Activity In Human Hepatocellular Carcinoma Cells Research Articles

Indomethacin and Diclofenac Hybrids with Oleanolic Acid Oximes Modulate Key Signaling Pathways in Pancreatic Cancer Cells.

Our earlier studies showed that coupling nonsteroidal anti-inflammatory drugs (NSAIDs) with oleanolic acid derivatives increased their anti-inflammatory activity in human hepatoma cells. The aim of this study was to evaluate their effect on the signaling pathways involved in inflammation processes in human pancreatic cancer (PC) cells. Cultured PSN-1 cells were exposed for 24 h (30 µM) to OA oxime (OAO) derivatives substituted with benzyl or morpholide groups and their conjugates with indomethacin (IND) or diclofenac (DCL). The activation of NF-κB and Nrf2 was assessed by the evaluation of the translocation of their active forms into the nucleus and their binding to specific DNA sequences via the ELISA assay. The expression of NF-κB and Nrf2 target genes was evaluated by R-T PCR and Western blot analysis. The conjugation of IND or DCL with OAO derivatives increased cytotoxicity and their effect on the tested signaling pathways. The most effective compound was the DCL hybrid with OAO morpholide (4d). This compound significantly reduced the activation and expression of NF-κB and enhanced the activation and expression of Nrf2. Increased expression of Nrf2 target genes led to reduced ROS production. Moreover, MAPKs and the related pathways were also affected. Therefore, conjugate 4d deserves more comprehensive studies as a potential PC therapeutic agent.

A network pharmacology approach to investigate the anticancer mechanism of cinobufagin against hepatocellular carcinoma via downregulation of EGFR-CDK2 signaling

Hepatocellular carcinoma (HCC) is one of the deadliest cancers with high mortality and poor prognosis, and the investigation on new approaches and effective drugs for HCC therapy is of great significance. In our study, we demonstrate that treatment with cinobufagin, a natural compound isolated from traditional chinese medicine Chansu, reduces proliferation and the colony formation capacity of the human hepatoma cells in vitro, in addition, cinobufagin induces mitotic arrest in human hepatoma cells. The results of a network pharmacology-based analysis show that EGFR, MAPK1, PTK2, CDK2, MAPK3, ESR1, CDK1, PRKCA, AR, and CSNK2A1 are the key targets involved in the anti-tumor activities of cinobufagin, additionally, several signaling pathways such as proteoglycans in cancer, pathways in cancer, HIF-1 signaling pathway, VEGF signaling pathway, ErbB signaling pathway, and PI3K-AKT signaling pathway are identified as the potential pathways involved in the inhibitory effects of cinobufagin against HCC. Furthermore, at the molecular level, we find that cinobufagin decreases EGFR expression and CDK2 activity in human hepatoma cells. Inhibition of EGFR or CDK2 expression could not only suppress the growth of tumor cells but also enhance the inhibitory effects of cinobufagin on the proliferative potential of human hepatoma cells. We also demonstrate that EGFR positively regulates CDK2 expression. Furthermore, EGFR inhibitor gefitinib or CDK2 inhibitor CVT-313 synergistically enhances anticancer effects of cinobufagin in human hepatoma cells. Taken together, these findings indicate that cinobufagin may exert antitumor effects by suppressing EGFR-CDK2 signaling, and our study suggests that cinobufagin may be a novel, promising anticancer agent for the treatment of HCC.

Geranylgeranylacetone decreases the production of hepatitis B virus-related antigen by comprehensive downregulation of mRNA transcription activity.

