Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0019204 (hepatocellular carcinoma)
 71,386 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Recent studies of our group have shown that the oxidation of the substituted anthraquinone skeleton is involved in the biotransformation of mitoxantrone. In this report the importance of this process with regard to the mode of action of the drug is investigated. This communication describes a new high performance liquid chromatography separation for mitoxantrone and its metabolites allowing the direct coupling of high performance liquid chromatography to mass spectrometry. Application of this technique to bile of mitoxantrone-treated pigs reveals the formation of several metabolites in addition to the drug-derived compounds found in urine. Seven biliary metabolites are identified as thioether derivatives of mitoxantrone and its side chain oxidation products. Independent synthesis and structural elucidation of 3 thioether conjugates of the drug provides unequivocal evidence that the hydroquinone moiety of mitoxantrone is the site of reaction with glutathione. Furthermore, the formation of the thioether conjugates in HepG2 hepatoma cells and in rat hepatocytes during cell incubations is demonstrated. Inhibition of cytochrome P-450 with metyrapone prevents the formation of the thioether conjugates and leads to a complete loss of the cytotoxicity of mitoxantrone in HepG2 cells and rat hepatocytes up to concentrations of 200 to 400 microM thereby indicating that mitoxantrone has a negligible effect by itself. Rat hepatocytes were found to be more susceptible for the oxidation-induced cytotoxicity than HepG2 cells. These results demonstrate that the acute cytotoxicity of mitoxantrone depends on prior oxidation of its 1,4-dihydroxy-5,8-diaminoanthraquinone moiety.
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PMID:Cytochrome P-450-induced cytotoxicity of mitoxantrone by formation of electrophilic intermediates. 822 49

The cytochrome P-450 3A gene family comprises the dominant forms of cytochrome P-450 found in human liver. We examined as a possible useful system for studying the regulation of cytochrome P-450 3A under controlled conditions in vitro, primary monolayer cultures of human hepatocytes and compared the results with those obtained from the study of cytochrome P-450 3A in the human hepatoblastoma cell line HepG2 or in the human hepatocellular carcinoma cell line TONG/HCC. Using 3A antibodies, 3A cDNAs and 3A3, 3A4, 3A5 and 3A7 isozyme-specific oligonucleotides as probes, we determined that primary human hepatocyte cultures routinely expressed a 3A3/4* immunoreactive protein and 3A mRNA. These gene products were well maintained for many days and were induced by treatment of the cultures with dexamethasone, phenobarbital, macrolide antibiotics, the HMG CoA reductase inhibitor lovastatin or an antifungal agent, clotrimazole. Of six donor livers examined, only two contained mRNA or protein for 3A5, a form found in only a few adult human subjects. In cultures prepared from one of these two livers, 3A5 mRNA was detectable for several days. In cultures of hepatocytes from the remaining four human livers that did not contain 3A5 mRNA or protein, we detected neither spontaneous nor inducible 3A5 proteins or mRNAs. HepG2 cells contained only 3A7 protein, a form found in human fetal liver, even after treatment with inducers. treatment of HepG2 cells with dexamethasone, macrolide antibiotics, phenobarbital and phenobarbital-like inducers or lovastatin produced dose-dependent induction of 3A7 mRNA and 3A7 immunoreactive protein. TONG/HCC cells contained 3A3, 3A4 and 3A5 mRNAs, but only 3A5 immunoreactive protein could be detected.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Regulation of human liver cytochromes P-450 in family 3A in primary and continuous culture of human hepatocytes. 822 33

