Critical role of farnesoid X receptor for hepatocellular carcinoma cell proliferation.

Farnesoid X receptor (FXR), a pivotal factor maintaining bile acid homeostasis, has been recently shown to be a critical factor required for liver regeneration. The elucidation of the mechanism how FXR controls the proliferation of hepatocellular carcinoma cells is useful to establish the therapy for liver cancer. Here, we show that FXR plays a crucial role in the proliferation of human hepatocellular carcinoma cell line, HepG2, Huh7 and HLE. The treatment of HepG2 with FXR siRNA elevates the level of p16/INK4a expression resulting in the inhibition of cell proliferation. By contrast, FXR activation reduces p16/INK4a expression and stimulates the cell proliferation. The ectopic expression of the active form of Ras that causes strong activation of extracellular signal-regulated kinase (ERK) leads to the decrease in FXR expression, suggesting that FXR expression is negatively regulated via Ras/ERK pathway. The elevation of p16/INK4a expression and the inhibition of cell proliferation by FXR knockdown are also observed in Huh7 and HLE. In this study, we have suggested a novel mechanism by which hepatocellular carcinoma cell proliferation is regulated: FXR stimulates cell proliferation by suppressing the p16/INK4a expression, whereas Ras/ERK pathway down-regulates the FXR expression, leading to the suppressed cell proliferation in hepatocellular carcinoma cell lines.

Human lactoferrin activates NF-kappaB through the Toll-like receptor 4 pathway while it interferes with the lipopolysaccharide-stimulated TLR4 signaling.

Lactoferrin (LF) has been implicated in innate immunity. Here we reveal the signal transduction pathway responsible for human LF (hLF)-triggered nuclear factor-kappaB (NF-kappaB) activation. Endotoxin-depleted hLF induces NF-kappaB activation at physiologically relevant concentrations in the human monocytic leukemia cell line, THP-1, and in mouse embryonic fibroblasts (MEFs). In MEFs, in which both tumor necrosis factor receptor-associated factor 2 (TRAF2) and TRAF5 are deficient, hLF causes NF-kappaB activation at a level comparable to that seen in wild-type MEFs, whereas TRAF6-deficient MEFs show significantly impaired NF-kappaB activation in response to hLF. TRAF6 is known to be indispensable in leading to NF-kappaB activation in myeloid differentiating factor 88 (MyD88)-dependent signaling pathways, while the role of TRAF6 in the MyD88-independent signaling pathway has not been clarified extensively. When we examined the hLF-dependent NF-kappaB activation in MyD88-deficient MEFs, delayed, but remarkable, NF-kappaB activation occurred as a result of the treatment of cells with hLF, indicating that both MyD88-dependent and MyD88-independent pathways are involved. Indeed, hLF fails to activate NF-kappaB in MEFs lacking Toll-like receptor 4 (TLR4), a unique TLR group member that triggers both MyD88-depependent and MyD88-independent signalings. Importantly, the carbohydrate chains from hLF are shown to be responsible for TLR4 activation. Furthermore, we show that lipopolysaccharide-induced cytokine and chemokine production is attenuated by intact hLF but not by the carbohydrate chains from hLF. Thus, we present a novel model concerning the biological function of hLF: hLF induces moderate activation of TLR4-mediated innate immunity through its carbohydrate chains; however, hLF suppresses endotoxemia by interfering with lipopolysaccharide-dependent TLR4 activation, probably through its polypeptide moiety.

Hypoxia downregulates farnesoid X receptor via a hypoxia-inducible factor-independent but p38 mitogen-activated protein kinase-dependent pathway.