Elimination of hepatitis B virus (HBV) is infrequently achieved with current therapies. Therefore, more effective anti-HBV therapy is needed. We previously reported that geranylgeranylacetone (GGA) showed anti-hepatitis C virus activity in human hepatoma cells. In this study, we examined the anti-HBV activity of GGA. We used HepG2.2.15.7 cells, PXB cells infected with HBV, Huh7 cells transfected with linear HBV, and PLC/PRF/5 cells as HBV-infected hepatocyte models. After GGA treatment, HBV-related antigen was measured by chemiluminescent immunoassay. HBV-related mRNA was examined by Northern blot. cccDNA and endoplasmic reticulum stress markers were measured by real-time polymerase chain reaction. The activities of HBV promoters and enhancer regions were examined using luciferase vectors. After GGA treatment, hepatitis B surface antigen and hepatitis B e antigen secretion was decreased in all HBV-infected hepatocyte models. HBV-related mRNA was also decreased by GGA treatment, although cccDNA levels were not affected. Additionally, the activity of HBV S1 and S2 promoter region and Enhancer 1/Enhancer 2/core promoter region was reduced by GGA treatment. The mRNA expression of the main transcription factors, hepatocyte nuclear factor 3 and 4 and CCAAT/enhancer binding protein, was also decreased. Further, the expression levels of endoplasmic reticulum stress markers were increased by GGA treatment, which reflected the change in HBV-related antigen secretion. Geranylgeranylacetone treatment reduces HBV-related protein levels by suppressing comprehensive downregulation of HBV promoter and enhancer activity, which might be caused by decreased hepatic transcription factor expression. GGA treatment may enhance anti-HBV effects in combination with other therapies.

A Pharmacological Chaperone Therapy for Acute Intermittent Porphyria.

Mutations in hydroxymethylbilane synthase (HMBS) cause acute intermittent porphyria (AIP), an autosomal dominant disease where typically only one HMBS allele is mutated. In AIP, the accumulation of porphyrin precursors triggers life-threatening neurovisceral attacks and at long-term, entails an increased risk of hepatocellular carcinoma, kidney failure, and hypertension. Today, the only cure is liver transplantation, and a need for effective mechanism-based therapies, such as pharmacological chaperones, is prevailing. These are small molecules that specifically stabilize a target protein. They may be developed into an oral treatment, which could work curatively during acute attacks, but also prophylactically in asymptomatic HMBS mutant carriers. With the use of a 10,000 compound library, we identified four binders that further increased the initially very high thermal stability of wild-type HMBS and protected the enzyme from trypsin digestion. The best hit and a selected analog increased steady-state levels and total HMBS activity in human hepatoma cells overexpressing HMBS, and in an Hmbs-deficient mouse model with a low-expressed wild-type-like allele, compared to untreated controls. Moreover, the concentration of porphyrin precursors decreased in liver of mice treated with the best hit. Our findings demonstrate the great potential of these hits for the development of a pharmacological chaperone-based corrective treatment of AIP by enhancing wild-type HMBS function independently of the patients’ specific mutation.

Acyclic retinoid and angiotensin-II receptor blocker exert a combined protective effect against diethylnitrosamine-induced hepatocarcinogenesis in diabetic OLETF rats

BackgroundInsulin resistance (IR) is closely associated with the progression of hepatocellular carcinoma (HCC). Acyclic retinoid (ACR) targets retinoid X receptor α and reportedly prevents HCC recurrence in clinical practice. Angiotensin-II receptor blocker (ARB) can also inhibit experimental hepatocarcinogenesis and HCC development. These are reported to suppress IR-based hepatocarcinogenesis; however, limited data are available regarding the combined effects of both these agents. This study aimed to investigate the combined chemopreventive effect of ACR and ARB on liver tumorigenesis on rats with congenital diabetes.MethodsMale diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) and non-diabetic Long-Evans Tokushima Otsuka (LETO) rats underwent 70% partial hepatectomy following a single intraperitoneal injection of diethylnitrosamine to induce hepatocarcinogenesis and the administration of ACR (peretinoin, 40 mg/kg/day), ARB (losartan, 30 mg/kg/day), and a combination of ACR and ARB. Six weeks thereafter, we assessed the size and number of the pre-neoplastic lesions (PNL) as well as the altered angiogenesis, oxidative stress, and chronic inflammation in the liver. Moreover, we assessed the effects exerted by ACR and ARB on in vitro cell growth in human HCC cell lines and human umbilical vascular endothelial cells (HUVECs).ResultsOLETF rats showed increase in the size and number of PNLs compared to LETO rats. ACR suppressed the augmentation in size and number of PNLs in the OLETF rats with suppression of cell growth, intrahepatic angiogenesis, lipid peroxidation, oxidative DNA damage, and proinflammatory cytokine production. Combining ACR with ARB enhanced the tumor-suppressive effect and ameliorated intrahepatic angiogenesis, lipid peroxidation, and proinflammatory status; however, cell growth and oxidative DNA damage remained unchanged. IR-mimetic condition accelerated in vitro proliferative activity in human HCC cells, while ACR inhibited this proliferation with G0/G1 arrest and apoptosis. Furthermore, ACR and ARB significantly attenuated the HUVECs proliferation and tubular formation under the IR-mimetic condition, and a combination of both agents demonstrated greater inhibitory effects on HUVEC growth than each single treatment.ConclusionsACR and ARB exert a combined inhibitory effect against IR-based hepatocarcinogenesis by the inhibition of cell growth, intrahepatic angiogenesis, and oxidative stress. Thus, this combination therapy appears to hold potential as a chemopreventive treatment therapy against HCC.

