Inhibitory Effect of Ascorbic Acid on the Proliferation and Invasion of Hepatoma Cells in Culture

Effect of ascorbic acid (AsA) on the proliferation and invasion of rat ascites hepatoma AH109A cells was investigated by measuring [³H]thymidine incorporation into acid-insoluble fraction of the cells and by co-culturing the hepatoma cells with rat mesentery-derived mesothelial cells, respectively. AsA suppressed the invasion of AH109A cells in a dose-dependent manner at concentrations of 62.5–500 μM, while it inhibited the proliferation of the cells at higher concentrations of 250 and 500 μM. Hepatoma cells previously cultured with hypoxanthine (HX) and xanthine oxidase (XO) or with hydrogen peroxide showed increased invasive activities. AsA suppressed the reactive oxygen species-potentiated invasive capacity by simultaneously treating AH109A cells with AsA, HX and XO or with AsA and hydrogen peroxide. Furthermore, AsA reduced the intracellular peroxide levels in AH109A cells. These results suggest that the antioxidative property of AsA may be involved in its anti-invasive action on hepatoma cells.     

Potentiation of invasive activity of hepatoma cells by reactive oxygen species is mediated by autocrine/paracrine loop of hepatocyte growth factor

We have already reported that reactive oxygen species (ROS) promote rat ascites hepatoma cell invasion beneath mesentery-derived mesothelial cell monolayer. To investigate the mechanism for this, we examined the involvement of motility factors, particularly hepatocyte growth factor (HGF). Rat ascites hepatoma cell line of AH109A expressed HGF and c-Met mRNAs. Treatment with ROS augmented amounts of HGF mRNA in AH109A and HGF concentration in the medium. ROS also induced HGF gene expression in mesothelial cells. Exogenously added HGF enhanced invasive activity of AH109A cells, but exerted no effect on proliferation. AH109A cells pretreated with ROS showed an increased invasive activity, which was cancelled by simultaneous pretreatment with anti-HGF antibody. These results suggest that the invasive activity of AH109A is mediated by the autocrine and paracrine pathways of HGF, and ROS potentiate invasive activity by inducing gene expression of HGF in AH109A and mesothelial cells.     

Inhibitory effect of gingerol on the proliferation and invasion of hepatoma cells in culture

Effect of [6]-gingerol, a major pungent component in ginger, on the proliferation of a rat ascites hepatoma AH109A cells was investigated by measuring [³H]thymidine incorporation into acid-insoluble fraction of the cultured cells and that on the invasion by co-culturing the hepatoma cells with rat mesentery-derived mesothelial cells. [6]-Gingerol inhibited both the proliferation and invasion of hepatoma cells in a dose-dependent manner at concentrations of 6.25–200 μM (proliferation) and 50–200 μM (invasion). [6]-Gingerol accumulated cells in S phase and elongated doubling time of hepatoma cells, and increased the rate of apoptosis. Hepatoma cells previously cultured with hypoxanthine (HX) and xanthine oxidase (XO) or with hydrogen peroxide showed increased invasive activities. [6]-Gingerol suppressed the reactive oxygen species-potentiated invasive capacity by simultaneously treating AH109A cells with [6]-gingerol, HX and XO or with [6]-gingerol and hydrogen peroxide. Furthermore, [6]-gingerol reduced the intracellular peroxide levels in AH109A cells. These results suggest that the suppression of hepatoma cell proliferation by [6]-gingerol may be due to cell cycle arrest and apoptosis induction. They also suggest that the anti-oxidative property of [6]-gingerol may be involved in its anti-invasive activity of hepatoma cells.     

Inhibitory effect of curcumin on the invasion of rat ascites hepatoma cells in vitro and ex vivo

Curcumin, a yellow pigment in turmeric, is a food factor withantioxidative activity. The effect of curcumin on the proliferation and invasion of the rat ascites hepatoma AH109Acells was studied in vitro and ex vivo assay systems. Especially, a co-culture system of the hepatoma cellswith mesothelial cells derived from rat mesentery was employed to investigate the invasive motility. Curcumin suppressed thehepatoma slipping motility in a dose-dependent manner up to 5 M and thereafter maintained the effect up to 20 M, whereas this substance exerted little influence on the proliferation of the hepatoma cells at the same concentrations. Sera obtained from rats orally given curcumin also inhibited the AH109A cellular invasive movement when added to the culturemedium. Hepatoma cells previously cultured with hypoxanthineand xanthine oxidase showed a highly invasive activity. Curcumin and curcumin-loaded rat sera suppressed this reactive oxygen species-potentiated invasive capacity by simultaneously treating AH109A cells with hypoxanthine, xanthine oxidase and either of curcumin samples. These resultssuggest that the antioxidative property of curcumin may beinvolved in its anti-invasive action.     