Farnesoid X receptor (FXR), a member of the nuclear receptor superfamily, has been shown to play pivotal roles in bile acid homeostasis by regulating the biosynthesis, conjugation, secretion and absorption of bile acids. Accumulating data suggest that FXR signaling is involved in the pathogenesis of liver and metabolic disorders. Here we show that FXR expression is significantly suppressed in HepG2 cells exposed to hypoxia. Concomitantly, the expression of the bile salt export pump, known as an FXR target gene product and responsible for the excretion of bile acids from the liver, is also decreased under hypoxia. Overexpression of hypoxia-inducible factor (HIF)-1alpha does not mimic the suppressive effect of hypoxia on FXR expression. Furthermore, simultaneous knockdown of HIF-1alpha, HIF-2alpha and HIF-3alpha fails to restore the FXR expression level under hypoxia, indicating that HIF is not involved in hypoxia-evoked FXR downregulation. Instead, we demonstrate that p38 mitogen-activated protein kinase is an indispensable factor for FXR downregulation under hypoxia. Thus, we propose a novel liver disorder model in which two signaling molecules, p38 mitogen-activated protein kinase and FXR, may contribute to the linkage of two pathogenic conditions, i.e. ischemia, a condition accompanying hypoxia, and cholestasis, a condition with intrahepatic accumulation of cytotoxic bile acids.

Novel insights into FGD3, a putative GEF for Cdc42, that undergoes SCF(FWD1/beta-TrCP)-mediated proteasomal degradation analogous to that of its homologue FGD1 but regulates cell morphology and motility differently from FGD1.

We previously demonstrated that FGD1, the Cdc42 guanine nucleotide exchange factor (GEF) responsible for faciogenital dysplasia, is targeted by the ubiquitin ligase SCF(FWD1/beta-TrCP) upon phosphorylation of two serine residues in its DSGIDS motif and subsequently degraded by the proteasome. Here we show that FGD3, which was identified as a homologue of FGD1 but has been poorly characterized, has conserved the same motif and is down-regulated similarly by SCF(FWD1/beta-TrCP). Although FGD3 and FGD1 share strikingly similar Dbl homology (DH) domains and adjacent pleckstrin homology (PH) domains, both of which are responsible for guanine nucleotide exchange, there also exist remarkable differences in their structures. Indeed, FGD1 and FGD3 induced significantly different morphological changes in HeLa Tet-Off cells: whereas FGD1 induced long finger-like protrusions, FGD3 induced broad sheet-like protrusions when the level of GTP-bound Cdc42 was significantly increased by the inducible expression of FGD3. Furthermore, FGD1 and FGD3 reciprocally regulated cell motility: when inducibly expressed in HeLa Tet-Off cells, FGD1 stimulated cell migration whereas FGD3 inhibited it. Thus we demonstrate that the highly homologous GEFs, FGD1 and FGD3 play different roles to regulate cellular functions but that their intracellular levels are tightly controlled by the same destruction pathway through SCF(FWD1/beta-TrCP).

[Nitrosamine formation from vegetables produced in Japan].

Many vegetables produced in Japan contain large amounts of nitrate that may be converted into nitrite in the oral cavity and afford carcinogenic nitrosamines in the stomach. On the other hand, vegetables contain ascorbate and other components that may affect the formation of nitrosamines. In this study, nitrosamine formation from vegetables with high nitrate content produced in Japan was examined under simulated oral cavity and stomach conditions. Extracts of chingensai, komatsuna and itomitsuba were digested with nitrate reductase and subsequently treated with an excess of morpholine at pH 3.0. The amount of N-nitrosomorpholine produced from each of the vegetable extracts was not affected by the vegetable components in the extracts. Addition of a large amount of ascorbate was required to decrease nitrosamine formation from the extract. The results indicated that nitrosamine formation from these vegetables could not be prevented by other components in the vegetables.

Interaction of nitric oxide with glutathione or cysteine generates reactive oxygen species causing DNA single strand breaks.

It was found that reactive oxygen species (ROS) were generated in the interactions of nitric oxide (NO) with glutathione (GSH) or cysteine (CySH) under aerobic conditions. When supercoiled DNA was incubated with a mixture of NO/GSH, NO/CySH, NOC-7 (a NO donor)/GSH or NOC-7/CySH under aerobic conditions, DNA single-strand breaks were observed on agarose gel electrophoresis. The strand breaks were inhibited by common ROS scavengers: superoxide dismutase+catalase, the spin trapping agent 5,5-dimethyl-1-pyrroline-N oxide (DMPO), ethanol, and EDTA. The strand breaks were also caused by incubation with a mixture of S-nitrosoglutathione (GSNO) with GSH or CySH, which was inhibited by ROS scavengers. In the reaction of NO/GSH, GSNO rapidly formed and then gradually decreased, and in the reaction of GSNO/GSH, GSNO was gradually decreased. The decrease inf GSNO was accelerated in the presence of superoxide+catalase. Hydroxyl radical was detected in the mixtures of NO with GSH or CySH under aerobic conditions, and thiyl radicals were detected in the mixtures of GSNO with GSH or CySH under anaerobic conditions as examined in electron spin resonance studies using DMPO as a spin trap. The results indicate that the interaction of NO with thiols in the presence of O2 generates ROS that caused DNA single-strand breaks.

