phytoestrogens and Liver-Neoplasms

Estrogenic industrial compounds such as bisphenol A (BPA) and nonylphenol typically bind estrogen receptor (ER) alpha and ERBeta and induce transactivation of estrogen-responsive genes/reporter genes, but their potencies are usually greater than or equal to 1,000-fold lower than observed for 17Beta-estradiol. Risk assessment of estrogenic compounds on the basis of their potencies in simple reporter gene or binding assays may be inappropriate. For example, selective ER modulators (SERMs) represent another class of synthetic estrogens being developed for treatment of hormone-dependent problems. SERMs differentially activate wild-type ERalpha and variant forms expressing activation function 1 (ER-AF1) and AF2 (ER-AF2) in human HepG2 hepatoma cells transfected with an estrogen-responsive complement C3 promoter-luciferase construct, and these in vitro differences reflect their unique in vivo biologies. The HepG2 cell assay has also been used in our laboratories to investigate the estrogenic activities of the following structurally diverse synthetic and phytoestrogens: 4 -hydroxytamoxifen; BPA; 2 ,4 ,6 -trichloro-4-biphenylol; 2 ,3 ,4 ,5 -tetrachloro-4-biphenylol; p-t-octylphenol; p-nonylphenol; naringenin; kepone; resveratrol; and 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane. The results show that synthetic and phytoestrogens are weakly estrogenic but induce distinct patterns of ER agonist/antagonist activities that are cell context- and promoter-dependent, suggesting that these compounds will induce tissue-specific (in vivo(ER agonist or antagonist activities. These results suggest that other receptors, such as the aryl hydrocarbon receptor, that also bind structurally diverse ligands may exhibit unique responses in vivo that are not predicted by standard in vitro bioassays.

Topics: Biological Assay; Carcinoma, Hepatocellular; Dose-Response Relationship, Drug; Endocrine System; Estrogens, Non-Steroidal; Humans; Isoflavones; Liver Neoplasms; Luciferases; Phytoestrogens; Plant Preparations; Promoter Regions, Genetic; Receptors, Estrogen; Risk Assessment; Structure-Activity Relationship; Transfection; Tumor Cells, Cultured

An individual's response to environmental exposures varies depending on their genotype, which has been termed the gene-environment interaction. The phenotype of cell exposed can also be a key determinant in the response to physiological cues, indicating that a cell-gene-environment interaction may exist. We investigated whether the cellular environment could alter the transcriptional response to environmental chemicals. Publicly available gene expression array data permitted a targeted comparison of the transcriptional response to a unique subclass of environmental chemicals that alter the activity of the estrogen receptor, xenoestrogens.. Thirty xenoestrogens were included in the analysis, for which 426 human gene expression studies were identified. Comparisons were made for studies that met the predefined criteria for exposure length, concentration, and experimental replicates. The cellular response to the phytoestrogen genistein resulted in remarkably unique transcriptional profiles in breast, liver, and uterine cell-types. Analysis of gene regulatory networks and molecular pathways revealed that the cellular context mediated the activation or repression of functions important to cellular organization and survival, including opposing effects by genistein in breast vs. liver and uterine cell-types. When controlling for cell-type, xenoestrogens regulate unique gene networks and biological functions, despite belonging to the same class of environmental chemicals. Interestingly, the genetic sex of the cell-type also strongly influenced the transcriptional response to xenoestrogens in the liver, with only 22% of the genes significantly regulated by genistein common between male and female cells.. Our results demonstrate that the transcriptional response to environmental chemicals depends on a variety of factors, including the cellular context, the genetic sex of a cell, and the individual chemical. These findings highlight the importance of evaluating the impact of exposure across cell-types, as the effect is responsive to the cellular environment. These comparative genetic results support the concept of a cell-gene-environment interaction.

Topics: Biomarkers, Tumor; Carcinoma, Hepatocellular; Gene Expression Regulation, Neoplastic; Gene Regulatory Networks; Gene-Environment Interaction; Genistein; Humans; Liver Neoplasms; Phytoestrogens; Tumor Cells, Cultured

Topics: Amino Acids; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Movement; Cell Proliferation; Energy Metabolism; Equol; Glycolysis; Hep G2 Cells; Humans; Liver Neoplasms; Metabolomics; Phytoestrogens; Proton Magnetic Resonance Spectroscopy

Phytoestrogens are popular alternatives to estrogen therapy however their effects on hemostasis in postmenopausal women are unknown. This chapter describes a protocol to determine the effect of the phytoestrogens genistein, daidzein and equol, on the expression of key genes from the hemostatic system in human hepatocyte cell models and to determine the role of estrogen receptors in mediating any response seen using in vitro culture systems and Taqman(®) gene expression analysis.

