Mouse Hepatoma Cell Line Research Articles

Analysis of PCDD/Fs and dioxin-like PCBs in atmospheric deposition samples from the Flemish measurement network: Optimization and validation of a new CALUX bioassay method

Since the CALUX (Chemically Activated LUciferase gene eXpression) bioassay is a fast, sensitive and inexpensive tool for the analysis of a high number of samples, validation of new methods is urgently needed. In this study, a new method for the analysis of PCDD/Fs and dioxin-like PCBs in atmospheric deposition samples with the CALUX bioassay was developed, optimized and validated. The method consists of 4 steps: filtration, extraction, clean up and bioassay analysis. To avoid the use of large amounts of toxic solvents, new techniques were used for filtration and extraction: a C18 filter was used instead of a liquid/liquid extraction and an Accelerated Solvent Extractor (ASE) was used instead of the traditional soxhlet extraction. After pre-oxidation of the sample extract, clean up was done using a multi-layer silica gel column coupled to a carbon column. The PCDD/F and PCB fractions were finally analyzed with the H1L7.5c1 and/or the H1L6.1c3 mouse hepatoma cell lines. The limit of quantification was 1.4 pg CALUX-BEQ m −2 d −1 for the PCBs and 5.6 pg CALUX-BEQ m −2 d −1 for the PCDD/Fs, when using the new sensitive H1L7.5c1 cell line. The GC–HRMS recovery for all PCDD/F congeners was between 55% and 112%, with a mean recovery of 90%. CALUX recoveries of spiked procedural blanks were between the accepted ranges of 80–120%. Repeatability and reproducibility were satisfactory and no interferences from metals were detected. The first results from the Flemish measurement program showed good correlation between CALUX and GC–HRMS.

Effects of small interfering RNA targeting heparanase-1 combined with heparin on invasiveness of mouse hepatocellular carcinoma cell lines

Heparanase-1 (HPA-1) can promote angiogenesis and metastasis of malignant tumors and plays an important role in the genesis and development of tumors. This study was to explore the effects of specific small interfering RNA (siRNA) targeting HPA-1 combined with heparin on invasiveness of mouse hepatocellular carcinoma cells. The expression of HPA-1 in Hca-F, Hca-P, and Hepa1-6 cells, which have high, low, and no metastatic potential, respectively, was analyzed by reverse transcription-polymerase chain reaction (RT-PCR), Western blot analysis and enzyme-linked immunosorbent assay (ELISA). After transfection with two specific siRNAs targeting HPA-1, siRNA-1 and siRNA-2, and treatment with heparin, invasiveness of Hca-F cells was observed by Matrigel invasion assay. HPA-1 was negative in Hepa1-6 cells while positive in both Hca-F and Hca-P cells. The expression levels of both HPA-1 mRNA and protein were obviously higher in Hca-F cells than in Hca-P cells. HPA-1 proteins could be secreted into culture supernatant of Hca-F and Hca-P cells, and the amount of secreted HPA-1 detected by Western blot analysis was larger in Hca-F cells than in Hca-P cells (1.34 ± 0.02 vs. 0.60 ± 0.01, P < 0.001), which was consistent with the results of ELISA. Both siRNA-1 and siRNA-2 downregulated the expression of HPA-1 and the siRNA-2 did more efficiently. The number of invasive Hca-F cells treated with siRNA-2 or heparin alone was larger than that of Hca-F cells treated with combination of them (9 ± 1 vs. 4 ± 1, P = 0.013; 15 ± 2 vs. 4 ± 1, P = 0.008), but smaller than that of untreated Hca-F cells (9 ± 1 vs. 22 ± 2, P = 0.006; 15 ± 2 vs. 22 ± 2, P = 0.026). The combined application of specific siRNA targeting HPA-1 and heparin is more effective in inhibiting the invasiveness of mouse hepatoma cells.