The FWD1/beta-TrCP-mediated degradation pathway establishes a 'turning off switch' of a Cdc42 guanine nucleotide exchange factor, FGD1.

FWD1/beta-TrCP is the F-box protein that functions as the receptor subunit of the SCF(FWD1/beta-TrCP) ubiquitin ligase and has been shown to be responsible for the degradation of important signaling molecules such as IkappaBs and beta-catenin. Protein substrates of FWD1/beta-TrCP contain a consensus DSGPsiXS motif (where Psi represents a hydrophobic residue and X represents any amino acid). Recognition by FWD1/beta-TrCP requires phosphorylation of the conserved serines in that motif. Here we show that FGD1, a Cdc42 guanine nucleotide exchange factor (GEF), is a novel target of the SCF(FWD1/beta-TrCP) ubiquitin ligase. A mutant FGD1 protein, FGD1(SA), in which both of the critical serine residues in the DSGPsiXS motif have been replaced by alanines, does not interact with FWD1/beta-TrCP and exhibits increased stability. Morphological changes induced by wild-type FGD1 (FGD1(WT)) are reduced by the co-expression of SCF(FWD1/beta-TrCP) whereas those induced by FGD1(SA) are not affected. FGD1(SA)-expressing cells show a higher level of cell motility than FGD1(WT)-expressing cells. We present a novel 'turning off' mechanism for the inactivation of FGD1, an upstream regulator for Cdc42.

Prevention of mutagen formation in heated meats and model systems.

Possible means for preventing mutagen formation in cooked meats and in heated model systems are described. One way to reduce mutagenicity in cooked meats is to control cooking temperature, time and method. Another way is to increase water content or to avoid loss of water in meats during cooking. Addition of an excessive amount of reducing sugars to meats before cooking is effective in minimizing mutagen formation, which may be due to suppression of generation of the pyrazine cation radical Maillard intermediate of heterocyclic amines. Addition of a small amount of ascorbate or erythorbate is also effective, which may be the result of scavenging the intermediary pyrazine cation radical.

[Defense of living body against oxidative damage].

Living bodies may experience oxidative stress induced by reactive oxygen species and heavy metal ions, which may damage components in the body and cause aging and disorders. In addition to the known defense systems against oxidative damage, the author describes new defense systems. Lipid peroxidation in living bodies, which has hitherto been thought to increase oxidative damage, was found to attenuate oxidative stress-induced DNA damage. Red blood cells become senescent due to oxidative stress during circulation, where membrane band 3 becomes aggregated to anti-band 3 IgG and macrophages attached through poly-N-acetyllactosaminyl sugar chains, and the sugar chain attachment to macrophages is stimulated by oxidative stress in macrophages. Oxidized protein hydrolase that preferentially hydrolyzes proteins damaged by oxidative stress was newly discovered, which may play an important role in saving cells from oxidative damage.

Coordination of oxidized protein hydrolase and the proteasome in the clearance of cytotoxic denatured proteins.

Intracellular accumulation of denatured proteins impairs cellular function. The proteasome is recognized as an enzyme responsible for the effective clearance of those cytotoxic denatured proteins. As another enzyme that participates in the destruction of damaged proteins, we have identified oxidized protein hydrolase (OPH) and found that OPH confers cellular resistance to various kinds of oxidative stress. In this study, we demonstrate the roles of the proteasome and OPH in the clearance of denatured proteins. The inhibition of proteasome activity results in the elevation of protein carbonyls in cells under oxidative stress. On the other hand, cells overexpressing OPH retain higher resistance to oxidative stress, even though the proteasome activity is inhibited. Furthermore, upon inhibition of the proteasome activity, OPH is recruited to a novel organelle termed the aggresome where misfolded or denatured proteins are processed. Thus, OPH and the proteasome coordinately contribute to the clearance of cytotoxic denatured proteins.