Topics: Carcinoma, Hepatocellular; Equol; Estradiol; Estrogen Receptor alpha; Gene Expression Regulation, Neoplastic; Genistein; Hep G2 Cells; Humans; Isoflavones; Liver Neoplasms; Phytoestrogens; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Taq Polymerase; Transfection; Workflow

Hepatocellular carcinoma (HCC) is the fifth most frequent cancer worldwide. Sorafenib is the only drug available that improves the overall survival of HCC patients. P-glycoprotein (P-gp), Multidrug resistance-associated proteins 2 and 3 (MRP2 and 3) and Breast cancer resistance protein (BCRP) are efflux pumps that play a key role in cancer chemoresistance. Their modulation by dietary compounds may affect the intracellular accumulation and therapeutic efficacy of drugs that are substrates of these transporters. Genistein (GNT) is a phytoestrogen abundant in soybean that exerts its genomic effects through Estrogen-Receptors and Pregnane-X-Receptor (PXR), which are involved in the regulation of the above-mentioned transporters. We evaluated the effect of GNT on the expression and activity of P-gp, MRP2, MRP3 and BCRP in HCC-derived HepG2 cells. GNT (at 1.0 and 10 μM) increased P-gp and MRP2 protein expression and activity, correlating well with an increased resistance to sorafenib cytotoxicity as detected by the methylthiazole tetrazolium (MTT) assay. GNT induced P-gp and MRP2 mRNA expression at 10 but not at 1.0 μM concentration suggesting a different pattern of regulation depending on the concentration. Induction of both transporters by 1.0 μM GNT was prevented by cycloheximide, suggesting translational regulation. Downregulation of expression of the miR-379 by GNT could be associated with translational regulation of MRP2. Silencing of PXR abolished P-gp induction by GNT (at 1.0 and 10 μM) and MRP2 induction by GNT (only at 10 μM), suggesting partial mediation of GNT effects by PXR. Taken together, the data suggest the possibility of nutrient-drug interactions leading to enhanced chemoresistance in HCC when GNT is ingested with soy rich diets or dietary supplements.

Topics: Antineoplastic Agents; Apoptosis; Blotting, Western; Carcinoma, Hepatocellular; Cell Proliferation; Drug Resistance, Neoplasm; Gene Expression Regulation, Neoplastic; Genistein; Humans; Liver Neoplasms; Membrane Transport Proteins; MicroRNAs; Niacinamide; Phenylurea Compounds; Phytoestrogens; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sorafenib; Tumor Cells, Cultured

In the present study, in order to investigate the anticancer effects of O-desmethylangolensin (O-DMA) on human hepatocellular carcinoma Hep3B cells, we first examined the antiproliferative effect of O-DMA. When Hep3B cells were treated with O-DMA at various concentrations (5-200 µM) for 24, 48 or 72 h, cell proliferation decreased significantly in a dose- and time-dependent manner. Moreover, O-DMA exposure at the IC50 concentration for 72 h arrested cells at the G2/M phase, which was accompanied by a reduction in CDK1, and an increase in cyclin A and B. Under the same conditions, O-DMA significantly increased the number of sub-G1 phase cells. Additionally, an Annexin V assay revealed that exposure to O-DMA affected the rate of cell apoptosis. O-DMA caused the downregulation of Bcl-2 and upregulation of Bax, which led to cytochrome c release from the mitochondria and activation of caspase-3. Taken together, these data suggest that O-DMA exhibits anticancer activity by arresting the cell cycle at G2/M phase and causing mitochondrial-dependent apoptosis in Hep3B cells.

Topics: Antineoplastic Agents; Apoptosis; Carcinoma, Hepatocellular; Caspase 3; CDC2 Protein Kinase; Cell Line, Tumor; Cell Proliferation; Cyclin A; Cyclin B; Cytochromes c; G2 Phase Cell Cycle Checkpoints; Humans; Isoflavones; Liver Neoplasms; Mitochondria; Phytoestrogens; Proto-Oncogene Proteins c-bcl-2