Structural basis for alpha fetoprotein‐mediated inhibition of caspase‐3 activity in hepatocellular carcinoma cells

Alpha-fetoprotein (AFP) is an early serum growth factor in the foetal liver development and hepatic carcinogenesis; However, the precise biological role of cytoplasmic AFP remains elusive. Although we recently demonstrated that cytoplasmic AFP might interact with caspase-3 and inhibit the signal transduction of apoptosis in human hepatocellular carcinoma (HCC) cells, the details of this interaction are not clear. To reveal the molecular relationship between AFP and caspase-3, we performed molecular docking, co-immunoprecipitation (Co-IP), laser confocal microscopy, site-directed mutagenesis and functional experiments to analyse the key amino acid residues in the binding site of caspase-3. The results of Co-IP, laser confocal microscopy and functional analyses were consistent with the computational model. We also used the model to explain why AFP cannot bind to caspase-8. These results provide the molecular basis for the AFP-mediated inhibition of caspase-3 activity in HCC cells. Altogether, we found that AFP interacts with caspase-3 through precise amino acids, namely loop-4 residues Glu-248, Asp-253 and His-257. The results further demonstrated that AFP plays a critical role in the inhibition of the apoptotic signal transduction that mediated by caspase-3. Thus, AFP might represent a novel biotarget for the therapy of HCC patients.

Downregulation of autophagy-related gene ATG5 and GABARAP expression by IFN-λ1 contributes to its anti-HCV activity in human hepatoma cells

Type-III interferon (IFN-λ), the most recently discovered family of IFNs, shares common features with type I IFNs, but also has many distinctive activities. It is not clear that whether IFN-λ has additional antiviral mechanisms. In this study, we investigated the effects of IFN-λ on autophagy, a cellular process closely related to hepatitis C virus (HCV) infection in human hepatoma Huh7 cells. Our results showed that IFN-λ1 treatment inhibit autophagic activity in Huh7 cells, as evidenced by the decreased expression of microtubule-associated protein 1 light chain 3B (LC3B)-II and conversion of LC3B-I to LC3B-II, decreased formation of GFP-LC3 puncta and accumulation of autophagosomes. IFN-λ1 could also inhibit HCV-induced or tunicamycin (a known inducer of autophagy with similar mechanism to HCV infection) -induced LC3B-II expression and autophagosome formation. Through PCR array, real time RT PCR, and western blot, two autophagy-related genes, ATG5 and GABARAP, were identified and verified to be down-regulated by IFN-λ1 treatment, either in HCV-uninfected Huh7 cells or in HCV JFH-1-infected cells. Overexpression of ATG5 and/or GABARAP could partly recover the IFN-λ1-inhibited HCV replication. Mechanism research demonstrated that IFN-λ1 could induce the expression of miR-181a and miR-214 (targeting ATG5 and GABARAP respectively), by which down-regulates ATG5 and GABARAP expression. Taken together, our results indicate that suppression of the autophagy response by IFN-λ1 contributes to IFN-λ1 anti-HCV activity. The results also provide a theoretical basis for improving the effectiveness of IFN treatment of HCV infection through inhibition of the HCV-induced autophagy response.

