenesis. However, the precise contribution of Cx32 in the development of NASH has not been established. In this study, we aimed to clarify the role of Cx32 and the chemopreventive effect of luteolin, an antioxidant flavonoid, on the progression of NASH and NASH-related hepatocarcinogenesis. Cx32 dominant negative transgenic (Cx32ΔTg) and wild-type (Wt) rats at 10 weeks of age were given diethylnitrosamine and fed methionine-choline-deficient diet (MCDD) or MCDD with luteolin for 12 weeks. MCDD induced steatohepatitis and fibrosis along with increased inflammatory cytokine expression and reactive oxygen species in the liver. These effects were more severe in Cx32ΔTg rats as compared with Wt rats, and significantly suppressed by luteolin in both genotypes. Concerning NASH-related hepatocarcinogenesis, the number of glutathione S-transferase placental form (GST-P)-positive foci was greater in Cx32ΔTg versus Wt rats, and significantly reduced by luteolin in Cx32ΔTg rats. Microarray analysis identified brain expressed, X-linked 1 (Bex1) as an upregulated gene in Cx32ΔTg rat liver. Quantitative RT-PCR and in situ hybridization revealed that increased Bex1 mRNA was localized in GST-P-positive foci in Cx32ΔTg rats, and the expression level was significantly decreased by luteolin. Moreover, Bex1 knockdown resulted in significant growth inhibition of the rat HCC cell lines. These results show that Cx32 and luteolin have suppressive roles in inflammation, fibrosis and hepatocarcinogenesis during NASH progression, suggesting a potential therapeutic application for NASH.

RNAi Knockdown of Hypoxia-Inducible Factor-1α Decreased the Proliferation, Migration, and Invasion of Hypoxic Hepatocellular Carcinoma Cells.

The obstruction of hepatic arterial blood flow results in tumor tissue hypoxia and elevated expression of hypoxia-inducible factor-1alpha (HIF-1α). Our study evaluated whether lentivirus-mediated short interference RNA against HIF-1α inhibits proliferation, invasion, and migration of hepatocellular carcinoma (HCC) cells under hypoxia. RNA interference knockdown of HIF-1α was achieved by HIF-1α-directed lentiviral shRNA, in a rat HCC cell line cultured under hypoxia condition for varying length of times. The expression levels of HIF-1α and vascular endothelial growth factor were examined using reverse transcription polymerase chain reaction and western blot analyses. Cell proliferation, migration, and invasion were measured by cell viability, transwell migration, and invasion assays, respectively. Inhibition of HIF-1α expression by shRNA suppressed vascular endothelial growth factor mRNA and protein levels under both normoxia and hypoxia. It also suppressed cell migration and invasion, which were enhanced under hypoxic conditions. RNAi knockdown of HIF-1α further suppressed hypoxia-mediated inhibition of the cell proliferation. These data suggest that shRNA of HIF-1α could antagonize the hypoxia-mediated increase in hepatic cancer cell migration and invasion, and synergize with hypoxia to inhibit the cell proliferation in HCC cells.

Why to compare absolute numbers of mitochondria

Prompted by pronounced structural differences between rat liver and rat hepatocellular carcinoma mitochondria, we suspected these mitochondrial populations to differ massively in their molecular composition. Aiming to reveal these mitochondrial differences, we came across the issue on how to normalize such comparisons and decided to focus on the absolute number of mitochondria. To this end, fluorescently stained mitochondria were quantified by flow cytometry. For rat liver mitochondria, this approach resulted in mitochondrial protein contents comparable to earlier reports using alternative methods. We determined similar protein contents for rat liver, heart and kidney mitochondria. In contrast, however, lower protein contents were determined for rat brain mitochondria and for mitochondria from the rat hepatocellular carcinoma cell line McA 7777. This result challenges mitochondrial comparisons that rely on equal protein amounts as a typical normalization method. Exemplarily, we therefore compared the activity and susceptibility toward inhibition of complex II of rat liver and hepatocellular carcinoma mitochondria and obtained significant discrepancies by either normalizing to protein amount or to absolute mitochondrial number. Importantly, the latter normalization, in contrast to the former, demonstrated a lower complex II activity and higher susceptibility toward inhibition in hepatocellular carcinoma mitochondria compared to liver mitochondria. These findings demonstrate that solely normalizing to protein amount may obscure essential molecular differences between mitochondrial populations.