INTRODUATION: Resveratrol, a phytoestrogen present at a high concentration in red wine, has been reported to possess many health benefit effects that are protective against age-related diseases. Protein S (PS), an important anticoagulant factor in the protein C (PC) anticoagulant pathway, is mainly synthesized by hepatocytes, and its plasma level is decreased in high-estrogen conditions such as pregnancy and oral contraceptive use. The aim of this study was to investigate whether resveratrol affects PS expression in HepG2 cells.. The secreted and intracellular levels of PS were determined by an enzyme-linked ligandsorbent assay and Western blotting. The mRNA expressions of PS, PC and β chain of C4b-binding protein (C4BP-β) were analyzed by reverse transcription-polymerase chain reaction. The PS gene promotor activities in HepG2 cells transiently expressing estrogen receptor (ER) α were examined by a luciferase reporter assay.. Resveratrol dose- and time-dependently down-regulated the PS expression in HepG2 cells at a transcriptional level, resulting in a significant decrease in secreted PS; however, the PC and C4BP-β mRNA expressions were not affected. This action of resveratrol was not mediated through either the ER signaling or those of mitogen-activated protein kinases and protein kinase C. Piceatannol, a hydroxylated metabolite of resveratrol, and genistein, an isoflavone found in soy products, also down-regulated the PS expression.. Resveratrol down-regulates the PS expression in HepG2 cells in an ER-independent manner, and the two phenolic hydroxyls at carbon-3 and -5 of resveratrol may be involved in this function.

Topics: Blood Proteins; Blotting, Western; Carcinoma, Hepatocellular; Dose-Response Relationship, Drug; Down-Regulation; Enzyme-Linked Immunosorbent Assay; Estrogen Receptor alpha; Gene Expression Regulation, Neoplastic; Genistein; Hep G2 Cells; Histocompatibility Antigens; Humans; Liver Neoplasms; Molecular Structure; Phytoestrogens; Promoter Regions, Genetic; Protein C; Protein S; Resveratrol; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Stilbenes; Structure-Activity Relationship; Time Factors; Transcription, Genetic; Transfection; Wine

Topics: Adult; Animals; Child; Climacteric; Female; Humans; Japan; Liver Neoplasms; Male; Mice; Mutagenicity Tests; Phytoestrogens; Phytotherapy; Soybean Proteins

The pregnane X receptor (PXR) mediates the induction of enzymes involved in steroid metabolism and xenobiotic detoxification. The receptor is expressed in liver and intestinal tissues and is activated by a wide range of compounds. The ability of a diverse range of dietary compounds to activate PXR-mediated transcription was assayed in HuH7 cells following transient transfection with human PXR (hPXR). The compounds investigated included phytochemicals such as lignans and phytoestrogens, organochlorine dietary contaminants such as polychlorinated biphenyls (PCBs) and triclosan and selected steroid, drug and herbal compounds. The hPXR activation at the top concentrations tested (10 microM) relative to the positive control 10 microM rifampicin ranged from 1.3% (trans-resveratrol) to 152% (ICI 182780). Hydroxylated compounds were marginally more potent than the parent compounds (tamoxifen activation was 74.6% whereas 4 hydroxytamoxifen activation was 84.2%) or significantly greater (vitamin D3 activation was 1.6%, while hydroxylated vitamin D3 activation was 55.6%). Enterolactone, the metabolite of common dietary lignans, was a medium activator of PXR (35.6%), compared to the lower activation of a parent lignan, secoisolariciresinol (20%). Two non-hydroxylated PCB congeners (PCB 118 and 153), which present a larger fraction of the PCB contamination of fatty foods, activated hPXR by 26.6% and 17%, respectively. The pesticide trans-nonachlor activation was 53.8%, while the widely used bacteriocide triclosan was a medium activator of hPXR at 46.2%. The responsiveness of PXR to activation by lignan metabolites suggests that dietary intake of these compounds may affect the metabolism of drugs that are CYP3A substrates. Additionally, the evidence that organochlorine chemicals, particularly the ubiquitous triclosan, activate hPXR suggests that these environmental chemicals may, in part, exhibit their endocrine disruptor activities by altering PXR-regulated steroid hormone metabolism with potential adverse health effects in exposed individuals.

Topics: Alkaline Phosphatase; Anti-Bacterial Agents; Carcinoma, Hepatocellular; Cell Line, Tumor; Cytochrome P-450 CYP3A; Cytochrome P-450 Enzyme Inhibitors; Cytochrome P-450 Enzyme System; Enzyme Inhibitors; Humans; Hydrocarbons, Chlorinated; Kinetics; Lignans; Liver Neoplasms; Phytoestrogens; Pregnane X Receptor; Quantitative Structure-Activity Relationship; Receptors, Cytoplasmic and Nuclear; Receptors, Steroid; Retinoid X Receptors; Rifampin; Transfection