Functional analysis of six human aryl hydrocarbon receptor variants in human breast cancer and mouse hepatoma cell lines

The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor that mediates the toxic responses of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD). The identification and functional analysis of AHR single nucleotide polymorphisms is important in understanding the functional diversity of this receptor as it might give rise to individuals with differing sensitivities to TCDD. In this study, the functional properties of six (I277V, P517S, R554K, V570I, Q666K, and R554K/V570I) human AHR variants were examined in human breast cancer cells (MCF-7 AHR100) and mouse hepatoma cells (Hepa c12) deficient in AHR. CYP1A1- or CYP1B1-regulated reporter gene assays in AHR100 or Hepa c12 cells exposed to TCDD revealed no significant differences in reporter gene activity among the different AHR variants. In contrast to previous findings describing the AHR-R554K/V570I variant to be transcriptionally deficient, no differences in TCDD-dependent increases in CYP1A1 and CYP1B1 mRNA levels were observed between AHR and AHR-R554K/V570I after 24 h or 48 h of exposure. Chromatin immunoprecipitation assays also revealed similar recruitment patterns of AHR and AHR-R554K/V570I to the 5′-regulatory regions of CYP1A1 and CYP1B1. Collectively, our findings show that none of the AHR variants examined exhibited an altered ability to regulate CYP1A1- or CYP1B1-driven transcription in AHR100 or Hepa c12 cell lines, and that AHR and AHR-R554K/V570I are functionally equivalent in the two cell lines examined.

Effects of Pegylated Interferon α2b on Metastasis of Hepatocellular Carcinoma

Interferon (IFN) has an anti-tumor activity in hepatocellular carcinoma (HCC) via anti-angiogenesis and induction of apoptosis. We have previously reported anti-metastatic effects of IFN combined chemotherapy on the outcome of HCC patients. The aim of this study was to investigate anti-metastatic effects of IFN. In vitro, pegylated interferon α2b (PEG-IFN-α2b) was administered to mouse MH134 cells (mouse HCC cell line, MH134), and anti-implantation effects were examined by evaluating the inhibition of cell invasion and cell proliferation. Expressions of vascular endothelial growth factor (VEGF) mRNA were also measured. In vivo, PEG-IFN-α2b was subcutaneously administered into MH134 cells and tumor growth was evaluated. In distant metastasis models, PEG-IFN-α2b was subcutaneously administered and MH134 cells were injected into the spleen. The number of liver metastases and microvessel densities (MVD) were counted. In vitro, the proliferation of MH134 cells was significantly suppressed by PEG-IFN-α2b dose-dependently. MH134 cells added with PEG-IFN-α2b exhibited significantly lower levels of invasion potential. In vivo, tumor size in mice treated with PEG-IFN-α2b significantly suppressed compared with control mice (mean 0.5 versus 5.0 cm, in diameter, P < 0.05) and also decreased number of liver metastases (19.3 versus 6.0, P < 0.05). Moreover, PEG-IFN-α2b significantly suppressed angiogenesis compared with the control. PEG-IFN-α2b in itself had remarkable anti-metastatic effects via inhibition of angiogenesis and cell adhesions.

Lipoxin A4 and Its Analogue Suppress the Tumor Growth of Transplanted H22 in Mice: The Role of Antiangiogenesis

Tumor angiogenesis plays an essential role in carcinogenesis, cancer progression, and metastasis. Some studies indicate that lipoxins, endogenous anti-inflammatory lipid mediators, might be involved in tumor angiogenesis; however, the governing mechanisms are still unknown. In the present study, we examined the effects of exogenous lipoxin A(4) (LXA(4)) in mouse hepatocarcinoma cell line (H22) and H22-bearing mice model. It was found that in H22 cells, LXA(4) inhibited the production of vascular endothelial growth factor and reduced hypoxia-inducible factor-1 alpha level. In addition, its analogue, BML-111, blocked the expression of vascular endothelial growth factor in serum and tumor sections from H22-bearing mice. H&E staining and immunostaining with antibodies against CD34 revealed that BML-111 suppressed tumor-related angiogenesis in vivo, but LXA(4) could not influence the proliferation of primary cultured human umbilical vein endothelial cells. The tumor growth was also inhibited by BML-111. We also found that BML-111 enhanced the in situ apoptosis while inhibiting macrophage infiltration in tumor tissue. The results provide new evidence that LXA(4) suppresses the growth of transplanted H22 tumor in mice through inhibiting tumor-related angiogenesis.