Receptor for activated C kinase 1 promotes hepatocellular carcinoma growth by enhancing mitogen-activated protein kinase kinase 7 activity

c-Jun N-terminal protein kinase (JNK) is a member of the mitogen-activated protein kinase (MAPK) superfamily. The activation of JNK is mediated by sequential protein phosphorylation through a MAPK module, namely, MAPK kinase kinase (MAP3K or MEKK) → MAPK kinase (MAP2K or MKK) → MAPK. Elevated levels of JNK activity have been frequently observed in hepatocellular carcinoma (HCC) and have been demonstrated to contribute to HCC growth by promoting HCC cell proliferation and resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)- or Fas-mediated apoptosis. Chronic inflammation contributes to the up-regulation of JNK activity in HCC. However, it remains unknown whether aberrant JNK activity also results from some cell intrinsic defect(s). Here, we show that receptor for activated C kinase 1 (RACK1), an adaptor protein implicated in the regulation of multiple signaling pathways, could engage in a direct interaction with MKK7, the JNK-specific MAP2K, in human HCC cells. Levels of RACK1 protein show correlation with the activity of the JNK pathway in human HCC tissues and cell lines. RACK1 loss-of-function or gain-of-function analyses indicate that RACK1 enhances MKK7/JNK activity in human HCC cells. Further exploration reveals that the interaction of RACK1 with MKK7 is required for the enhancement of MKK7/JNK activity by RACK1. RACK1/MKK7 interaction facilitates the association of MKK7 with MAP3Ks, thereby enhancing MKK7 activity and promoting in vitro HCC cell proliferation and resistance to TRAIL- or Fas-mediated apoptosis as well as in vivo tumor growth. Overexpressed RACK1 augments JNK activity and thereby promotes HCC growth through directly binding to MKK7 and enhancing MKK7 activity.

Repeated dosing of piperine induced gene expression of P‐glycoprotein via stimulated pregnane‐X‐receptor activity and altered pharmacokinetics of diltiazem in rats

This study investigated the effect of piperine on the gene expression of P-glycoprotein (P-gp) as well as pregnane-X-receptor (PXR) activity and also its implication on the bioavailability of diltiazem, a P-gp substrate. The effect of piperine on the systemic exposure of diltiazem was examined in rats after the intravenous and oral administration of diltiazem with/without 2 week pretreatment with piperine. Compared with the control group given diltiazem (20 mg/kg) alone, the pretreatment with piperine (10 or 20 mg/kg, once daily for 2 weeks) decreased the oral exposure of diltiazem by 36-48% in rats. Consequently, the bioavailability of oral diltiazem was significantly lower (p < 0.05) after the 2 week pretreatment with piperine. The pretreatment with piperine for 2 weeks also reduced the systemic exposure of desacetyldiltiazem, a major active metabolite of diltiazem by approximately 73%, accompanied by a significant decrease in the metabolite-parent ratio. In contrast to the oral pharmacokinetics, piperine did not affect the intravenous pharmacokinetics of diltiazem in rats. Immunoblot analysis indicated that the protein expression level of intestinal P-gp was significantly enhanced after the 2 week pretreatment with piperine in rats. In addition, piperine increased the PXR reporter activity in human hepatoma cells. Taken together, the 2 week pretreatment with piperine significantly induced intestinal P-gp expression in conjunction with stimulated PXR activity and decreased the oral exposure of diltiazem and desacetyldiltiazem in rats.