Role for Putative Hepatocellular Carcinoma Stem Cell Subpopulations in Biological Response to Incomplete Thermal Ablation: In Vitro and In Vivo Pilot Study

To investigate the potential role for CD44(+) and CD90(+) hepatocellular carcinoma (HCC) cellular subpopulations in biological response to thermal ablation-induced heat stress. This study was approved by the institutional animal care committee. The N1S1 rat HCC cell line was subjected to sublethal heat stress (45 °C) or control (37 °C) for 10 min, costained with fluorescent-labeled antibodies against CD44, CD90, and 7-AAD after a 48-h recovery and analyzed by flow cytometry to assess the percentage of live CD44(+) and CD90(+) HCC cells (n = 4). Experiments were repeated with pretreatment of N1S1 cells with a dose titration of the dual PI3K-mTOR inhibitor BEZ235 or vehicle control (n = 3). Rats bearing orthotopic N1S1 tumors were subjected to ultrasound-guided partial laser ablation (n = 5) or sham ablation (n = 3), euthanized 24 h after ablation, and liver/tumor analyzed for immunohistochemical staining of CD44 and CD90. Differences between groups were compared with an unpaired t test. Sublethal heat stress induced a significant increase in the relative proportion of live CD44(+) and CD90(+) HCC cells compared to the control group: CD44(+)CD90(-) (5.3-fold; p = 0.0001), CD44(-)CD90(+) (2.4-fold; p = 0.003), and CD44(+)CD90(+) (22.0-fold; p < 0.03). Inhibition of PI3K-mTOR prevented heat stress-induced enrichment of the population of live CD44(+) HCC cells (p < 0.01), but not CD90(+) cells (p > 0.10). Immunohistochemical analysis demonstrated preferential localization of clusters of CD44(+) cells at both the tumor margin and ablation margin. These studies provide experimental evidence supporting a role for HCC cells expressing the putative stem cell marker CD44 in HCC response to heat stress.

Abstract B247: The MEK inhibitor BAY 869766 inhibits tumor growth and metastatic spread, prolongs the survival and acts synergistically with standard of care drugs in models of hepatocellular carcinoma and pancreatic cancer.

Abstract Introduction: BAY 869766 (RDEA119) is an orally available, allosteric MEK1/2 inhibitor, targeting the central switch in the MAP kinase pathway. BAY 869766 demonstrates potent in vitro and in vivo activity in different human cancer cell lines and preclinical models. BAY 869766 is currently in clinical phase I/II studies in the indications HCC (hepatocellular carcinoma) and pancreatic cancer (PaCa). In the described preclinical studies BAY 869766 was studied in two orthotopic, syngeneic HCC and PaCa models and five subcutaneous PaCa xenografts as a single agent and combination therapy. In contrast to subcutaneous xenografts, orthotopic syngeneic models provide additional important information about microenvironment-dependent tumor growth, metastatic spread, immune response and survival. Results: To establish a syngeneic, orthotopic HCC model, the rat HCC cell line MH3924a was first evaluated in vitro. BAY 869766 induced antiproliferative effects in MH3924a HCC cells in monotherapy and showed synergistic effects in combination with sorafenib. In the corresponding rat allograft model efficacy of BAY 869766 in mono- and combination therapy with sorafenib was confirmed. Primary tumor growth and metastatic spread were strongly reduced. These effects were accompanied by a reduction of pERK levels, proliferation index and microvessel density. Finally, the median overall survival in combination treatment was increased by 100% in comparison with vehicle treatment. To characterize BAY 869766 in an orthotopic syngenic PaCa model, the Panc02 mouse allograft model was established. BAY 869766 was tested in mono- and combination therapy with Gemcitabine. Especially, combination treatment led to significant lower tumor weight, reduction of metastatic spread and increase of median survival by 135%. To test BAY 869766 in further clinical relevant pancreatic cancer models, five patient-derived pancreatic cancer tumor samples were subcutaneously implanted in mice and subsequently treated with BAY 869766. Significant tumor growth inhibition in these patient-derived pancreatic cancer xenograft models was observed (Treatment/Control ratios from 23,5% − 44%). Conclusion: These preclinical results support the clinical development of the MEK inhibitor BAY 869766 in HCC and pancreatic cancer. Inhibition of Ras-Raf-MEK-Erk-signaling, direct antiproliferative effects on tumor cells, reduction of metastatic spread and anti-angiogenic properties were demonstrated in these studies. In combination with the respective SoC drugs a significant benefit in survival could be achieved in the orthotopic models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B247.