Phenobarbital is an inducer of xenobiotic-metabolizing enzymes, such as cytochrome P-450, glutathione S-transferases (GSTs) and NAD(P)H:quinone reductase, as well as being a promoter of hepatocarcinogenesis. The molecular mechanisms regulating these biological activities are, however, unknown. In this paper we show that induction by phenobarbital of GST Ya and quinone reductase gene expression is mediated by regulatory elements, EpRE and ARE respectively, which are composed of two adjacent AP-1-like binding sites. EpRE was recently found to be activated by a Fos/Jun heterodimeric complex (AP-1). Here we show that phenobarbital induces an increase in AP-1 binding activity in nuclear extracts of cultured hepatoma cells. Furthermore, we observe that the induction of chloramphenicol acetyltransferase (CAT) activity from an EpRE Ya-cat gene construct and of AP-1 binding activity by phenobarbital is inhibited by the thiol compounds N-acetyl-L-cysteine and glutathione. These results suggest that the phenobarbital induction of AP-1 activity, leading to the AP-1-mediated transcriptional activation of the GST Ya and quinone reductase genes, may involve production of reactive oxygen species and an increase in intracellular oxidant levels, which is prevented by thiol compounds. In view of the involvement of AP-1 in the control of cell proliferation and transformation, the induction by phenobarbital of AP-1 binding activity observed here provides a possible molecular mechanism for the tumour-promoting activity of this drug.
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PMID:Phenobarbital induction of AP-1 binding activity mediates activation of glutathione S-transferase and quinone reductase gene expression. 845 90

In this study we examined the interactions of liver microsomes with the antibiotic calvatic acid and with structural analogues, some of which had shown antimicrotubular properties. These drugs decreased cytochrome P-450 content differently according to the substitutions on the azoxy function and the ethoxycarbonyl derivatives were found to be the most effective ones. The decrease in cytochrome P-450 could be prevented by addition of cysteine or GSH, suggesting an involvement of sulphydryl groups. Furthermore, chromatographic analyses showed that ethoxycarbonyl derivatives were completely metabolized, and this would explain the different behaviour of these compounds towards microtubular protein when they were incubated with purified bovine brain protein or with liver or hepatoma extracts.
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PMID:Interactions between calvatic acid and related compounds with rat liver microsomes. 898 28

The mechanism by which nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, prevents swelling-activated organic osmolyte efflux was examined in the human hepatoma cell line Hep G2. When swollen in hypotonic medium, Hep G2 cell exhibited a regulatory volume decrease that was associated with the release of intracellular taurine, an amino acid found at a concentrations of 22.0 +/- 2.5 nmol/mg protein (approximately 5 mM) in these cells. Rate coefficients for swelling-activated [3H]taurine uptake and efflux were unaffected when extracellular taurine was increased from 0.1 to 25 mM, indicating that taurine is released via a channel. Taurine efflux was rapidly activated after cell swelling and immediately inactivated when cells were returned to normal size by restoration of isotonicity. Swelling-activated taurine efflux was not altered by replacement of extracellular Na+ with choline+ or K+ but was inhibited when cellular ATP levels were decreased with a variety of chemical agents, consistent with an ATP-regulated channel previously described in other cell types. NDGA inhibited swelling-activated [3H]taurine efflux in Hep G2 cells at concentrations of 50-150 microM; however, these same concentrations of NDGA also lowered cell ATP levels. Likewise, ketoconazole, an inhibitor of cytochrome P-450 monoxygenases, inhibited [3H]taurine efflux only at concentrations at which cell ATP levels were also lowered. In contrast, other inhibitors of cyclooxygenase (indomethacin, 100 microM) or of lipoxygenases (caffeic acid, 100 microM), as well as arachidonic acid itself (100 microM), had no effect on either taurine efflux or cell ATP. The present findings characterize a swelling-activated, ATP-sensitive osmolyte channel in Hep G2 cells and demonstrate that inactivation of the channel by NDGA is related to the ability of this drug to deplete cellular ATP.
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PMID:Nordihydroguaiaretic acid depletes ATP and inhibits a swelling-activated, ATP-sensitive taurine channel. 917 31