Novel Multiple Apoptotic Mechanism of Shikonin in Human Glioma Cells

Shikonin is the main naphthoquinone compound of the root of Lithospermum erythrorhizon. Our previous study demonstrated that shikonin possesses anticancer activity in human hepatoma cells. However, the anticancer mechanism of shikonin in human glioma cells is unclear at present. In the present study, we demonstrated that shikonin induces apoptosis in three human glioma cell lines: U87MG, Hs683, and M059K cells. Cell cycle, generation of reactive oxygen species (ROS), depletion of glutathione (GSH), and disruption of mitochondrial transmembrane potential in shikonin-treated cells were determined by flow cytometry. Apoptosis-related proteins, catalase, and superoxide dismutase-1 (SOD-1) were determined by Western blot testing. N-acetylcysteine (NAC), pifithrin-α (PFT-α), or cyclosporin A were applied to evaluate the molecular mechanism of shikonin in apoptosis. Shikonin induces the generation of ROS, depletion of GSH, disruption of mitochondrial transmembrane potential, upregulation of p53, and cleavage of PARP [poly(ADP-ribose) polymerase] in U87MG glioma cells. Moreover, shikonin causes catalase downregulation and SOD-1 upregulation as well as decreased Bcl-2 and increased Bax expression. Pretreatment with NAC, PFT-α, or cyclosporin A causes the recovery of shikonin-induced apoptosis. The ROS generation and GSH depletion induced by shikonin trigger mitochondrial transmembrane potential disruption. ROS production was partially dependent on the upregulation of p53 upon shikonin treatment. These studies are the first to show that shikonin-induced apoptosis occurs through multiple pathways in human glioma cells. We conclude that shikonin may be used as a potential chemotherapeutic agent against human glioma.

Interaction of HIF and USF Signaling Pathways in Human Genes Flanked by Hypoxia-Response Elements and E-box Palindromes

Rampant activity of the hypoxia-inducible factor (HIF)-1 in cancer is frequently associated with the malignant progression into a harder-to-treat, increasingly aggressive phenotype. Clearly, anti-HIF strategies in cancer cells are of considerable clinical interest. One way to fine-tune, or inhibit, HIF's transcriptional outflow independently of hydroxylase activities could be through competing transcription factors. A CACGTG-binding activity in human hepatoma cells was previously found to restrict HIF's access to hypoxia response cis-elements (HRE) in a Daphnia globin gene promoter construct (phb2). The CACGTG factor, and its impact on hypoxia-responsive human genes, was analyzed in this study by genome-wide computational scans as well as gene-specific quantitative PCR, reporter and DNA-binding assays in hepatoma (Hep3B), cervical carcinoma (HeLa), and breast carcinoma (MCF7) cells. Among six basic helix-loop-helix transcription factors known to target CACGTG palindromes, we identified upstream stimulatory factor (USF)-1/2 as predominant phb2 CACGTG constituents in Hep3B, HeLa, and MCF7 cells. Human genes with adjacent or overlapping HRE and CACGTG motifs included with lactate dehydrogenase A (LDHA) and Bcl-2/E1B 19 kDa interacting protein 3 (BNIP3) hypoxia-induced HIF-1 targets. Parallel recruitment of HIF-1α and USF1/2a to the respective promoter chromatin was verified for all cell lines investigated. Mutual complementing (LDHA) or moderating (BNIP3) cross-talk was seen upon overexpression or silencing of HIF-1α and USF1/2a. Distinct (LDHA) or overlapping (BNIP3) promoter-binding sites for HIF-1 and USFs were subsequently characterized. We propose that, depending on abundance or activity of its protein constituents, O(2)-independent USF signaling can function to fine-tune or interfere with HIF-mediated transcription in cancer cells.

Bisphenol A alters human CYP3A4 enzyme expression and activity

Bisphenol A (BPA) is an endocrine-disrupting chemical largely used in consumer products and has been reported to interact with the pregnane X receptor (PXR). Human CYP3A4 enzyme has an essential role in hydroxylation of steroid hormones and is regulated at the molecular level by PXR. The purpose of our study is to describe the effect of BPA on CYP3A4 transcriptional expression and enzyme activity in the human hepatoma cell line, DPX2. Laboratory-based study. The human hepatoma cell line DPX2, engineered to stably harbor human PXR was exposed to BPA at increasing relevant concentrations followed by total RNA isolation. CYP3A4 mRNA levels were subsequently determined by reverse transcription, and quantitative real-time polymerase chain reaction (RT-PCR). Results were expressed as the mean fold induction.DMSO served as control. CYP3A4 enzyme activity assays were conducted directly in DPX2 cells cultured in 96-well plates. The cells were treated with various concentrations of BPA for 24 hours and CYP3A4 activity was measured with a luminescent CYP3A4 substrate (P450-Glo CYP3A4 Assay with with luciferin IPA). Treatment of human DPX2 cells with BPA induced a significant increase in CYP3A4 mRNA expression at 5,10, and 50μM compared to control (P<0.05). BPA also induced CYP3A4 enzyme activity at concentrations relevant to human exposure, but at higher concentrations, a decrease in enzyme activity occurs.Tabled 1BPA effects on CYP3A4 mRNA expression and enzyme activity, respectively.TreatmentMean fold induction in CYP3A4 mRNA expressionP value mRNA expressionMean fold induction in CYP3A4 enzyme activityP value enzyme activityDMSO11BPA 1μM3.10.611.4P>0.05BPA 5μM8.2P<0.0012P<0.05BPA 10μM11.4P<0.0013.7P<0.05BPA 50μM10P<0.0013.7P<0.05BPA 100μM3.30.371.7P<0.05 Open table in a new tab BPA, in relevant exposure ranges, demonstrates induction of CYP3A4 mRNA expression and activity in human hepatoma cells in vitro which in vivo may affect endocrine function by altering steroid hormone metabolism/action.