Enhancement of cytotoxic and pro-apoptotic effects of 2-aminophenoxazine-3-one on the rat hepatocellular carcinoma cell line dRLh-84, the human hepatocellular carcinoma cell line HepG2, and the rat normal hepatocellular cell line RLN-10 in combination with 2-deoxy-D-glucose

The cytotoxic and pro-apoptotic effects of a single dose of 2-aminophenoxazine-3-one (Phx-3) or 2-deoxyglucose (2-DG) or of a combined dose of Phx-3 and 2-DG were studied in the rat hepatocellular carcinoma cell line dRLh-84, the human hepatocellular carcinoma cell line HepG2 and the rat normal hepatocellular cell line RLN-10. The number of viable cells decreased in a dose-dependent manner, when dRLh-84, HepG2 or RLN-10 cells were treated with 2-DG (0.5-20mM) or Phx-3 (1-50µM) alone at 37˚C for 48h. When these cells were treated with 10mM 2-DG and different concentrations of Phx-3, the number of viable cells decreased dose-dependently and in an additive manner for these agents. A single dose of 2 or 10µM Phx-3 induced apoptotic morphology characterized by nuclear condensation and cell shrinkage in dRLh-84, HepG2 and RLN-10 cells, while a single dose of 10mM 2-DG did not. When Phx-3 (2 or 10µM) treatment was combined with 2-DG (10mM) treatment in these three cell lines, the cells with apoptotic morphology increased extensively, which was confirmed by flow cytometric analysis. In addition, autophagic morphology characterized by cytosolic vacuole formation was significantly increased in the hepatocellular carcinoma cell lines dRLh-84 and HepG2 but not in the normal hepatocellular cell line RLN-10 after a single dose of Phx-3 or 2-DG or a combined dose of Phx-3 and 2-DG. Furthermore, when dRLh-84 and HepG2 cells were treated with Phx-3 alone or a combined dose of Phx-3 and 2-DG, depolarization of the mitochondria was extensive, but that of the normal cell line RLN-10 was not. These results may imply that the mechanism for the apoptosis of hepatocellular carcinoma cells caused by Phx-3 alone or a combined dose of Phx-3 and 2-DG differs from that of the normal cell line RLN-10. The present results demonstrate that Phx-3 alone may be beneficial for targeting liver cancer and that its anticancer activity may be enhanced by 2-DG. However, a combined dose of Phx-3 and 2-DG may exert adverse effects on normal liver cells, as evidenced by the cytotoxic and pro-apoptotic effects of the combined treatment in the rat normal hepatocellular cell line RLN-10.

Sorafenib inhibits growth and metastasis of hepatocellular carcinoma by blocking STAT3

To investigate the inhibitory role and the underlying mechanisms of sorafenib on signal transducer and activator of transcription 3 (STAT3) activity in hepatocellular carcinoma (HCC). Human and rat HCC cell lines were treated with sorafenib. Proliferation and STAT3 dephosphorylation were assessed. Potential molecular mechanisms of STAT3 pathway inhibition by sorafenib were evaluated. In vivo antitumor action and STAT3 inhibition were investigated in an immunocompetent orthotopic rat HCC model. Sorafenib decreased STAT3 phosphorylation at the tyrosine and serine residues (Y705 and S727), but did not affect Janus kinase 2 (JAK2) and phospha-tase shatterproof 2 (SHP2), which is associated with growth inhibition in HCC cells. Dephosphorylation of S727 was associated with attenuated extracellular signal-regulated kinase (ERK) phosphorylation, similar to the effects of a mitogen-activated protein kinase (MEK) inhibitor U0126, suggesting that sorafenib induced S727 dephosphorylation by inhibiting MEK/ERK signaling. Meanwhile, sorafenib could also inhibit Akt phosphorylation, and both the phosphatidylinositol-3-kinase (PI3K) inhibitor LY294002 and Akt knockdown resulted in Y705 dephosphorylation, indicating that Y705 dephosphorylation by sorafenib was mediated by inhibiting the PI3K/Akt pathway. Finally, in the rat HCC model, sorafenib significantly inhibited STAT3 activity, reducing tumor growth and metastasis. Sorafenib inhibits growth and metastasis of HCC in part by blocking the MEK/ERK/STAT3 and PI3K/Akt/STAT3 signaling pathways, but independent of JAK2 and SHP2 activation.