Retinoic acid stimulates the expression of tissue-type plasminogen activator (t-PA) in vascular endothelial cells in vitro and enhances t-PA levels in plasma and tissues in vivo. Compared with the in vivo situation, high retinoic acid concentrations are required to induce optimally t-PA expression in vitro. These findings led us to study retinoic acid metabolism in cultured human endothelial cells. For comparison, these studies were also performed in the human hepatoma cell line, HepG2, and key experiments were repeated with human primary hepatocytes. Both hepatocyte cultures gave very similar results. Human endothelial cells were shown to possess an active retinoic acid metabolizing capacity, which is quantitatively comparable to that of hepatocytes, but different from that of hepatocytes in several qualitative aspects. Our results demonstrate that all-trans-retinoic acid is quickly metabolized by both endothelial cells and hepatocytes. All-trans-retinoic acid induces its own metabolism in endothelial cells but not in hepatocytes. 9-cis-Retinoic acid is degraded slowly by endothelial cells, whereas hepatocytes metabolize 9-cis-retinoic acid very quickly. Furthermore, our data show that hepatocytes, but not endothelial cells, detectably isomerise all-trans-retinoic acid to 9-cis-retinoic acid and vice versa. In both endothelial cells and hepatocytes all-trans-retinoic acid metabolism was inhibitable by the cytochrome P-450 inhibitors liarozole (10 microM) and ketoconazole (10 microM), albeit to different extents and with different specificities. In the presence of the most potent retinoic acid metabolism inhibitor in endothelial cells, liarozole, at least 10-fold lower all-trans-retinoic acid concentrations were required than in the absence of the inhibitor to obtain the same induction of t-PA. In conclusion, our results clearly demonstrate that all-trans-retinoic acid and 9-cis retinoic acid are actively but differently metabolized and isomerised by human endothelial cells and hepatocytes. The rapid metabolism of retinoic acid explains the relatively high concentrations of retinoic acid required to induce t-PA in cultured endothelial cells.
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PMID:Differences in metabolism and isomerization of all-trans-retinoic acid and 9-cis-retinoic acid between human endothelial cells and hepatocytes. 926 2

The ligand-activated aromatic hydrocarbon receptor (AHR) dimerizes with the AHR nuclear translocator (ARNT) to form a functional complex that transactivates expression of the cytochrome P-450 CYP1A1 gene and other genes in the dioxin-inducible [Ah] gene battery. Previous work from this laboratory has shown that the activity of the CYP1A1 enzyme negatively regulates this process. To study the relationship between CYP1A1 activity and Ah receptor activation we used CYP1A1-deficient mouse hepatoma c37 cells and CYP1A1- and AHR-deficient African green monkey kidney CV-1 cells. Using gel mobility shift and luciferase reporter gene expression assays, we found that c37 cells that had not been exposed to exogenous Ah receptor ligands already contained transcriptionally active AHR-ARNT complexes, a finding that we also observed in wild-type Hepa-1 cells treated with Ellipticine, a CYP1A1 inhibitor. In CV-1 cells, transient expression of AHR and ARNT leads to high levels of AHR-ARNT-dependent luciferase gene expression even in the absence of an agonist. Using a green fluorescent protein-tagged AHR, we showed that elevated reporter gene expression correlates with constitutive nuclear localization of the AHR. Transcriptional activation of the luciferase reporter gene observed in CV-1 cells is significantly decreased by (i) expression of a functional CYP1A1 enzyme, (ii) competition with chimeric or truncated AHR proteins containing the AHR ligand-binding domain, and (iii) treatment with the AHR antagonist alpha-naphthoflavone. These results suggest that a CYP1A1 substrate, which accumulates in cells lacking CYP1A1 enzymatic activity, is an AHR ligand responsible for endogenous activation of the Ah receptor.
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PMID:Constitutive activation of the aromatic hydrocarbon receptor. 941 99

The rat hepatoma cell line McA RH7777 was cloned into alpha-fetoprotein-producing (AFP+) and non-producing (AFP-) sublines. A monoclonal antibody (MAb A2/3) reacting with an antigen (Ag A2/3) present only in AFP- clones or AFP- cells in mixed clones was obtained. Ag A2/3 was absent from the liver of embryonic, fetal, newborn and adult rats, but it was present in gastric and intestinal mucosa of adult rats. Ag A2/3 was found to be a heavy metal-inducible protein: Cd2+ and Pb2+ strongly induced the expression of Ag A2/3 in vivo in the liver of adult rats, while xenobiotics and CCl4 were not active in this respect. In vitro Cd2+ and Pb2+ induced Ag A2/3 expression in several AFP+ clones, leading to a simultaneous marked decrease of AFP+ cells from such clones. The effect of Cd2+ in the induction of Ag A2/3 and suppression of AFP was reversible. SDS PAGE revealed one protein band with an m.w. close to 45,000, which was not sensitive to mercaptoethanol. Despite its inducible properties, Ag A2/3 was shown not to belong to metallothioneins, cytochrome P-450, glutathion-transferase or heat shock proteins families, well-known as being inducible cell stress proteins. Expression of Ag A2/3 could be one of the factors determining the high amplitude of AFP production by individual liver tumors. The nature of Ag A2/3 and its alternative expression with respect to AFP remain to be studied.
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PMID:Inducible protein in rat hepatomas with expression alternative to alpha-fetoprotein. 945 96