Effects of oxygen on the reactivity of nitrogen oxide species including peroxynitrite.

This paper describes the O(2)-dependent control of the reactivity of nitrogen oxide species for the production of biologically important nitrated and nitrosated compounds. In this study, the effects of O(2) on the reactivity of NO, NO(2), and ONOO(-)/ONOOH for nitration of tyrosine (Tyr) and nitrosation of glutathione (GSH) and morpholine (MOR) were examined. NO produced S-nitrosoglutathione (GSNO) and N-nitrosomorpholine (NMOR) through the formation of N(2)O(3) under aerobic conditions, and NO(2) produced 3-nitrotyrosine (3-NO(2)Tyr), GSNO, and NMOR. Transnitrosation from GSNO to MOR was observed only in the presence of O(2). Although preformed ONOO(-)/ONOOH produced all the products under aerobic conditions, the formation of 3-NO(2)Tyr and GSNO was markedly reduced and the formation of NMOR was enhanced under anaerobic conditions. The reactivity of the CO(2) adduct of ONOO(-) was similarly dependent on O(2). 3-NO(2)Tyr was produced effectively by reaction with ONOO(-)/ONOOH at the O(2) concentration of 270 microM and by reaction with its CO(2) adduct at O(2) concentrations greater than 5 microM. Generation of.OH from ONOO(-)/ONOOH was suppressed under anaerobic conditions. The reactivity of ONOO(-)/ONOOH and.OH generation from ONOO(-) were reversibly controlled by the O(2) concentration.

Protective effect of supplementation of fish oil with high n-3 polyunsaturated fatty acids against oxidative stress-induced DNA damage of rat liver in vivo.

The present study was undertaken to know the effect of supplementation of fish oil with high n-3 polyunsaturated fatty acids (PUFA) on oxidative stress-induced DNA damage of rat liver in vivo. Male Wistar rats were fed a diet containing fish oil or safflower oil with high n-6 PUFA at 50 g/kg of diet and an equal amount of vitamin E at 59 mg/kg of diet for 6 weeks. Livers of rats fed fish oil were rich in n-3 PUFA, whereas those of rats fed safflower oil were rich in n-6 PUFA. Ferric nitrilotriacetate was intraperitoneally injected to induce oxidative stress. The degree of lipid peroxidation of the liver was assessed by the levels of phospholipid hydroperoxides and thiobarbituric acid-reactive substances (TBARS), and the degree of oxidative DNA damage was assessed by comet type characterization in alkaline single-cell gel electrophoresis and 8-hydroxy-2'-deoxyguanosine levels. The levels of TBARS of the livers of the fish oil diet group increased to a greater extent than those of the safflower oil diet group, whereas the levels of the hydroperoxides of the livers of both diet groups increased to a similar extent. The vitamin E level of livers of the fish oil diet group was remarkably decreased. The degree of DNA damage of both diet groups was increased, but the increased level of the fish oil diet group was remarkably lower than that of the safflower oil diet group. The above results indicate that fish oil supplementation does not enhance but appears to protect against oxidative stress-induced DNA damage and suggest that lipid peroxidation does not enhance but lowers the DNA damage.

Is nitric oxide (NO) an antioxidant or a prooxidant for lipid peroxidation?

Antioxidant and prooxidant effects of nitric oxide (NO) on lipid peroxidation in aqueous and non-aqueous media were examined. In an aqueous solution, NO did not induce peroxidation of unoxidized methyl linoleate (ML) and suppressed the radical initiator-induced oxidation of ML. NO suppressed the Fe(II) ion-induced oxidation of mouse liver microsomes. NO reduced the O2 consumption during the radical initiator-induced oxidation of linoleic acid in an aqueous medium. NO conversion into NO2- in an aqueous medium was not affected by unoxidized ML and was slightly reduced by peroxidizing ML. On the other hand, as well as pure NO2, NO induced peroxidation of unoxidized ML in n-hexane in a dose-dependent fashion. NO did not suppress the radical initiator-induced oxidation of ML in n-hexane. Nitrogen oxide species (NO2 or N2O3) formed by autoxidation was dramatically lost in n-hexane in the presence of unoxidized ML. The results indicated that NO terminated lipid peroxidation in an aqueous medium, whereas NO induced lipid peroxidatiton in a non-aqueous medium. Hence, NO showed both antioxidant and prooxidant effects on lipid peroxidation depending on the solvents.