Abstract 4706: Overexpression of the components of RNA-induced silencing complex: Contribution to hepatocellular carcinoma

Abstract Objectives: There is virtually no treatment for advanced hepatocellular carcinoma (HCC). As such novel targets need to be identified to develop effective therapies to combat this fatal disease. The oncogene Astrocyte elevated gene-1 (AEG-1) is overexpressed in more than 90% of human HCC patients and plays a seminal role in hepatocarcinogenesis. AEG-1 is localized on the cell membrane as well as in the cytoplasm, nucleus and endoplasmic reticulum. In each location AEG-1 interacts with specific proteins thereby modulating diverse intracellular processes which contributes pleotrophic oncogenic properties to AEG-1. Identification of AEG-1 interacting proteins would facilitate better understanding of AEG-1 function and help develop novel and targeted therapies for HCC. Methods: Yeast two-hybrid assay and co-immunoprecipitation (co-ip) followed by mass spectrometry were used to identify AEG-1-interacting proteins. Co-ip and double immunofluorescence analyses were performed to confirm protein-protein interaction. A Renilla luciferase reporter plasmid containing a miRNA-recognition site in the 3’-untranslated region was used in a transfection based assay to monitor RNA-induced silencing complex (RISC) activity. Immunohistochemistry in tissue microarray was performed to check protein expression. Nude mice xenograft studies were done to check tumorigenesis. Results: We identified Staphylococcal nuclease domain containing 1 (SND1), a nuclease functioning in the RNA-induced silencing complex (RISC), as an AEG-1 interacting protein. We documented that AEG-1 interacts with SND1 and Argonaute 2 (Ago2), the major nuclease in RISC, and thus AEG-1 itself is a component of RISC. Knocking down AEG-1 or SND1 abrogates and overexpression of AEG-1 or SND1 augments RISC activity in human HCC cells. In 109 human HCC samples SND1 was overexpressed in ∼74% cases compared to normal liver. Correspondingly, significantly higher RISC activity was observed in human HCC cells compared to normal hepatocytes. We hypothesized that increased RISC activity, conferred by AEG-1 or SND1 overexpression, might facilitate function of oncomiRs resulting in increased degradation of tumor suppressor mRNAs. Indeed, overexpression of AEG-1 or SND1 downregulated while knockdown of AEG-1 or SND1 upregulated the mRNA level of tumor suppressor PTEN, a target of several oncomiRs overexpressed in HCC. Inhibition of SND1 enzymatic activity significantly abrogated cell growth and proliferation of human HCC cells. Stable overexpression of SND1 augmented while stable knockdown of SND1 inhibited tumor growth by human HCC cells in nude mice xenograft assay. Conclusion: We unravel a novel mechanism that overexpression of AEG-1 and SND1 leading to increased RISC activity might contribute to hepatocarcinogenesis. Targeted inhibition of SND1 enzymatic activity might be developed as an effective therapy for HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4706. doi:10.1158/1538-7445.AM2011-4706

FOXO1 stimulates ceruloplasmin promoter activity in human hepatoma cells treated with IL-6