Differential expression of Axl in hepatocellular carcinoma and correlation with tumor lymphatic metastasis

Protein kinases play important roles in tumor development and progression. A variety of members of the signal transduction enzymes serve as targets for therapeutic intervention in cancer. The dysregulation of Axl receptor and its ligand growth arrest-specific 6 (Gas6) is implicated in the pathogenesis of several cancers. In this study, the differential expressions of Axl were investigated in mouse hepatocarcinoma cell lines Hca-F and Hca-P, which have high- and low-metastatic potential to lymph nodes. Experimental inhibition of Axl by siRNA assessed further the metastatic potential of Axl. The results showed that down-regulation of Axl expression attenuated Hca-F cells proliferation, migration, and invasion in vitro, as well as inhibited metastasis to peripheral lymph nodes in vivo. Further analysis demonstrated that the addition of exogenous Gas6 mediated the migration and invasion of Hca-F cells both in vitro and in vivo through Axl. Furthermore, Gas6 stimulation of Axl in Hca-F cells resulted primarily in the down-regulation of Cyr61, a member of the CCN protein family involved in tumor progression. These data suggest that Axl acts as a tumor lymphatic metastasis-associated gene, and may function partly through the regulation of Cyr61.

Triple expression of B7-1, B7-2 and 4-1BBL enhanced antitumor immune response against mouse H22 hepatocellular carcinoma

Costimulatory signals are essential for T-cell activation and hence play a very important role in antitumor immunity. B7 and 4-1BBL which belongs to tumor necrosis factor (TNF) family provide costimulatory interaction for T-cell activation and function. This study investigated the role of B7 and 4-1BBL in the amplification of tumor immunity by transduction of the B7-1, B7-2 and 4-1BBL into mouse hepatocellular carcinoma cell line H22. The tumorigenicity of H22 variants expressing either B7-1, B7-2 (H22/B7-1/B7-2) or 4-1BBL was compared with an H22 variant expressing B7-1, B7-2 and 4-1BBL (H22/B7-1/B7-2/4-1BBL). The study next investigated whether the combination of B7-1/B7-2 and 4-1BBL cell injection induced cytotoxic T lymphocyte (CTL) response and IL-2/IFN-γ secretion. The immune mechanisms underlying this combination treatment were then analyzed. Syngeneic BALB/c mice injected with H22/B7-1/B7-2/4-1BBL cells that expressed elevated levels of B7-1, B7-2 and 4-1BBL showed a tumor development frequency of 50% compared with 100% in mice injected with the H22 parental line, H22/neo, H22/B7-1/B7-2 and H22/4-1BBL. Mice inoculated with H22 tumor cells expressing B7-1, B7-2 and 4-1BBL developed a strong cytotoxic T lymphocyte response and long-term immunity against wild-type tumor, suggesting a synergistic effect between the B7 and 4-1BBL costimulatory pathways. Results showed that H22/B7-1/B7-2/4-1BBL tumor vaccines probably protect the infiltrating lymphocytes from apoptosis and induce NF-κB activation to improve T-cell-mediated antitumor response. In this study, the antitumor consequences of using B7-1, B7-2 and 4-1BBL gene transfer have demonstrated the therapeutic potential of gene therapy approach for hepatocellular carcinoma.

Roles of COMM-domain-containing 1 in stability and recruitment of the copper-transporting ATPase in a mouse hepatoma cell line

A novel function of COMMD1 {COMM [copper metabolism MURR1 (mouse U2af1-rs1 region 1)]-domain-containing 1}, a protein relevant to canine copper toxicosis, was examined in the mouse hepatoma cell line Hepa 1-6 with multi-disciplinary techniques consisting of molecular and cellular biological techniques, speciation and elemental imaging. To clarify the function of COMMD1, COMMD1-knockdown was accomplished by introducing siRNA (small interfering RNA) into the cells. Although COMMD1-knockdown did not affect copper incorporation, it inhibited copper excretion, resulting in copper accumulation, which predominantly existed in the form bound to MT (metallothionein). It is known that the liver copper transporter Atp7b (ATP-dependent copper transporter 7beta), localizes on the trans-Golgi network membrane under basal copper conditions and translocates to cytoplasmic vesicles to excrete copper when its concentration exceeds a certain threshold, with the vesicles dispersing in the periphery of the cell. COMMD1-knockdown reduced the expression of Atp7b, and abolished the relocation of Atp7b back from the periphery to the trans-Golgi network membrane when the copper concentration was reduced by treatment with a Cu(I) chelator. The same phenomena were observed during COMMD1-knockdown when another Atp7b substrate, cis-diamminedichloroplatinum, and its sequestrator, glutathione ethylester, were applied. These results suggest that COMMD1 maintains the amount of Atp7b and facilitates recruitment of Atp7b from cytoplasmic vesicles to the trans-Golgi network membrane, i.e. COMMD1 is required to shuttle Atp7b when the intracellular copper level returns below the threshold.