Gene expression profiles during activation of cultured rat hepatic stellate cells by tumoral hepatocytes and fetal bovine serum

Hepatic stellate cells (HSCs) transdifferentiate to become extracellular matrix-producing myofibroblasts during liver injury. Myofibroblasts can also promote invasion and metastasis of hepatocellular carcinoma (HCC). In this study, we determined gene expression changes in two different models of HSC activation, induction-activated HSCs (iHSCs) and culture-activated HSCs (cHSCs). Hepatic stellate cells were isolated by density centrifugation and exposed to conditioned medium (CM) from the rat HCC cell line C5F, and fetal bovine serum (FBS). Expression of 27,100 genes in quiescent HSCs, cHSCs and iHSCs was analyzed by microarray and was confirmed on a subset of genes by real-time RT-PCR and Western blot. One thousand nine hundred sixty-seven genes were differentially expressed in cHSCs and iHSCs, including genes that encode proinflammatory factors, adhesion molecules, cell surface receptors, signaling transduction and immune factors such as Il1a, Vcam1, Ccl6, Ilr7, PRAP, osteopontin, Gp39, Raf1, Rac2, Adam17, Wnt6, MMP-9, and Cfd. C5F-CM-induced activation only partially reproduced the gene expression changes observed during FBS culture activation. iHSCs showed specific gene expression, suggesting that HCC cells can specifically induce HSC activation. Induction- activated HSCs' gene expression patterns were partially similar to and different from that of cHSCs. iHSCs might play an important role in invasion and metastasis of HCC. This study provided theoretical foundations for investigating the biology of HSCs in HCC.

Analysis of differential gene expression in rat hepatic stellate cells activated by culture or hepatocellular carcinoma cell induction

Hepatic stellate cells (HSC) in hepatocellular carcinoma (HCC) transdifferentiate into extracellular matrix-producing myofibroblasts. Activated HSC can promote invasion and metastasis of HCC. To understand the differences of HSC in normal liver and HCC, we compared the gene expression patterns in HCC cell induction-activated and culture-activated rat HSC. HSC were isolated by density centrifugation and exposed to conditioned medium from rat HCC cell line C5F. Expression of 22 012 genes in quiescent HSC, culture-activated HSC and HCC induction-activated HSC was analyzed by cDNA microarray and confirmed by real-time RT-PCR and Western blot. 1672 genes were differentially expressed in culture-activated HSC, including proinflammatory factors, cell adhesion molecules, cell surface receptors, signaling transduction molecules and immune factors. 711 genes were differentially expressed in HCC induction-activated HSC. Some of them were identical to those in culture-activated HSC. HCC Induction-activated HSC showed specific gene expression patterns, including Raf1, Rac2, Adam17, Wnt6, MMP-9 and TNF, suggesting that HCC cells can specifically induce HSC activation. The gene expression patterns in HCC induction-activated HSC are different from those in culture-activated HSC. HCC induction-activated HSC may play a major role in the invasion and metastasis of HCC. In vivo activation should be considered as the standard for the study of HSC biology. HCC induction-activated HSC should be considered as the standard for HSC biology studies.

High mobility group box associated with cell proliferation appears to play an important role in hepatocellular carcinogenesis in rats and humans

To identify genes important in hepatocellular carcinogenesis, especially processes involved in malignant transformation, we focused on differences in gene expression between adenomas and carcinomas by DNA microarray. Eighty-one genes for which expression was specific in carcinomas were analyzed using Ingenuity Pathway Analysis software and Gene Ontology, and found to be associated with TP53 and regulators of cell proliferation. In the genes associated with TP53, we selected high mobility group box (HMGB) for detailed analysis. Immunohistochemistry revealed expression of HMGBs in carcinomas to be significantly higher than in other lesions among both human and rat liver, and a positive correlation between HMGBs and TP53 was detected in rat carcinomas. Knock-down of HMGB 2 expression in a rat hepatocellular carcinoma cell line by RNAi resulted in inhibition of cell growth, although no effects on invasion were evident in vitro. These results suggest that acquisition of malignant potential in the liver requires specific signaling pathways related to high cell proliferation associated with TP53. In particular, HMGBs appear to have an important role for progression and cell proliferation associated with loss of TP53 function in rat and in human hepatocarcinogenesis.

