alitretinoin and Carcinoma--Hepatocellular

Retinoid X receptor α [RXRα; nuclear receptor (NR)2B1] is a crucial regulator in the expression of a broad array of hepatic genes under both normal and pathologic conditions. During inflammation, RXRα undergoes rapid post-translational modifications, including c-Jun N-terminal kinase (JNK)-mediated phosphorylation, which correlates with a reduction in RXRα function. A small ubiquitin-like modifier (SUMO) acceptor site was recently described in human RXRα, yet the contributors, regulators, and consequences of SUMO-RXRα are not well understood. Inflammation and other stressors alter nuclear receptor function in liver and induce SUMOylation of several NRs as part of proinflammatory gene regulation, but linkages between these two pathways in liver, or for RXRα directly, remain unexplored. We sought to determine if inflammation induces SUMOylation of RXRα in human liver-derived (HuH-7) cells. Lipopolysaccharide, interleukin-1β, and tumor necrosis factor α (TNFα) rapidly and substantially stimulated SUMOylation of RXRα. Two RXRα ligands, 9-cis retinoic acid (9cRA) and LG268, induced SUMOylation of RXRα, whereas both inflammation- and ligand-induced SUMOylation of RXRα require the K108 residue. Pretreatment with 1,9-pyrazoloanthrone (SP600125), a potent JNK inhibitor, abrogates TNFα- and 9cRA-stimulated RXRα SUMOylation. Pretreatment with SUMOylation inhibitors markedly augmented basal expression of several RXRα-regulated hepatobiliary genes. These results indicate that inflammatory signaling pathways rapidly induce SUMOylation of RXRα, adding to the repertoire of RXRα molecular species in the hepatocyte that respond to inflammation. SUMOylation, a newly described post-translational modification of RXRα, appears to contribute to the inflammation-induced reduction of RXRα-regulated gene expression in the liver that affects core hepatic functions, including hepatobiliary transport.

Topics: Alitretinoin; Carcinoma, Hepatocellular; Cell Line, Tumor; Hepatocytes; Humans; Inflammation Mediators; Interleukin-1beta; JNK Mitogen-Activated Protein Kinases; Ligands; Liver; Liver Neoplasms; Organic Chemicals; Protein Processing, Post-Translational; Receptors, Cytoplasmic and Nuclear; Retinoid X Receptor alpha; Signal Transduction; Small Ubiquitin-Related Modifier Proteins; Sumoylation; Tretinoin; Tumor Necrosis Factor-alpha

The farnesoid X receptor/retinoid X receptor-alpha (FXR/RXRalpha) complex regulates bile salt homeostasis, in part by modulating transcription of the bile salt export pump (BSEP/ABCB11) and small heterodimer partner (SHP/NR0B2). FXR is activated by bile salts, RXRalpha by the vitamin A derivative 9-cis retinoic acid (9cRA). Cholestasis is associated with vitamin A malabsorption. Therefore, we evaluated the role of vitamin A/9cRA in the expression of human and mouse bile salt export pump (hBSEP/mBsep), small heterodimer partner (hSHP/mShp), and mouse sodium-dependent taurocholate co-transporting polypeptide (mNtcp). HBSEP and hSHP transcription were analyzed in FXR/RXRalpha-transfected HepG2 cells exposed to chenodeoxycholic acid (CDCA) and/or 9cRA. BSEP promoter activity was determined by luciferase reporter assays, DNA-binding of FXR and RXRalpha by pull-down assays. Serum bile salt levels and hepatic expression of Bsep, Shp, and Ntcp were determined in vitamin A-deficient (VAD)/cholic acid (CA)-fed C57BL/6J mice. Results indicated that 9cRA strongly repressed the CDCA-induced BSEP transcription in HepG2 cells, whereas it super-induced SHP transcription; 9cRA reduced DNA-binding of FXR and RXRalpha. The 9cRA repressed the CDCA-induced BSEP promoter activity irrespective of the exact sequence of the FXR-binding site. In vivo, highest Bsep messenger RNA (mRNA), and protein expression was observed in CA-fed VAD mice. Shp transcription was highest in CA-fed vitamin A-sufficient mice. Ntcp protein expression was strongly reduced in CA-fed VAD mice, whereas mRNA levels were normal. CA-fed control and VAD mice had similarly increased serum bile salt levels.. We showed that 9cRA has opposite effects on bile salt-activated transcription of FXR/RXRalpha target genes. Vitamin A deficiency in CA-fed mice leads to high BSEP expression. Clearance of serum bile salts may, however, be limited because of post-transcriptional reduction of Ntcp. The molecular effects of vitamin A supplementation during cholestasis need further analysis to predict a therapeutic effect.