Effects of green, oolong and black teas and related components on the proliferation and invasion of hepatoma cells in culture

The effects of teas and related components on the proliferation and invasion of cancer cells were examined by employing both in vitro proliferation and invasion assay systems. Powdered green, oolong and black tea extracts dose-dependently inhibited proliferation and invasion of a rat ascites hepatoma cell line of AH109A but did not affect the proliferation of the normal mesentery-derived mesothelial cells (M-cells) isolated from rats; higher concentrations of powdered oolong and black teas could restrain the proliferation of another tumor cell line of L929. The AH109A cells were found to penetrate underneath the monolayer of M-cells in the presence of 10% calf serum. When each rat serum obtained at 0.5, 1, 2, 3 and 5 h after oral intubation of each tea extract was added to the culture media instead of calf serum at a concentration of 10%, both the invasion and proliferation of AH109A were significantly suppressed. These ex vivo results suggest the potential bioavailability of effective tea components in rats. Furthermore, (–)-epigallocatechin gallate, (–)-epicatechin gallate and (–)-epigallocatechin from green tea as well as the mixture of theaflavin and theaflavin gallates from black tea were shown to be the most effective components against the invasion and proliferation of AH109A. These results show that the inhibitory effects of the teas and related components against AH109A cells are due to the cell-specific and higher sensitivity of the cell line to tea components. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effect of apple polyphenol extract on hepatoma proliferation and invasion in culture and on tumor growth, metastasis, and abnormal lipoprotein profiles in hepatoma-bearing rats

The effect of apple polyphenol extract (APE) on the proliferation and invasion of a rat ascites hepatoma cell line of AH109A was examined in vitro. APE suppressed both the hepatoma proliferation and invasion in a dose-dependent manner up to 200 mug/ml. Serum obtained from rats orally given APE also inhibited hepatoma proliferation and invasion when added to the culture medium. Subsequently, the effect of dietary APE on growth and the metastasis of AH109A hepatomas were investigated in vivo. APE reduced the growth and metastasis of solid hepatomas and significantly suppressed the serum lipid peroxide level in rats transplanted with AH109A. APE also suppressed the serum very-low-density lipoprotein + low-density lipoprotein (VLDL + LDL)-cholesterol level. These in vitro and in vivo findings suggest that APE has anti-hepatoma activities.     

Inhibitory effects of theanine and sera from theanine-fed rats on receptor-mediated cancer cell invasion beneath mesothelial-cell monolayers

To investigate the bioavailability and mode of action of theanine against cancer, we examined in vitro and ex vivo effects of theanine on invasion of a rat ascites hepatoma cell line of AH109A. Theanine dose-dependently inhibited the invasion of AH109A cells across rat mesentery-derived mesothelial-cell (M-cell) monolayers without restraining AH109A cell proliferation in vitro. Rat sera obtained after oral intubation of theanine also inhibited the invasion. A competitive N-methyl-D-aspartate (NMDA) type glutamate receptor antagonist, (±) 2-amino-5-phosphonopentanoic acid (AP-5), dose-dependently counteracted the theanine-mediated in vitro and ex vivo inhibition of AH109A invasion. A competitive non-NMDA type glutamate receptor antagonist, 6,7-dinitroquinoxaline 2,3-dione (DNQX), did not affect this inhibition by theanine in vitro. These results suggest that the inhibition of AH109A invasion by theanine may be mediated by the NMDA receptor of AH109A. This revised version was published online in July 2006 with corrections to the Cover Date.     

Assay systems for screening food components that have anti-proliferative and anti-invasive activity to rat ascites hepatoma cells: In vitro and ex vivo effects of green tea extract

We have developed in vitro and ex vivo assay systems for screening food components and natural substances that suppress the proliferation and/or invasion of a rat ascites hepatoma cell line of AH109A and have used them to study the effect of green tea extract. AH109A cells were found to penetrate underneath the monolayer of primary cultured mesothelial cells isolated from Donryu rat mesentery in the presence of 10% newborn bovine serum. Green tea extract inhibited this AH109A penetration in a dose dependent manner and also inhibited AH109A proliferation in vitro dose-dependently. Green tea tannin, the major polyphenolic substances in green tea extract, also inhibited the proliferation and invasion of AH109A in vitro in a dose dependent manner. When rat serum obtained 0.5 h after oral intubation of green tea extract was added to the culture media instead of newborn bovine serum at a concentration of 10%, the invasion of AH109A was significantly inhibited as compared to control rat serum (before green tea extract intubation); the inhibitory effect lasted for 1 h and disappeared 3 h after oral intubation of green tea extract, but those rat sera showed no inhibition of AH109A proliferation. These results suggest that green tea extract has an inhibitory effect on the invasion of AH109A both in vitro and ex vivo, but on the proliferation of AH109A only in vitro, and that these assay systems are effective for the screening of food components which inhibit tumor cell proliferation and invasion.     