Diabetes mellitus and prognosis after curative therapy for hepatocellular carcinoma: Alas, still grave for those who are hyperglycemic

Diabetes mellitus and prognosis after curative therapy for hepatocellular carcinoma: Alas, still grave for those who are hyperglycemic

Free Fatty Acids Induce JNK-dependent Hepatocyte Lipoapoptosis

Elevated serum free fatty acids (FFAs) and hepatocyte lipoapoptosis are features of non-alcoholic fatty liver disease. However, the mechanism by which FFAs mediate lipoapoptosis is unclear. Because JNK activation is pivotal in both the metabolic syndrome accompanying non-alcoholic fatty liver disease and cellular apoptosis, we examined the role of JNK activation in FFA-induced lipoapoptosis. Multiple hepatocyte cell lines and primary mouse hepatocytes were treated in culture with monounsaturated fatty acids and saturated fatty acids. Despite equal cellular steatosis, apoptosis and JNK activation were greater during exposure to saturated versus monounsaturated FFAs. Inhibition of JNK, pharmacologically as well as genetically, reduced saturated FFA-mediated hepatocyte lipoapoptosis. Cell death was caspase-dependent and associated with mitochondrial membrane depolarization and cytochrome c release indicating activation of the mitochondrial pathway of apoptosis. JNK-dependent lipoapoptosis was associated with activation of Bax, a known mediator of mitochondrial dysfunction. As JNK can activate Bim, a BH3 domain-only protein capable of binding to and activating Bax, its role in lipoapoptosis was also examined. Small interfering RNA-targeted knock-down of Bim attenuated both Bax activation and cell death. Collectively the data indicate that saturated FFAs induce JNK-dependent hepatocyte lipoapoptosis by activating the proapoptotic Bcl-2 proteins Bim and Bax, which trigger the mitochondrial apoptotic pathway.

Adenovirus-mediated CD40 ligand gene therapy in a rat model of orthotopic hepatocellular carcinoma

Unresectable hepatocellular carcinoma (HCC) lacks effective therapy and entails very poor prognosis. In this study, we have explored a gene-therapeutic approach to stimulate antitumor immunity by adenoviral-mediated transfer of CD40 ligand to treat HCC in rats. In vitro infection of a rat HCC cell line (McA-RH7777) with adenoviral vector expressing CD40 ligand (AdCMVmCD40L) induced CD40L expression in a dose-dependent manner. Expression of CD40L in McA-RH7777 cells did not alter their growth rate in vitro, but it abrogated their tumorigenicity when CD40L-expressing cells were implanted into the liver of syngenic Buffalo rats. In vivo gene therapy of established orthotopic HCC nodules (6.5 mm in diameter) in Buffalo rats by intratumor injection of AdCMVmCD40L vector led to complete tumor eradication and long-term survival in 69.5% of treated animals. Therapy with AdCMVmCD40L induced strong lymphocytic infiltration of the tumoral tissue and increased apoptosis of malignant cells. The observed antitumoral effect was mediated by CD8+ T cells and was associated with increased interleukin (IL)-12 serum levels and enhanced natural killer (NK) activity. Animals that eliminated the tumor after in vivo gene therapy developed protective antitumor immunity being resistant to rechallenge with neoplastic cells. Toxicity of the therapy with AdCMVmCD40L was slight, with only a transient increase in the level of serum transaminases and minor lymphocyte infiltration of normal liver tissue. These data demonstrate that intratumoral administration of AdCMVmCD40L may provide an efficient and safe treatment for HCC. (HEPATOLOGY 2001;34:72-81.)

Establishment of rat hepatocellular carcinoma cell lines with differing metastatic potential in nude mice

Establishment of rat hepatocellular carcinoma cell lines with differing metastatic potential in nude mice