Topics: Alitretinoin; Animals; ATP Binding Cassette Transporter, Subfamily B, Member 11; ATP-Binding Cassette Transporters; Carcinoma, Hepatocellular; Cell Line, Tumor; Chenodeoxycholic Acid; Cholic Acid; DNA-Binding Proteins; Gene Expression Regulation; Humans; Male; Mice; Mice, Inbred C57BL; Organic Anion Transporters, Sodium-Dependent; Receptors, Cytoplasmic and Nuclear; Response Elements; Retinoid X Receptor alpha; Symporters; Transcription Factors; Tretinoin; Vitamin A; Vitamin A Deficiency

The concentration of type I interferon receptor (IFN-Rc) in the liver is a crucial factor in determining the efficacy of interferon (IFN) therapy in patients with chronic hepatitis C. Retinoic acids (RAs) can enhance the expression of type I IFN-Rc expression. The aim of this study was to investigate whether RAs increase the anti-hepatitis C virus (HCV) effect of IFN through an increase in IFN-Rc. The hepatocellular carcinoma cell line HuH-7 was treated with 10(-7) mol/L all-trans RA (ATRA) and 9-cis RA (9-CRA). Expression of type I IFN-Rc was investigated at both the mRNA and protein levels with the use of real-time quantitative polymerase chain reaction and flow cytometry, respectively. We investigated the anti-HCV effect, using in vitro HCV transfection, by monitoring the level of HCV RNA in the culture medium. ATRA and 9-CRA enhanced the expression of type I IFN-Rc at both the mRNA and protein levels. After IFN-alpha treatment, the activity of 2,5'-oligoadenylate synthetase was enhanced by RAs, and this enhancement was abolished when blocking antibodies had previously been bound to the surface receptors. IFN treatment decreased the concentration of HCV RNA, and this effect was enhanced by treatment with RAs. Our findings suggest that RAs enhance the anti-HCV replication effect of IFN-alpha through up-regulation of type I IFN-Rc in HuH-7 cells.

Topics: 2',5'-Oligoadenylate Synthetase; Alitretinoin; Antibodies; Antiviral Agents; Carcinoma, Hepatocellular; Drug Interactions; Gene Expression; Hepacivirus; Humans; Interferon-alpha; Interferons; Liver Neoplasms; Membrane Proteins; Receptor, Interferon alpha-beta; Receptors, Interferon; RNA, Viral; Transfection; Tretinoin; Tumor Cells, Cultured; Virus Replication

Acyclic retinoid (AR; all trans-3,7,11,15-tetramethyl-2,4,6,10,14-hexadecapentaenoic acid) prevented hepatocarcinogenesis in animal models and in a randomized clinical trial by eradicating premalignant and latent malignant clones of transformed cells from the liver. We investigated the possible mechanism of this clonal deletion at the cellular level.. Human hepatoma-derived cell lines, PLC/PRF/5, HuH-7, and JHH-7, were treated in vitro with AR. Secretion of albumin and that of lectin-reactive isoform of alpha-fetoprotein (AFP-L3) were measured as markers of differentiation and dedifferentiation of the cells, respectively. Telomerase reverse transcriptase (TERT) mRNA expression and telomerase activity were measured by reverse transcriptase polymerase chain reaction (RT-PCR) and stretch PCR assay, respectively. Caspase activities were measured by colorimetric protease assay. Mitochondrial membrane permeability transition was examined by Rhodamine staining.. Production of albumin was recovered while that of AFP-L3 was reduced after exposure of the cells to 10 microM AR for 2 days. This differentiation was maintained for another 2 days without retinoid. In parallel, both TERT mRNA expression and telomerase activity were down-regulated. The cells subsequently died due to apoptosis after 4-6 experimental days. Serial increases in mitochondrial membrane permeability and caspase-9 and -3 activities induced apoptosis.. AR first induces differentiation and reduces telomerase activity. Subsequent apoptosis may contribute to the eradication of the clone.