Estrogen treatment is thought to lower low-density lipoprotein (LDL) cholesterol levels by increasing clearance through hepatic LDL receptors. This study aimed to determine the effect of estrogens and phytoestrogens on LDL receptor activity in a human hepatoma cell line, HepG2.. HepG2 cells in culture were incubated for 24 h with estrogen or phytoestrogen and LDL receptor activity was measured by examining the cellular binding of colloidal gold-labelled LDL.. 17Beta-estradiol significantly increased LDL receptor activity whereas estriol had negligible effects. Incubation with the isoflavonoids, formononetin, biochanin A and daidzein, caused significant elevations in receptor activity at concentrations above 40 microM. Coumestrol, a coumestan with a high level of estrogenic activity, caused a 3-fold increase in receptor activity at a concentration of 50 microM. Of the phytoestrogenic mammalian lignans enterolactone and enterodiol, only enterolactone displayed the ability to significantly upregulate LDL receptor activity at 50 microM.. This study suggests that the LDL receptor-stimulating effect of natural estrogens is mainly due to estradiol and that the cholesterol-lowering effect of diets high in phytoestrogens may be due in part to their ability to increase hepatic LDL receptor activity.

Topics: Carcinoma, Hepatocellular; Cell Line; Cholesterol, LDL; Dose-Response Relationship, Drug; Estradiol; Estrogens; Gene Expression Regulation; Humans; Liver; Liver Neoplasms; Phytoestrogens; Receptors, LDL; Tumor Cells, Cultured

Diets containing the soya-derived phytoestrogens, genistein and daidzein, decrease plasma cholesterol in humans and experimental animals. The mechanisms responsible for the hypocholesterolaemic effects of these isoflavones are unknown. The present study was conducted to determine if genistein and daidzein regulate hepatocyte cholesterol metabolism and apolipoprotein (apo) B secretion in cultured human hepatoma (HepG2) cells. ApoB secretion was decreased dose-dependently by up to 63% and 71% by genistein and daidzein (100 microM; P<0.0001) respectively. In contrast, no effect on apoAI secretion was observed. Cellular cholesterol synthesis was inhibited 41% by genistein (100 microM; P<0.005) and 18% by daidzein (100 microM; P<0.05), which was associated with significant increases in 3-hydroxy-3-methylglutaryl-CoA reductase mRNA. Cellular cholesterol esterification was decreased 56% by genistein (100 microM; P<0.04) and 29% by daidzein (100 microM; P<0.04); however, mRNA levels for acyl-CoA:cholesterol acyltransferase (ACAT) 1 and ACAT2 were unaffected. At 100 microM, both isoflavones equally inhibited the activities of both forms of ACAT in cells transfected with either ACAT1 or ACAT2. Genistein (100 microM) and daidzein (100 microM) significantly decreased the activity of microsomal triacylglycerol transfer protein (MTP) by 30% and 24% respectively, and significantly decreased MTP mRNA levels by 35% and 55%. Both isoflavones increased low-density lipoprotein (LDL)-receptor mRNA levels by 3- to 6-fold (100 microM; P<0.03) and significantly increased the binding, uptake and degradation of (125)I-labelled LDL, suggesting that enhanced reuptake of newly secreted apoB-containing lipoproteins contributed to the net decrease in apoB secretion. These results indicate that genistein and daidzein inhibit hepatocyte apoB secretion through several mechanisms, including inhibition of cholesterol synthesis and esterification, inhibition of MTP activity and expression and increased expression of the LDL-receptor.

Topics: Apolipoproteins B; Carcinoma, Hepatocellular; Cholesterol Esters; Dose-Response Relationship, Drug; Estrogens, Non-Steroidal; Genistein; Glycine max; Humans; Hydroxymethylglutaryl CoA Reductases; Isoflavones; Kinetics; Liver Neoplasms; Phytoestrogens; Plant Preparations; RNA, Messenger; Transcription, Genetic; Tumor Cells, Cultured

Differences in ligand-activation of estrogen receptor alpha (ER(alpha)) were investigated in human HepG2 liver carcinoma and U2 osteogenic sarcoma cells transfected with wild-type ER (ER-wt) and variants expressing only activation function 1 (ERAF1) or AF2 (ER-AF2). The estrogen-responsive C3-luc construct containing the complement C3 gene promoter linked to a bacterial luciferase reporter gene was used to determine ligand-induced wild-type or variant ER activation. The quality pattern of ER-dependent responses was similar in both cell lines for a series of weakly estrogenic hydroxy and dihydroxyaromatic compounds including p-octylphenol, p-nonylphenol, 2',4',6'-trichloro-4-biphenylol, 2',3',4', 5'-tetrachloro-4-biphenylol, bisphenol A and 2, 2'-bis(p-hydroxyphenyl)-1,1,1-trichloroethane. However, some significant quantitative differences in these compounds were also observed. The weakly estrogenic pesticide, kepone, and the phytoestrogens, resveratrol (a trihydroxystilbene) and naringen (a flavanone), induced distinctly different patterns of responses; induction by these compounds was not observed in either cell line cotransfected with ER-wt or ER-AF2. In contrast, naringen activated ER-AF1 in HepG2 cells and resveratrol activated ER-AF1 in U2 cells. In HepG2 cells cotreated with E2 plus the estrogenic compounds, only BPA and resveratrol exhibited ER(alpha) antagonist activity. Structure-dependent differences in ER(alpha) activation and inhibition are consistent with the increasingly complex patterns of ER action in various tissues and indicate that the estrogenic activity of an individual compound can only be determined by using an extensive testing protocol.