FOXO1, a member of the winged-helix family of transcription factors, is a ubiquitously expressed protein involved in regulating a variety of cellular processes including glucose homeostasis, apoptosis, cell cycle control, muscle differentiation, and angiogenesis. In addition to these biological functions, FOXO1 is a key player in the oxidative stress response by stimulating the expression of metal-containing anti-oxidant proteins such as manganese superoxide dismutase, selenoprotein P, and catalase. Evidence in the literature suggests that FOXO1 may also be capable of regulating the expression of the anti-oxidant protein Ceruloplasmin (Cp), a six-copper-containing protein synthesized and secreted mainly by the liver. In the present report, we demonstrate that FOXO1 stimulates Cp promoter activity in conjunction with the cytokine IL-6. Through deletional analysis and in vitro binding studies, we determine the DNA sequence responsible for the FOXO1-dependent regulation of the Cp proximal promoter. Finally, we demonstrate that FOXO1 is capable of enhancing the expression of endogenous Cp in human hepatic carcinoma cells treated with IL-6. These results allow us to identify FOXO1 as a regulator of Cp expression to promote the anti-oxidant pathway in response to IL-6 signaling.

Down-regulation of CYLD as a trigger for NF-κB activation and a mechanism of apoptotic resistance in hepatocellular carcinoma cells

The cylindromatosis gene (CYLD) was identified as a tumor suppressor gene, which is mutated in familial cylindromatosis (Brooke-Spiegler syndrome), an autosomal-dominant predisposition to multiple tumors of the skin appendages. CYLD is a deubiquitinating enzyme acting as a negative regulator of the nuclear factor κB (NF-κB) signaling pathway by removing lysine-63-linked polyubiquitin chains from NF-κB activating proteins. In order to investigate the role of CYLD in apoptotic signaling in human hepatocellular carcinoma (HCC) cells, we first studied the expression levels of CYLD in HCC tissues. CYLD expression was lower in HCC both at protein and mRNA levels compared to the surrounding non-malignant tissue. In order to further study the role of CYLD in the apoptotic sensitivity of HCC cells, CYLD was specifically down-regulated in HCC cell lines via RNA interference. The specific down-regulation of CYLD resulted in increased resistance towards treatment with doxorubicin, 5-fluorouracil and cisplatin. In addition, the down-regulation of CYLD in HCC cells decreased the sensitivity towards tumor necrosis factor-α-induced apoptosis. The CYLD knockdown also led to the degradation of the NF-κB inhibitor, IκB-α, resulting in enhanced NF-κB activity in HCC cells. Finally, we found that CYLD expression was triggered by the multikinase inhibitor, sorafenib, by the inhibition of Raf-1, as well as by the blockage of the pro-survival kinases, MEK (U0126) and the epidermal growth factor receptor (AG1478). In summary, we show that CYLD is down-regulated in human HCC and is involved in the apoptotic resistance of HCC cells. Our data identify the reconstitution of CYLD expression as an attractive approach for overcoming resistance to treatment in HCC.

Hepatitis C virus core protein enhances Telomerase activity in Huh7 cells

Hepatitis C is an oncogenic virus although the mechanisms responsible for this behavior are not clear. We studied the effects of hepatitis C virus (HCV) core protein expression on Telomerase, an enzyme closely associated with cellular immortalization and neoplasia. The aim of this study was to investigate the effects of HCV core protein on the regulation of Telomerase activity in human hepatoma cells. Regulation and expression of human Telomerase reverse transcriptase (TERT) was compared in Huh7 cells stably transfected with HCV core protein or cells expressing vector alone. Telomerase activity was measured using Quantitative Telomerase Detection (QTD) and telomere length was measured by fluorescence in situ hybridization (FISH). Transient transfection and luciferase assay were used to evaluate TERT promoter activity. Telomerase activity was increased twofold in Huh7 cells expressing HCV core protein compared to controls (P < 0.01). This was accompanied by a 1.4-fold increase of TERT mRNA and 1.9-fold increase in TERT protein (P < 0.01 in either case). Cellular fractionation and immunocytochemical studies showed increased localization of TERT in the nucleus of core-expressing cells as compared to controls. FISH assay confirmed that telomeres of HCV core-expressing Huh7 cells were relatively longer than those of control cells (0.22 + 0.05 vs. 0.12 + 0.03, P < 0.01). TERT promoter activity was enhanced about 30% in HCV core-expressing Huh7 cells compared to control cells (P < 0.02). HCV core protein is associated with increased Telomerase activity in hepatoma cells. These findings suggest that enhancement of Telomerase activity by HCV core protein may contribute to the oncogenicity of HCV.