Topics: Alitretinoin; Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Caspases; Cell Differentiation; Cell Membrane Permeability; DNA-Binding Proteins; Down-Regulation; Gene Expression Regulation, Enzymologic; Humans; Liver Neoplasms; Mitochondria; RNA, Messenger; Telomerase; Tretinoin; Tumor Cells, Cultured

The bile salt excretory pump (BSEP, ABCb11) is critical for ATP-dependent transport of bile acids across the hepatocyte canalicular membrane and for generation of bile acid-dependent bile secretion. Recent studies have demonstrated that the expression of this transporter is sensitive to the flux of bile acids through the hepatocyte, possibly at the level of transcription of the BSEP gene. To determine the mechanisms underlying the regulation of BSEP by bile acids, the promoter of the BSEP gene was cloned. The sequence of the promoter contained an inverted repeat (IR)-1 element (5'-GGGACA T TGATCCT-3') at base pairs -63/-50 consisting of two nuclear receptor half-sites organized as an inverted repeat and separated by a single nucleotide. This IR-1 element has been shown in several recent studies to serve as a binding site for the farnesoid X receptor (FXR), a nuclear receptor for bile acids. FXR activity requires heterodimerization with RXR alpha, and when bound by bile acids, the complex effectively regulates the transcription of several genes involved in bile acid homeostasis. Gel mobility shift assays demonstrated specific binding of FXR/RXR alpha heterodimers to the IR-1 element in the BSEP promoter. In HepG2 cells, co-transfection of FXR and RXR alpha is required to attain full transactivation of the BSEP promoter by bile acids. Two FXR transactivation-deficient mutants (an AF-2 deletion and a W469A point mutant) failed to transactivate, indicating that the effect of bile acids is FXR-dependent. Further, mutational analysis confirms that the FXR/RXR alpha heterodimer activates transcription through the IR-1 site in the human BSEP promoter. These results demonstrate a mechanism by which bile acids transcriptionally regulate the activity of the bile salt excretory pump, a critical component involved in the enterohepatic circulation of bile acids.

Topics: Alitretinoin; Amino Acid Substitution; ATP Binding Cassette Transporter, Subfamily B, Member 11; ATP-Binding Cassette Transporters; Base Sequence; Bile Acids and Salts; Binding Sites; Carcinoma, Hepatocellular; DNA Primers; DNA-Binding Proteins; Gene Expression Regulation; Genes, Reporter; Hepatocytes; Humans; Liver Neoplasms; Molecular Sequence Data; Mutagenesis; Mutagenesis, Site-Directed; Point Mutation; Promoter Regions, Genetic; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Recombinant Proteins; Retinoid X Receptors; Sequence Deletion; TATA Box; Transcription Factors; Transcription, Genetic; Transcriptional Activation; Transfection; Tretinoin; Tumor Cells, Cultured