Inhibition of hepatoma cell invasion beneath mesothelial-cell monolayer by sera from tea- and related component-treated rats and their modes of action

The bioavailability and action of teas on the invasion of a rat ascites hepatoma cell line, AH109A, were determined and their modes of action were by co-culturing the cancer cells with a rat mesentery-derived mesothelial-cell (M-cell) monolayer in the presence of sera from rats orally given teas and their component, (-)-epigallocatechin gallate (EGCG). The rat sera obtained 2 and 5 hr after oral intubation of a low concentration of green, oolong, or black tea, or EGCG significantly inhibited AH109A invasion underneath the M-cell monolayer. These sera showed a time-dependent and significant inhibitory effect on the AH109A invasion. The 2-hr sera and 2.5 μM EDTA in the medium completely eliminated the enhancement of AH109A invasion induced by a reactive oxygen species (ROS)-generating system. These results show that the inhibition of relevant ROS-potentiated invasion of AH109A cells across the M-cell monolayer may be due to the antioxidative action of EGCG, the in vivo metabolites, and tea-induced changes in the endogenous substances. The results suggest that the drinking of tea in daily life may have certain preventive and therapeutic effects against cancer cell invasion. This revised version was published online in July 2006 with corrections to the Cover Date.     

Restoration by Prostaglandins E2 and F2 α of Resveratrol-Induced Suppression of Hepatoma Cell Invasion in Culture

In our previous study, resveratrol, a polyphenolic compound in grapes with antioxidative property, and resveratol-loaded rat serum (RS) were found to suppress the invasion of AH109A cells, an ascite hepatoma cell line. The aim of the present study was to investigate whether and which prostaglandins (PGs) would be involved in the invasion of AH109A cells and its suppression by resveratrol and resveratrol-loaded RS, using an in vitro invasion assay system. Not only PGE₂ but also PGF_{2 α} stimulated the spontaneous invasion of AH109A cells.They also canceled the resveratrol-induced suppression of hepatoma cell invasion. Results obtained suggest an involvement of PGs, especially PGE₂, in the invasion of hepatoma cells.     

Isolation and Characterization of Subpopulations of Rat Ascites Hepatoma Cell Line of AH109A with Different Metastatic Potentials

Rat ascites hepatoma cell line of AH109A proved to be divided into two subpopulations with different invasive and metastatic potentials, when cultured in the medium containing allogeneic rat sera. One population adheres to the culture dish, actively extending pseudopodia, and the other remains in a floating state. Utilizing this character, we have separated these two populations. After three successive separation steps, adhesive AH109A cells and floating AH109A cells were obtained. Adhesive AH109A cells proliferated more rapidly and invaded more actively than did floating AH109A cells. Adhesive AH109A cells metastasized mainly to lung, while floating AH109A cells to mesentery, when intravenously injected into tail veins. Histological studies revealed that adhesive AH109A cells showed lymphatic metastases to lung. These results suggest that the two populations separated from parental AH109A cells provide good models for the study of tumor invasion and tissue-specific metastasis and that adhesive AH109A cells can be used for the creation of lymphatic metastasis model of rats.     

Studies on Hot Water-soluble and Dilute Alkali-soluble Polysaccharides from the Fruit Body of Mushroom (Psalliota campestris (Linn) Fr.)

The effect of apple polyphenol extract (APE) on the proliferation and invasion of a rat ascites hepatoma cell line of AH109A was examined in vitro. APE suppressed both the hepatoma proliferation and invasion in a dose-dependent manner up to 200 μg/ml. Serum obtained from rats orally given APE also inhibited hepatoma proliferation and invasion when added to the culture medium. Subsequently, the effect of dietary APE on growth and the metastasis of AH109A hepatomas were investigated in vivo. APE reduced the growth and metastasis of solid hepatomas and significantly suppressed the serum lipid peroxide level in rats transplanted with AH109A. APE also suppressed the serum very-low-density lipoprotein + low-density lipoprotein (VLDL + LDL)-cholesterol level. These in vitro and in vivo findings suggest that APE has anti-hepatoma activities.     