Effect of supplementation of n-3 polyunsaturated fatty acids on oxidative stress-induced DNA damage of rat hepatocytes.

The effect of supplementation of n-3 polyunsaturated fatty acids (PUFA) on oxidative stress-induced DNA damage of rat hepatocytes was examined. Male Wistar rats were fed a diet containing safflower oil (control n-6 PUFA diet) or fish oil (n-3 PUFA diet) in 50 g/kg of dried diet and an equal amount of vitamin E in 59 mg/kg of dried diet for 6 weeks. The liver of rats fed safflower oil was rich in n-6 PUFA, whereas that of rats fed fish oil was rich in n-3 PUFA. Isolated hepatocytes were treated in vitro with ADP/Fe (II) ion or hydrogen peroxide at 37 degrees C for 30 min to induce oxidative stress. The degree of lipid peroxidation was assessed by the levels of phospholipid hydroperoxides and thiobarbituric acid-reactive substances. The degree of oxidative DNA damage was assessed based on comet-type characterization in alkaline single-cell gel electrophoresis and 8-hydroxy-deoxyguanosine levels. In both ADP/Fe(II) ion and hydrogen peroxide oxidation, the degree of lipid peroxidation of hepatocytes increased in both diet groups, and the level of increase in the fish oil diet group was slightly higher than that in the safflower oil diet group. In ADP/Fe(II) ion oxidation, the degree of DNA damage increased in both diet groups, but there were no significant differences in the level of increase. In contrast, in hydrogen peroxide oxidation, the degree of DNA damage increased in both diet, and the increase in the fish oil diet group was significantly lower than that in the safflower oil diet group. It is unlikely that an n-3 PUFA-rich diet enhances oxidative stress-induced hepatocyte DNA damage as compared with the control n-6 PUFA-rich diet.

Suppression of free radical-induced DNA strand breaks by linoleic acid and low density lipoprotein in vitro.

The results of the present study have shown that unoxidized linoleic acid (LA) and low density lipoprotein (LDL) suppressed free radical-induced supercoiled plasmid DNA strand breaks. Unoxidized LA suppressed DNA strand breaks induced by free radicals generated from hydrogen peroxide/Fe(II) ion, 2'-azobis(2-amidinopropane)hydrochloride (AAPH), and 4-(hydroxymethyl)benzene diazonium salt. Thiobarbituric acid reactive substances (TBARS) of LA were increased on treatment with the radical generators. The intensities of the electron spin resonance (ESR) signals of the spin adducts of the radicals were reduced by unoxidized LA. Although LA hydroperoxide caused DNA strand breaks as has already been shown, its strand breaking activity was observed only at the higher concentrations. Unoxidized LDL inhibited ascorbic acid/Cu(II) ion-, ascorbic acid/Fe(II) ion-, peroxynitrite- and AAPH-induced DNA strand breaks. The TBARS of LDL were increased by treatment with the agents. LDL oxidized with Cu(II) ion did not cause DNA strand breaks. The results indicate that the potency of the free radicals to cause DNA strand breaks was attenuated by the fatty acid and the lipoprotein through lipid peroxidation.

Evidence that reactive oxygen species do not mediate NF-kappaB activation.

It has been postulated that reactive oxygen species (ROS) may act as second messengers leading to nuclear factor (NF)-kappaB activation. This hypothesis is mainly based on the findings that N-acetyl-L-cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC), compounds recognized as potential antioxidants, can inhibit NF-kappaB activation in a wide variety of cell types. Here we reveal that both NAC and PDTC inhibit NF-kappaB activation independently of antioxidative function. NAC selectively blocks tumor necrosis factor (TNF)-induced signaling by lowering the affinity of receptor to TNF. PDTC inhibits the IkappaB-ubiquitin ligase activity in the cell-free system where extracellular stimuli-regulated ROS production does not occur. Furthermore, we present evidence that endogenous ROS produced through Rac/NADPH oxidase do not mediate NF-kappaB signaling, but instead lower the magnitude of its activation.