Inhibition of JNK1 expression decreases migration and invasion of mouse hepatocellular carcinoma cell line in vitro

c-Jun N-terminal kinase (JNK) is located in focal adhesion plaque (FAP). JNK is necessary to growth, morphogenesis, and differentiation of cells; especially JNK1 has a close relation with tumors. In this study, we silenced JNK1 by using short hairpin RNA (shRNA) and examined the effect on migration and invasion of mouse hepatocellular carcinoma (HCC) cell line Hca-F in vitro. Three shRNA expression vectors (JNK1shRNA-1, JNK1shRNA-2, and JNK1shRNA-3) were constructed and transfected to Hca-F cells stably. The most effective shRNA was selected by detecting the expression levels of mRNA and protein. Transwell assay was performed to detect the ability of migration and invasion of cells. A negative control sequence (JNK1shRNA control) and non-transfected normal Hca-F cells were treated as control groups. The "Results" showed that the expression vectors of pSilencer-JNK1shRNA were constructed and transfected to Hca-F cells successfully. The most effective shRNA was JNK1shRNA-2. The expressions of mRNA and protein of JNK1 in Hca-F cells after transfection of JNK1shRNA-2 were decreased significantly compared with the other groups (all, P<0.01; all, P<0.05). The ability of migration and invasion was decreased after down-regulation of JNK1 expression (all, P<0.05). These results suggest that the inhibition of JNK1 expression can decrease ability of migration and invasion of mouse hepatocellular carcinoma cell line in vitro. JNK1 plays an important role in lymphatic metastasis of HCC. It may be a new target for gene therapy of lymphatic metastasis of HCC.

Effects of silencing chloride intracellular channel 1 gene expression on the proliferation and invasion of mouse hepatocellular carcinoma cell lines

To study the effects of silencing CLIC1 gene expression on the proliferation and invasion of Hca-F cells. The mouse CLIC1 cDNA sequence was retrieved from NCBI. Three shRNA sequences were designed and cloned into pGPU6/GFP/Neo plasmids. The plasmids were transfected into Hca-F cells with Lipofectamine 2000. Cell Counting-8 (CCK-8) kit and transwell chamber were used to study the effects of CLIC1 on the proliferation and invasion of Hca-F cells. The pGPU6/GFP/Neo-shRNA-3 plasmid effectively repressed the expression of CLIC1 mRNA. Inhibition of CLIC1 gene expression led to decreased cell proliferation and reduced invasion. CLIC1 is essential for the proliferation and invasion of Hca-F cells.

Differential regulation of the dioxin-induced Cyp1a1 and Cyp1b1 genes in mouse hepatoma and fibroblast cell lines

The xenobiotic metabolizing enzymes Cyp1a1 and Cyp1b1 can be induced by the environmental contaminant 2,3,7,8-tetrachlorodibenzo-ρ-dioxin (dioxin) via the aryl hydrocarbon receptor (AhR). These genes are differentially induced by dioxin in different mouse cell lines. In the mouse hepatoma cell line Hepa1c1c7 (Hepa-1), the Cyp1a1 gene is induced to very high levels by dioxin, but the levels of Cyp1b1 mRNA are extremely low and are not inducible by dioxin. The reverse is the case for the mouse embryonic fibroblast cell line C3H10T1/2, in which Cyp1b1 is induced to very high levels by dioxin, but the levels of Cyp1a1 mRNA are extremely low and not inducible by dioxin. However, dioxin treatment leads to the recruitment of AhR to the enhancer regions of both genes in both cell lines. Somatic cell hybrid clones generated between the two cell lines display high levels of induction of both genes in response to dioxin. Strong reactivation of the Cyp1a1 gene was also observed in C3H10T1/2 cell line after treatment with the DNA methyl transferase inhibitor, 5-aza-2′-deoxycytidine (5-AzadC) and the histone deacetylase inhibitor, trichostatin-A (TSA). However, only modest reactivation of Cyp1b1 was observed in Hepa-1 cells after 5-AzadC or TSA treatment. These data demonstrate that the presence or absence of binding of AhR to regulatory regions is not responsible for determining the differences in levels of induction of the two genes in these cell lines and indicate that DNA methylation plays a major role in silencing of Cyp1a1 gene expression in C3H10T1/2 cells, but appears to play only a minor role in silencing Cyp1b1 gene expression in Hepa-1 cells, which likely occurs principally because Hepa-1 cells lack a factor required for high levels of induction of this gene.