Topics: Animals; Complement C3; Estrogen Receptor alpha; Estrogens, Non-Steroidal; Female; Genes, Reporter; Genetic Variation; Humans; In Vitro Techniques; Isoflavones; Kinetics; Ligands; Liver Neoplasms; Luciferases; Mice; Osteosarcoma; Phytoestrogens; Plant Preparations; Promoter Regions, Genetic; Receptors, Estrogen; Transfection; Tumor Cells, Cultured; Uterus

Carcinogenic aromatic amines, including the heterocyclic amines, may pose a significant health risk to humans. To determine the potential for chemoprotective intervention against the carcinogenicity of these arylamines and to better understand their mechanism of action, a range of agents, most of them natural dietary constituents, was examined in vitro for their ability to modulate the N-hydroxylation of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and 4-aminobiphenyl (ABP), an initial step in their bioactivation. Experiments were conducted with rat and human liver microsomes. The agents (diallyl sulfide, indole-3-carbinol, alpha-angelicalactone, cafestol/kahweol palmitates, cafestol, kahweol, benzylisothiocyanate, genistin, formononetin, daidzin, equol, biochanin A, Oltipraz, tannic acid, quercetin, ethoxyquin, green tea, and black tea) comprised a variety of chemical classes that included sulfur-containing compounds, antioxidants, flavonoids, phytoestrogens, diterpenes, and polyphenols. Several of these agents, quercetin, ethoxyquin, and black tea, were found to strongly inhibit PhIP N-hydroxylation in rat liver microsomes, resulting in a nearly 85-90% decrease in activity at 100 microM or 0.2%. Tannic acid and green tea, in addition to these agents, were also strong inhibitors of ABP N-hydroxylation. In human liver microsomes, each of these agents was strongly inhibitory (approx 85-95% at 100 microM or 0.02%) of PhIP and ABP N-hydroxylation. Theaflavins and polyphenols were judged to be the primary inhibiting components in the teas, the theaflavins showing the most potent effect. These results demonstrate that chemoprotective agents can inhibit the bioactivation of carcinogenic arylamines, and this is likely to be one of the mechanisms of protection.

Topics: Aminobiphenyl Compounds; Animals; Anticarcinogenic Agents; Antioxidants; Carcinogens; Diterpenes; Estrogens, Non-Steroidal; Flavonoids; Humans; Imidazoles; Isoflavones; Liver Neoplasms; Male; Microsomes, Liver; Phenols; Phytoestrogens; Plant Preparations; Polymers; Rats; Rats, Inbred F344

Phytoestrogens are defined as plant substances that are structurally or functionally similar to estrogen. They are present in many foods and their higher consumption in certain populations has been correlated with protection against many diseases including coronary heart disease, breast cancer and endometrial and ovarian cancer. In this report, ten phytoestrogens with diverse chemical structures were studied for their binding to the human estrogen receptor and their transcription activation properties in yeast and mammalian cells. Our results showed that some of these compounds bind with relatively high affinity to the estrogen receptor and activate the receptor in the yeast and mammalian cell system. In addition, none of these compounds showed anti-estrogenic activity. We conclude that the yeast system accurately predicts the estrogenic activity of compounds with diverse chemical structures in mammalian cells. In addition, our data with phytoestrogens that do not show transcription activation properties raise the possibility that these compounds may exert their biological effects through pathways different from the classical estrogen signalling mechanism.

Topics: beta-Galactosidase; Binding, Competitive; Carcinoma, Hepatocellular; Cloning, Molecular; Estradiol; Estrogens, Non-Steroidal; Humans; Isoflavones; Kinetics; Liver Neoplasms; Phytoestrogens; Plant Preparations; Radioligand Assay; Receptors, Estrogen; Recombinant Fusion Proteins; Recombinant Proteins; Saccharomyces cerevisiae; Transcriptional Activation; Transfection; Tumor Cells, Cultured