Cholesterol synthesis in mice is suppressed but lipofuscin formation is not affected by long-term feeding of n-3 fatty acid-enriched oils compared with lard and n-6 fatty acid-enriched oils.

Hypocholesterolemic activity of dietary polyunsaturated fatty acids is observed after relatively short-term but not long-term feedings, and their long-term feedings are suspected to accelerate aging through tissue accumulation of lipid peroxides and age pigments (lipofuscin). To define the long-term effects of fats and oils in more detail, female mice were fed a conventional basal diet supplemented with lard (Lar), high-linoleic (n-6) safflower oil (Saf), rapeseed oil (Rap), high-alpha-linolenic (n-3) perilla oil (Per), or a mixture of ethyl docosahexaenoate and soybean oil (DHA/Soy) from 17 weeks to 71 weeks of age. The DHA/Soy and Per groups had decreased serum cholesterol levels compared with the Lar and Saf groups, but the difference between the Lar and Saf groups was not significant. The 3-hydroxy-3-methyglutary-CoA (HMG-CoA) reductase activity in the liver was also significantly lower in the Per and DHA/Soy groups. However, no significant difference in lipofuscin contents in the brain and liver was observed among the 5 dietary groups, despite significant differences in peroxidizability indices of the dietary and/or tissue lipids. These results indicate that n-3 fatty acid-rich oils are hypocholesterolemic by suppressing hepatic HMG-CoA reductase activity compared with animal fats and high-linoleic (n-6) oil, but tissue lipofuscin contents are not affected by a long-term feeding of fats and oils with different degree of unsaturation in mice.

Overexpression of oxidized protein hydrolase protects COS-7 cells from oxidative stress-induced inhibition of cell growth and survival.

Oxidized protein hydrolase (OPH) preferentially degrades oxidatively damaged proteins in vitro and is widely distributed in various cells and tissues. The role of OPH in intact cells exposed to oxidative stress was examined. For this purpose, using COS-7, a cell line derived from African green monkey kidney, COS-7-OPH cells that stably overexpressed OPH were established. When COS-7-OPH cells were exposed to oxidative stress induced by H(2)O(2) and paraquat, accumulation of protein carbonyls in the cells was apparently lower than that of parental COS-7 cells, and COS-7-OPH cells were significantly resistant to the oxidative stress compared with parental COS-7 cells. The majority of overexpressed OPH in the cells was found to be located uniformly in cytosol, and its location was not altered by H(2)O(2)-induced oxidative stress. Above results indicate that OPH in intact cells plays a preventive role against oxidative stress and suggest that OPH relieves cells from accumulation of oxidatively damaged proteins.

Increased urinary hydrogen peroxide levels caused by coffee drinking.

Experiments with volunteers in Singapore have demonstrated that coffee drinking increases urinary hydrogen peroxide levels (Long, Halliwell, Free Rad. Res., 32, 463-467 (2000)). We re-examined the effect of coffee drinking of healthy Japanese subjects on urinary hydrogen peroxide levels. A cup of brewed or canned coffee commercially available in Japan generated 120-420 micro mol hydrogen peroxide in incubation in a neutral medium at 37 degrees C for 6 h. The increased levels were higher than those obtained from a cup of green tea extract or a glass of red wine. After the subject drank a cup of coffee, apparent hydrogen peroxide levels (micro mol/g creatinine) in urine collected 1-2 h after coffee drinking increased 3-10-fold compared to the levels before coffee drinking. The increased urinary hydrogen peroxide levels are likely derived mainly from 1,2,4-benzenetriol excreted in urine, because the major component that generates hydrogen peroxide is found to be 1,2,4-benzenetriol, and storing urine collected after coffee drinking increased hydrogen peroxide levels in a time-dependent fashion. Total hydrogen peroxide equivalent levels excreted in 3 h-urine after coffee drinking were estimated to be 0.5-10% that of coffee consumed. A residual amount of hydrogen peroxide may be retained or consumed in human bodies.