Stearoyl-CoA desaturase (SCD) is a regulatory enzyme involved in the synthesis of the monounsaturated fatty acids palmitoleate and oleate. The regulation of SCD is of physiological importance because the ratio of saturated fatty acids to unsaturated fatty acids is thought to modulate membrane fluidity. Differential display analysis of retinal pigment epithelial (ARPE-19) cells identified SCD as a gene regulated by retinoic acid. Two SCD transcripts of 3.9 and 5.2 kilobases in size were found to be expressed in these cells by Northern blot analysis. All-trans-retinoic acid (all-trans-RA) increased SCD mRNA expression in a dose- and time-dependent manner; an approximately 7-fold increase was observed with 1 microm all-trans-RA at 48 h. SCD mRNA expression was also increased by 9-cis-retinoic acid (9-cis-RA) as well as 4-(E-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-1-propenyl)benzoic acid (TTNPB), a retinoic acid receptor (RAR)-specific agonist. AGN194301, a RAR alpha-specific antagonist, suppressed the SCD expression induced by all-trans-RA, TTNPB, and 9-cis-RA. These results indicate the involvement of RAR alpha in the induction of SCD expression by retinoic acid. However, AGN194204, a RXR (retinoid X receptor) pan agonist, also increased SCD mRNA expression. This increase was not blocked by AGN194301, suggesting that an RAR-independent mechanism may also be involved. Thus, SCD expression in retinal pigment epithelial cells is regulated by retinoic acid, and the regulation appears to be mediated through RAR and RXR.

Topics: Alitretinoin; Animals; Antineoplastic Agents; Benzoates; Blotting, Northern; Carcinoma, Hepatocellular; Cell Line; Cells, Cultured; Chlorocebus aethiops; COS Cells; Fatty Acids, Unsaturated; Gene Expression Regulation, Enzymologic; Gingiva; HeLa Cells; Humans; Kinetics; Liver Neoplasms; Pigment Epithelium of Eye; Polymerase Chain Reaction; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoid X Receptors; Retinoids; RNA, Messenger; Stearoyl-CoA Desaturase; Tetrahydronaphthalenes; Transcription Factors; Transcription, Genetic; Tretinoin; Tumor Cells, Cultured

In hepatoma Huh-7 cells, inhibition of sphingosine kinase (SphK) activity by N,N-dimethylsphingosine (DMS) resulted in up-regulated production of liver-specific serum proteins including albumin and alpha-fetoprotein (AFP). The changes in these protein levels coincided well with those of two liver-enriched transcription factors, hepatocyte nuclear factor (HNF)-1 and -4, which regulate a number of liver-specific genes at the transcriptional level. Moreover, DMS induced the expression of retinoic acid receptor-alpha and retinoid X receptor-alpha. In DMS-treated cells, 9-cis retinoic acid (RA) further enhanced HNF-4alpha and albumin expression but it inhibited AFP accumulation. These results suggest that activation of SphK disengages cells from their liver-specific phenotype, and that 9-cis RA further induces differentiation of hepatoma cells when SphK activity is inhibited.

Topics: Alitretinoin; alpha-Fetoproteins; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Carcinoma, Hepatocellular; Cell Differentiation; DNA-Binding Proteins; Enzyme Inhibitors; Hepatocyte Nuclear Factor 1; Hepatocyte Nuclear Factor 1-alpha; Hepatocyte Nuclear Factor 1-beta; Hepatocyte Nuclear Factor 4; Hepatocytes; Humans; Liver; Lysophospholipids; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Nuclear Proteins; Phosphoproteins; Phosphotransferases (Alcohol Group Acceptor); Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; Retinoid X Receptors; Serum Albumin; Sphingosine; Transcription Factors; Tretinoin; Tumor Cells, Cultured