Abstract A41: Regulation of Nrf2-dependent gene expression in aging

Abstract Nrf2 is a transcription factor which controls induction of antioxidant and detoxification enzymes, and, therefore, plays an essential role in protection against chemical carcinogens. Here, we reported that expression of Nrf2 and its dependent genes was affected by age and became compromised in aged mouse livers. Detail analysis revealed that age-dependent Nrf2 expression was regulated primarily at transcriptional level. Accordingly, we cloned and characterized the promoter region of mouse Nrf2 gene. We identified multiple Sp1 binding sites and confirmed their binding activity by gel-shift and chromatin immunoprecipitation assay. We also showed that the identified Sp1 sites were functional since knockdown of Sp1 expression impaired Nrf2 promoter activity as well as expression of Nrf2-dependent genes. We then compared Sp1 expression and its binding activity to Nrf2 promoter in young and aged mouse liver tissues. While no significant change in Sp1 expression was found, Sp1 binding activity was severely impaired in aged tissues. Given that Sp1-binding sites are GC rich elements, we analyzed their methylation status in Nrf2 promoter, and found that Sp1 sites were highly methylated in aged tissues. In support of the role of methylation, treatment of a mouse hepatoma cell line with demethylase inhibitors indeed reduced Sp1 binding to Nrf2 promoter. We also cloned and analyzed human Nrf2 promoter. Multiple Sp1 binding sites were identified and shown to be equally functional as in mouse Nrf2 promoter. Thus, we propose that age-dependent expression of Nrf2 and its downstream genes is regulated by methylation. A strategy that targets methylation would help to restore Nrf2-mediated protection against oxidative stress or chemical carcinogens in aged tissues. Citation Information: Cancer Prev Res 2010;3(1 Suppl):A41.

Immunological killing effect of recombicant adenovirus vector rAD-mTERT-m4-1BBL on mouse hepatoma cell line Hepa1-6 cells co-cultured with T lymphocytes

To study the immunological suppressing effect of recombinant adenovirus vector rAD-mTERT promotor-m4-1BBL (rAD-mTERT) on mouse hepatoma cell line Hepa1-6 cells in co-culture with T lymphocytes. Adding recombinant adenovirus rAD, rAD-CMV-m4-1BBL (rAD-CMV) and rAD-mTERT to Hepa1-6 and L929 cells, respectively, to observe the effect of these adenoviruses on growth and apoptosis of these cells in co-culture with T lymphocytes. Adding adenovirus significantly suppressed the growth and slightly increased apoptosis of the two types of cells (P < 0.05). rAD-mTERT promotor-m4-1BBL showed only pro-apoptotic effect on Hepa1-6 cells. When co-cultured with T lymphocytes, rAD-CMV-m4-1BBL showed promoting effect on apoptosis of the cells. Compared with that of T cells pre-co-culture, CD4(+) and CD8(+) T cells were proliferated, and the ratio of CD4/CD8 was significantly reduced (from 1.27 to 1.08). Adding the recombinant adenoviruses only suppresses the cell growth, but not promotes their apoptosis. In co-culture with T lymphocytes, recombinant adenovirus vector rAD-mTERT promotor-m4-1BBL can targetingly suppress the growth and induce apoptosis of Hepa1-6 cells. The apoptosis is induced through the immunological killing effect of T lymphocytes.

Aryl hydrocarbon nuclear translocator (hypoxia inducible factor 1β) activity is required more during early than late tumor growth

c4 is a derivative of the mouse hepatoma cell line, Hepa-1, that harbors a mutation in the aryl hydrocarbon receptor nuclear translocator gene (Arnt, or hypoxia inducible factor 1beta [HIF-1beta]) leading to loss of activity. Clone 3 cells were generated by introducing a doxycycline-repressible Arnt expression vector into c4 cells. Clone 3 cells were injected subcutaneously into immunosuppressed mice, which were treated with doxycyline (a) throughout the growth of the subsequent tumor xenografts, or (b) from day 7 through to the end of the experiment (day 30), or not treated (c). Tumors in all groups grew exponentially between days 14 and 30, and at rates that were indistinguishable from each other. However, tumors in group a were smaller than those of the other two groups throughout the measurable growth period, while tumor volumes in groups b and c were not significantly different from each other. The degrees of vascularity and apoptosis did not correlate with the differences in degrees of growth between the different groups. Thus, Arnt is required during the early stages of growth of the tumors but less in later stages. Since Arnt does not detectably effect the growth kinetics of Hepa-1 cells either during hypoxia or normoxia, this requirement is unlikely to reflect a direct effect of Arnt on cell proliferation, and is therefore probably a consequence of altered interaction(s) between the tumor cells and the host. These studies suggest that Arnt (and HIF-1alpha/HIF-2alpha) inhibitors will be particularly effective against smaller tumors, including micrometastases.