Blockade of Endogenous Reactive Oxygen Species by N-Acetyl-L-Cysteine Suppresses the Invasive Activity of Rat Hepatoma Cells by Modulating the Expression of Hepatocyte Growth Factor Gene

We have already reported that exogenously added reactive oxygen species (ROS) could potentiate the invasive activity of rat hepatoma cell line of AH109A by activating autocrine loop of hepatocyte growth factor (HGF)-c-Met pathway. In this report, we examined the involvement of endogenous ROS in the invasive activity of hepatoma cells by using a cell-permeable antioxidant, N-acetyl-L-cysteine (NAC). NAC could certainly scavenge intracellular ROS when directly added to the media at the concentration of 1 or 5 mM and could significantly suppress hepatoma cell invasion, although it showed a little effect on hepatoma cell proliferation at these concentrations. NAC also decreased the content of HGF mRNA and the secretion of HGF at these concentrations, leading to suppression of their invasion. In the present study, blockade of endogenous ROS by NAC proved to efficiently suppress the invasive activity of hepatoma cells by down-regulating HGF gene expression, suggesting the importance of endogenous ROS in cellular signaling of tumor cell invasion.     

Anti-proliferative effect of pterostilbene on rat hepatoma cells in culture

Pterostilbene, a methoxylated analogue of resveratrol, is a natural compound primarily found in blueberries and several types of grapes. However, little is known about the effect of pterostilbene on the proliferation of hepatoma cells and its modes of actions. This study was undertaken to characterize its ability to suppress the proliferation of hepatoma AH109A cells and the possible mechanism(s) involved. Pterostilbene showed a significant and dose-dependent effect on the anti-proliferative activity against AH109A cells. Pterostilbene exerted little or no effect on the proliferation of rat L6 myoblasts and rat skin fibroblasts. Pterostilbene-loaded rat sera could significantly inhibit the proliferation of AH109A cells, which suggests that pterostilbene could be absorbed through gastrointestinal tract and retain its anti-proliferative activity. Pterostilbene arrested the cell cycle of AH109A cells at G0/G1 phase and reduced the protein expression of cyclin-dependent kinase 4 and cyclin-dependent kinase 6 dose-dependently. We also found that pterostilbene could significantly increase the intracellular peroxide level of AH109A cells, which may be involved in its anti-proliferative activity.     

Inhibitory effect of serum from rats administered with coffee on the proliferation and invasion of rat ascites hepatoma cells

The action of coffee on the proliferation and invasion of a rat ascites hepatoma cell line of AH109A was investigated using in vitro and ex vivo assay systems. When rats were given oral intubation of instant coffee powder solution, the sera of those rats had the potent inhibitory activity on both the proliferation and invasion of AH109A. The activity of rat serum was both time- and dose-dependent. The instant coffee powder also inhibited the proliferation and invasion of AH109A in vitro. These results indicate that coffee has anti-proliferative and anti-invasive activity both in vitro and ex vivo. They also suggest that some anti-proliferative and anti-invasive material(s), which may be the ingredient(s) of coffee or their metabolites, appear in rat serum when rats are given oral intubation of coffee, although a possibility that host defense systems may be activated by the oral intubation of coffee cannot be ruled out. This revised version was published online in June 2006 with corrections to the Cover Date.     

Upregulation of GPR109A in Parkinson’s Disease

Background

Anecdotal animal and human studies have implicated the symptomatic and neuroprotective roles of niacin in Parkinson’s disease (PD). Niacin has a high affinity for GPR109A, an anti-inflammatory receptor. Niacin is also thought to be involved in the regulation of circadian rhythm. Here we evaluated the relationships among the receptor, niacin levels and EEG night-sleep in individuals with PD.

Methods and Findings

GPR109A expression (blood and brain), niacin index (NAD-NADP ratio) and cytokine markers (blood) were analyzed. Measures of night-sleep function (EEG) and perceived sleep quality (questionnaire) were assessed. We observed significant up-regulation of GPR109A expression in the blood as well as in the substantia nigra (SN) in the PD group compared to age-matched controls. Confocal microscopy demonstrated co-localization of GPR109A staining with microglia in PD SN. Pro and anti-inflammatory cytokines did not show significant differences between the groups; however IL1-β, IL-4 and IL-7 showed an upward trend in PD. Time to sleep (sleep latency), EEG REM and sleep efficiency were different between PD and age-matched controls. Niacin levels were lower in PD and were associated with increased frequency of experiencing body pain and decreased duration of deep sleep.

Conclusions

The findings of associations among the GPR109A receptor, niacin levels and night-sleep function in individuals with PD are novel. Further studies are needed to understand the pathophysiological mechanisms of action of niacin, GPR109A expression and their associations with night-sleep function. It would be also crucial to study GPR109A expression in neurons, astrocytes, and microglia in PD. A clinical trial to determine the symptomatic and/or neuroprotective effect of niacin supplementation is warranted.     