Retinoic acid (RA) induces apoptosis in Hep3B human hepatoma cells. 9-Cis-RA (c-RA) had a similar effect as all-trans-RA (t-RA) in inducing cell death in Hep3B cells. RA-induced Hep3B-cell death was associated with inhibited expression of the hepatocyte nuclear factor 4 (HNF-4) gene. Palmitoyl-CoA ((C16:0)-CoA), the reported HNF-4 ligand, prevented RA-induced apoptosis. The effect of (C16:0)-CoA was specific, since palmitic acid and co-enzyme A had no effect in preventing RA-induced apoptosis. Bovine serum albumin (BSA) also prevented RA-induced apoptosis. However, in contrast to BSA, which induced cell growth, (C16:0)-CoA alone had no effect on cell growth. Investigating the possible role of HNF-4 in apoptosis, the reported HNF-4 antagonist (C18:0)-CoA was employed, and it also prevented RA-induced apoptosis. By transient transfection, overexpression of HNF-4 did not prevent RA-induced apoptosis. The induction and prevention of apoptosis caused by RA and (C16:0)-CoA were associated, respectively with the induction and inhibition of the expression of transforming growth factor beta (TGFbeta), which is known to play a role in apoptosis. Furthermore, RA and (C16:0)-CoA can regulate AP-1, which is a key regulator of the TGFbeta gene. Our data indicate that fatty acyl-CoAs can prevent RA-induced apoptosis and that TGFbeta, rather than HNF-4, may play a role in these regulatory processes. Our data also suggest that (C16:0)-CoA and (C18:0)-CoA are not the agonist and antagonist for HNF4, respectively in the Hep3B cell system.

Topics: Alitretinoin; Apoptosis; Basic Helix-Loop-Helix Leucine Zipper Transcription Factors; Blotting, Northern; Carcinoma, Hepatocellular; Culture Media, Serum-Free; DNA-Binding Proteins; Dose-Response Relationship, Drug; Down-Regulation; Fatty Acids; Hepatocyte Nuclear Factor 4; Humans; Ligands; Liver Neoplasms; Palmitoyl Coenzyme A; Phosphoproteins; RNA, Messenger; Time Factors; Transcription Factors; Transfection; Transforming Growth Factor beta; Tretinoin; Tumor Cells, Cultured

The expression of acute-phase protein genes is controlled by many factors, such as IL-1, IL-6, glucocorticoids, thyroid hormone (T3), and retinoic acids. We studied the interaction of T3, glucocorticoids, all-trans retinoic acid (RA), and 9-cis retinoic acid (9cRA) on the expression of the rat alpha 1-acid glycoprotein (AGP) gene in vitro. Dexamethasone (Dex) activated AGP gene expression in a rat liver derived cell line, RLN-10. Although T3, RA, and 9cRA by themselves had no effect on AGP production, they reduced the response to Dex of the AGP gene.

Topics: Alitretinoin; Animals; Carcinoma, Hepatocellular; Cell Line; Dexamethasone; Down-Regulation; Gene Expression Regulation; Liver; Orosomucoid; Rats; RNA, Messenger; Time Factors; Tretinoin; Triiodothyronine; Tumor Cells, Cultured

The role of retinoic acids (RA) on liver fatty acid-binding protein (L-FABP) expression was investigated in the well differentiated FAO rat hepatoma cell line. 9-cis-Retinoic acid (9-cis-RA) specifically enhanced L-FABP mRNA levels in a time- and dose-dependent manner. The higher induction was found 6 h after addition of 10(-6) M 9-cis-RA in the medium. RA also enhanced further both L-FABP mRNA levels and cytosolic L-FABP protein content induced by oleic acid. The retinoid X receptor (RXR) and the peroxisome proliferator-activated receptor (PPAR), which are known to be activated, respectively, by 9-cis-RA and long chain fatty acid (LCFA), co-operated to bind specifically the peroxisome proliferator-responsive element (PPRE) found upstream of the L-FABP gene. Our result suggest that the PPAR-RXR complex is the molecular target by which 9-cis-RA and LCFA regulate the L-FABP gene.

Topics: Alitretinoin; Animals; Carcinoma, Hepatocellular; Carrier Proteins; Dimerization; Drug Synergism; Fatty Acid-Binding Protein 7; Fatty Acid-Binding Proteins; Fatty Acids; Gene Expression Regulation; Liver; Microbodies; Myelin P2 Protein; Neoplasm Proteins; Nerve Tissue Proteins; Rats; Receptors, Cytoplasmic and Nuclear; Receptors, Retinoic Acid; Retinoid X Receptors; RNA, Messenger; Transcription Factors; Tretinoin; Tumor Cells, Cultured