Functional Characterization of a Novel Interferon-inducible Protein, Mouse ISG12(b2) (135.66)

Abstract Interferons (IFNs) play crucial roles in the host defense against virus. Many interferon-stimulated genes (ISGs) induced by viral infection exert antiviral effects. ISG12(b2) was dramatically induced in liver from dengue-infected mice as well as in Hepa 1-6 (mouse hepatoma cell line) following dengue virus infection. We have biochemically and functionally characterized mouse ISG12(b2). ISG12(b2) is mainly localized to the mitochondria. Overexpression of ISG12(b2) in Hepa 1-6 resulted in the changes in mitochondrial morphology, the mitochondrial membrane potential, the accumulation of Sub-G1 population, the activation of caspase-8, caspase-9, and caspase-3, and the increase of the PARP cleavage. Knockdown of ISG12(b2) by siRNA followed by dengue virus infection in Hepa 1-6 revealed the attenuation of caspase activation and PARP cleavage. Taken together, our results suggest that ISG12(b2) is involved in the activation of the mitochondrial apoptotic pathway, which may subsequently eliminate virus-infected cells.

Knockdown of Caveolin-1 by siRNA Inhibits the Transformation of Mouse Hepatoma H22 Cells In Vitro and In Vivo

Caveolin-1 (Cav-1) is a main structural protein of caveolae and plays important roles in signal transduction and tumorigenesis. We previously showed that Cav-1 was highly expressed in mouse hepatoma cell lines and positively correlated with cell invasion capability. Thus, interfering with the expression and activity of Cav-1 might be a potential way to intervene with hepatoma progression. We used RNA interference to study the biological effects of silencing Cav-1 expression in hepatoma H22 cells, to validate its potential as a therapeutic target. Using small-interfering RNAs (siRNAs) targeting the mRNA region of Cav-1, we effectively suppressed Cav-1 mRNA and protein levels. This resulted in the decreased transformation ability of H22 cells in vitro and in vivo. In addition, downregulation of Cav-1 expression promoted the apoptosis of H22 cells in vitro and in vivo. These results suggest that the use of siRNA could be an efficient molecular therapeutic method for hepatoma with high expression of Cav-1.

SiRNA‐targeted COL8A1 inhibits proliferation, reduces invasion and enhances sensitivity to D‐limonence treatment in hepatocarcinoma cells

The COL8A1 (collagen type VIII, alpha-1) gene, which encodes the alpha 1 chain of collagen, type VIII, may modulate migration, proliferation and adherence of various cells. Only very sparse information exists on COL8A1 expression in hepatocarcinoma. To investigate the possible role of COL8A1 in the mouse hepatocarcinoma cell line Hca-F with highly metastatic potential in the lymph nodes, we used an RNA interference (RNAi) approach to silence COL8A1 expression. The results showed that a small interfering RNA (siRNA) targeted against COL8A1 significantly impeded Hca-F cells proliferation and colony formation in soft agar. This reduction of COL8A1 expression also led to the decreased invasion of Hca-F cells dramatically in vitro. Furthermore, the downregulation of COL8A1 expression also sensitized cells to the action of D-limonene. These data together provide insights into the function of COL8A1 and suggest that COL8A1 might represent a new potential target for gene therapy in hepatocarcinoma.