Monoterpene indole alkaloids from the leaves of Tabernaemontana elegans induce apoptosis activity in human hepatoma cells

Monoterpene indole alkaloids from the leaves of Tabernaemontana elegans induce apoptosis activity in human hepatoma cells

HINT1 inhibits β‐catenin/TCF4, USF2 and NFκB activity in human hepatoma cells

In this study we explored the relevance of Hint, a novel tumor suppressor gene, to human hepatoma. The human hepatoma cell lines Hep3B and HepG2 express very low levels of the HINT1 protein but the Huh7 cells express a relatively high level. In Hep3B and HepG2 cells, but not in Huh7 cells, the promoter region of Hint1 is partially methylated and treatment with 5-azadcdeoxycytidine increased expression of the HINT1 protein and Hint1 mRNA in Hep3B and HepG2 cells. Increased expression of HINT1 in HepG2 cells markedly inhibited their growth. It also inhibited the transcriptional activities of beta-catenin/TCF4, and USF2, and inhibited the expression of endogenous cyclin D1 and TGFbeta2. Furthermore, HINT1 co-immunoprecipitated with USF2 in extracts of Hep2 cells. HINT1 also inhibited NFkappaB transcription factor reporter activity and inhibited translocation of the endogenous p65 protein to the nucleus of HepG2 cells. Therefore, decreased expression of the Hint1 gene through epigenetic silencing may play a role in enhancing the growth of a subset of human hepatoma by increasing the expression of genes controlled by the transcription factors beta-catenin, USF2, and NFkappaB.

Cholesterol regulates ACAT2 gene expression and enzyme activity in human hepatoma cells

Acyl-coenzyme A:cholesterol acyltransferase (ACAT) catalyzes the synthesis of cholesteryl esters from cholesterol and long-chain fatty acids. The two ACAT enzymes, ACAT1 and ACAT2, lack sterol regulatory elements in their promoters and have not been thought to be transcriptionally regulated by cellular cholesterol. However, Cynomolgus monkeys respond to high-cholesterol diet with increased hepatic ACAT2 mRNA expression. Also, a decrease in hepatic ACAT2 mRNA expression has been observed during statin treatment in humans. Thus, we hypothesized that cholesterol may exert transcriptional regulation on the human ACAT2 gene. To test this, we studied two human hepatoma cell lines (HuH7 and HepG2) under conditions of cholesterol loading or depletion and analyzed ACAT gene expression, enzymatic activity, and cellular cholesterol mass. We show a dose-dependent increase of ACAT2 mRNA expression, an increased enzymatic activity of ACAT2, and increased esterified cholesterol mass upon cholesterol loading. These results suggest that human ACAT2 is transcriptionally regulated by cholesterol.

Group I but not Group II NPV induces antiviral effects in mammalian cells

Nucleopolyhedrovirus (NPV) is divided into Group I and Group II based on the phylogenetic analysis. It has been reported that Group I NPVs such as Autographa californica multiple NPV (AcMNPV) can transduce mammalian cells, while Group II NPVs such as Helicoverpa armigera single NPV (HaSNPV) cannot. Here we report that AcMNPV was capable of stimulating antiviral activity in human hepatoma cells (SMMC-7721) manifested by inhibition of Vesicular Stomatitis virus (VSV) replication. In contrast, the HaSNPV and the Spodoptera exigua multiple NPV (SeMNPV) of group II had no inhibitory effect on VSV. Recombinant AcMNPV was shown to induce interferons alpha/beta even in the absence of transgene expression in human SMMC-7721 cells, while it mediated transgene expression in BHK and L929 mammalian cells without an ensuing antiviral activity.