There has been increasing interest in the development of a hepatocyte bioreactor for the treatment of acute hepatic failure; however, little is known about the effect of hepatocyte byproducts on the viability of the cells in the bioreactor environment. We investigated the effects of increasing concentrations of bile on the growth and viability of the human hepatoma cell line Hep G2 and on the cytochrome P-450 content and dependent mixed function oxidase (MFO) activities, reduced glutathione (GSH) content, and glutathione S-transferase (GST) activity of primary cultures of rat hepatocytes. Our purpose was to determine whether or not it would be necessary to pretreat the plasma from patients with acute liver failure to remove elevated bile concentrations which might be toxic to the hepatocytes in an artificial liver device. Bile was found to inhibit Hep G2 cell growth at concentrations as low as 0.1% and to decrease viability at concentrations above 0.5%. The cytochrome P-450 and GSH contents and the activities of the MFO system and of GST were decreased in the primary cultures of hepatocytes following 24 h treatment with concentrations of bile at and above 0.5%. The MFO activities associated with different cytochrome P-450 isoenzymes decreased to different extents in the presence of bile with the O-dealkylation of pentoxyresorufin being more labile than that of ethoxyresorufin. Our data indicate that elevated bile concentrations are cytotoxic to liver cells, and it may be necessary to pretreat patient plasma to decrease its bile content to protect the cells during the clinical operation of a hepatocyte bioreactor device.
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PMID:Cytotoxicity of bile in human Hep G2 cells and in primary cultures of rat hepatocytes. 979 80

Chlomethiazole (CMZ) is a sedative and anticonvulsant drug that has been shown to be an efficient transcriptional inhibitor of expression of rat hepatic ethanol-inducible cytochrome P-450 2E1 (CYP2E1). Recent results have shown that human CYP2E1 expression in vivo is almost completely inhibited in control subjects and in alcoholic patients treated with CMZ. In the present investigation, we evaluated the mode of action of CMZ on CYP2E1 expression in Fao rat hepatoma cells. Transcriptional activity of the CYP2E1 gene was monitored using reverse transcription-polymerase chain reaction-based quantification of CYP2E1 heterologous nuclear RNA (hnRNA) against a mimic DNA standard, mRNA was detected by Northern blotting, enzyme protein was detected by Western blotting, and CYP2E1-dependent catalytic activity was detected by assay of chlorzoxazone-6-hydroxylation. Six hours after CMZ treatment, the levels of both CYP2E1 protein and catalytic activity were concomitantly reduced at an IC50 value of about 5 microM. Ethanol treatment of the cells caused a 2-fold induction of CYP2E1 protein levels, which was inhibited by CMZ. Change of medium unexpectedly caused an increase in CYP2E1 gene transcription 4 h later, as monitored by quantitative determination of CYP2E1 hnRNA. However, CMZ failed to influence the expression of CYP2E1 hnRNA or mRNA both constitutively and after medium change, indicating no effect on gene transcription or mRNA synthesis/stability. Cycloheximide treatment of the cells did not abolish the inhibitory action of CMZ, further indicating an action at the post-translational level; in addition, CMZ inhibited CYP2E1 expression in V79 cells with stably expressed CYP2E1 under the control of the SV40 promoter. The data indicate that the CYP2E1 gene is transcriptionally activated in response to medium change and that CMZ, apart from a transcriptional inhibitor of CYP2E1 expression, acts in addition as an efficient high-affinity post-translational inhibitor of CYP2E1, probably due to an allosteric destabilization of the enzyme. This indicates a very rapid and effective CMZ-mediated inhibition of CYP2E1 in vivo.
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PMID:Post-translational inhibition of cytochrome P-450 2E1 expression by chlomethiazole in Fao hepatoma cells. 1021 62


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