Niacin noncompetitively inhibits DGAT2 but not DGAT1 activity in HepG2 cells

Niacin is a widely used lipid-regulating agent in dyslipidemic patients. Previously, we have shown that niacin inhibits triacylglycerol synthesis. In this report, using HepG2 cells, we have examined the effect of niacin on the mRNA expression and microsomal activity of diacylglycerol acyltransferase 1 and 2 (DGAT1 and DGAT2), the last committed but distinctly different enzymes for triglyceride synthesis. Addition of niacin to the DGAT assay reaction mixture dose-dependently (0-3 mM) inhibited DGAT activity by 35-50%, and the IC(50) was found to be 0.1 mM. Enzyme kinetic studies showed apparent K(m) values of 8.3 microM and 100 microM using [(14)C]oleoyl-CoA and sn-1,2-dioleoylglycerol as substrates, respectively. A decrease in apparent V(max) was observed with niacin, whereas the apparent K(m) remained constant. A Lineweaver-Burk plot of DGAT inhibition by niacin showed a noncompetitive type of inhibition. Niacin selectively inhibited DGAT2 but not DGAT1 activity. Niacin inhibited overt DGAT activity. Niacin had no effect on the expression of DGAT1 and DGAT2 mRNA. These data suggest that niacin directly and noncompetitively inhibits DGAT2 but not DGAT1, resulting in decreased triglyceride synthesis and hepatic atherogenic lipoprotein secretion, thus indicating a major target site for its mechanism of action.     

Niacin inhibits surface expression of ATP synthase beta chain in HepG2 cells: implications for raising HDL

Niacin is an effective agent for raising HDL, but its cellular target sites are largely unknown. We examined effects of niacin on the surface expression of ATP synthase beta chain, a newly described HDL/apolipoprotein A-I (apoA-I) receptor for HDL endocytosis, in HepG2 cells. A significant amount of immunodetectable beta chain was observed on the surface of HepG2 cells, which was competitively displaced by apoA-I. Niacin treatment reduced the surface expression of beta chain in HepG2 cells by approximately 27%, and decreased (125)I-labeled HDL uptake up to approximately 35%. However, nicotinamide, a niacin metabolite that does not have clinical lipid effects, exhibited weaker inhibition on the beta chain cell surface expression, and failed to show inhibitory action on (125)I-labeled HDL uptake. Furthermore, anti-beta chain antibody significantly reduced (125)I-labeled HDL uptake and abolished the inhibitory effect of niacin. Niacin did not change beta chain mRNA expression. These data suggest that niacin inhibits cell surface expression of the ATP synthase beta chain, leading to reduced hepatic removal of HDL protein, thus implicating a potential cellular target for niacin action to raise HDL.     

Triglyceride modulation by acifran analogs: activity towards the niacin high and low affinity G protein-coupled receptors HM74A and HM74

Niacin is known to exert profound beneficial effects on cholesterol levels in humans, although its use is somewhat hampered by the gram quantities necessary to exert effects and the prevalence of compliance-limiting skin flushing side effects that occur. Recently, two G protein-coupled receptors (GPCRs) for niacin were identified and characterized as high (HM74A; GPR109A) and low (HM74; GPR109B) affinity receptors based on the binding affinities of niacin. These receptors also bind acifran (AY-25,712), which is known to modulate lipid levels like niacin, with similar affinities. Twelve analogs of acifran were chemically synthesized. One analogue demonstrated a dose-dependent decrease in serum triglycerides in rats within 3h of oral administration. Next, the acifran analogs were assessed for their activity towards the high and low affinity niacin receptors expressed in CHO-K1 cells. Constructs expressing HM74A or HM74 were stably transfected into CHO-K1 cells and shown to elicit phosphorylation of p42 and p44 mitogen-activated protein kinase (ERK1/ERK2) phosphorylation upon addition of niacin or acifran. The presence of functionally coupled GPCRs was further confirmed using Pertussis toxin, which completely inhibited the ability of either niacin or acifran to elicit phospho-ERK1/ERK2. The EC(50) of p-ERK1/ERK2 for niacin for the high and low affinity receptors was 47nM and indeterminate (i.e., >100microM), respectively, while the EC(50) for acifran was 160 and 316nM, respectively. Two chemical analogs of acifran demonstrated robust phosphorylation of ERK1/ERK2. Collectively, these data suggest that the synthesis of acifran analogs may be a suitable path for developing improved HM74A agonists.