Activation of peroxisome proliferator‐activated receptor‐α in mice induces expression of the hepatic low‐density lipoprotein receptor

Background and purpose:Mutations in the low-density lipoprotein receptor (LDLR) gene cause familial hypercholesterolaemia in humans and deletion of the LDLR induces lesion development in mice fed a high-fat diet. LDLR expression is predominantly regulated by sterol regulatory element-binding protein 2 (SREBP2). Fenofibrate, a peroxisome proliferator-activated receptor α (PPARα) ligand, belongs to a drug class used to treat dyslipidaemic patients. We have investigated the effects of fenofibrate on hepatic LDLR expression.Experimental approach:The effects of fenofibrate on hepatic LDLR expression (mRNA and protein) and function were evaluated by both in vitro (with AML12 cells) and in vivo experiments in mice.Key results:Fenofibrate increased LDLR expression and LDL binding in a mouse hepatoma cell line, AML12 cells. Fenofibrate restored sterol-inhibited hepatocyte LDLR expression. Mechanistic studies demonstrated that induction of LDLR expression by fenofibrate was dependent on PPARα and sterol regulatory elements (SRE). Specifically, fenofibrate induced LDLR expression by increasing maturation of SREBP2 and phosphorylation of protein kinase B (Akt) but had no effect on SREBP cleavage-activating protein. In vivo, a high-fat diet suppressed LDLR expression in mouse liver while elevating total and LDL cholesterol levels in plasma. However, fenofibrate restored LDLR expression inhibited by high-fat diets in the liver and reduced LDL cholesterol levels in plasma.Conclusions and implications:Our data suggest that fenofibrate increased hepatic LDLR expression in mice by a mechanism involving Akt phosphorylation and LDLR gene transcription mediated by SREBP2.

High‐performance liquid chromatography/nano‐electrospray ionization tandem mass spectrometry, two‐dimensional difference in‐gel electrophoresis and gene microarray identification of lymphatic metastasis‐associated biomarkers

The potential biomarkers for the lymphatic metastatic process of mouse hepatocarcinoma were investigated by using two-dimensional difference in-gel electrophoresis (2D DIGE), high-performance liquid chromatography/nano-electrospray ionization tandem mass spectrometry (HPLC/nESI-MS/MS) and GeneChip. 2D DIGE was performed to screen and quantify the differentially expressed proteins between two well-established mouse hepatocarcinoma cell lines, Hca-F with 75% and Hca-P with 25% metastasis rate of lymph node potentials. The protein spots in the gel were visualized by the highly sensitive Deep Purple (GE Healthcare) fluorescent stain. Protein identification was obtained for gel spots by HPLC/nESI-MS/MS analysis with high quality. GeneChip microarray was performed to identify genes differentially expressed at the mRNA level. Seventeen genes including the chloride intracellular channel l, caspase 3, fructose bisphosphatase 2, glutamate dehydrogenase 1, V-crk sarcoma virus CT10 oncogene homolog, N-myc downstream regulated gene1, villin2, gelsolin, enoyl coenzyme A hydratase 1, transketolase, vimentin, annexins A5 and A7, keratin complex2 basic gene7 and gene8, lactamase (bata 2) and Ero1-like protein were found abnormally regulated and expressed concordantly both at the protein and mRNA levels between the two cell lines. More than half of these genes were for the first time revealed to be involved directly in hepatocarcinoma due to the lymphatic metastasis. The interdisciplinary combination of HPLC/nESI-MS/MS with 2D DIGE and GeneChip techniques opens up the possibility for the biomarker discovery of disease with high confidence.

Protective role of interleukin-18 against Fas-mediated liver injury

Interleukin (IL)-18 plays an important role in the pathogenesis of several liver diseases as well as Fas-mediated apoptosis. However, the effects of IL-18 on Fas-mediated liver injury have not been well elucidated. Therefore, we examined the effects of IL-18 on Fas-mediated apoptosis in in vitro and in vivo experiments. We found that recombinant IL-18 protected mouse hepatocellular carcinoma cell lines, BNL5, from Fas-mediated apoptosis in a dose-dependent manner with up-regulation of both nuclear factor (NF) kappaB and X-linked inhibitors of apoptosis (XIAP). IL-18 transgenic (Tg) mice were also protected from Fas-mediated liver injury and this was further confirmed by histological study and TUNEL staining. In IL-18 Tg mice, up-regulation of XIAP and down-regulation of caspase 3 were observed after injection of anti-Fas, which was consistent with the in vitro findings. These results suggest that IL-18 suppresses Fas-mediated apoptosis of hepatocytes by up-regulation of NFkappaB and XIAP, following inhibition of caspase-3 activity. This observation raises the possibility that IL-18 could be a therapeutic strategy for Fas-mediated liver injury as a negative regulator of XIAP.