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.     

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.     

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.     

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 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.     

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.     

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.     

Anti-invasive activity of niacin and trigonelline against cancer cells

The effects of niacin, namely, nicotinic acid and nicotinamide, and trigonelline on the proliferation and invasion of cancer cells were studied using a rat ascites hepatoma cell line of AH109A in culture. Niacin and trigonelline inhibited the invasion of hepatoma cells at concentrations of 2.5-40 microM without affecting proliferation. Hepatoma cells previously cultured with a reactive oxygen species (ROS)-generating system showed increased invasive activity. Niacin and trigonelline suppressed this ROS-potentiated invasive capacity through simultaneous treatment of AH109A cells with the ROS-generating system. The present study indicates for the first time the anti-invasive activities of niacin and trigonelline against cancer cells.     

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.     

Anti-cancer activities of tea epigallocatechin-3-gallate in breast cancer patients under radiotherapy

The purpose of this study was to test the hypothesis that administration of epigallocatechin-3-gallate (EGCG), a polyphenol present in abundance in widely consumed tea, inhibits cell proliferation, invasion, and angiogenesis in breast cancer patients. EGCG in 400 mg capsules was orally administered three times daily to breast cancer patients undergoing treatment with radiotherapy. Parameters related to cell proliferation, invasion, and angiogenesis were analyzed while blood samples were collected at different time points to determine efficacy of the EGCG treatment. Compared to patients who received radiotherapy alone, those given radiotherapy plus EGCG for an extended time period (two to eight weeks) showed significantly lower serum levels of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and reduced activation of metalloproteinase-9 and metalloproteinase-2 (MMP9/MMP2). Addition of sera obtained from patients treated with combination of radiotherapy and EGCG feeding for 2-8 weeks to in vitro cultures of highly-metastatic human MDA-MB-231 breast cancer cells resulted in the following significant changes: (1) suppression of cell proliferation and invasion; (2) arrest of cell cycles at the G0/G1 phase; (3) reduction of activation of MMP9/MMP2, expressions of Bcl-2/Bax, c-Met receptor, NF-κB, and the phosphorylation of Akt. MDA-MB-231 cells exposed to 5-10 μM EGCG also showed significant augmentation of the apoptosis inducing effects of γ-radiation, concomitant with reduced NF-κB protein level and AKT phosphorylation. These results provide hitherto unreported evidence that EGCG potentiated efficacy of radiotherapy in breast cancer patients, and raise the possibility that this tea polyphenol has potential to be a therapeutic adjuvant against human metastatic breast cancer.     

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.     

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.     

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 effects of chlorogenic acid and its related compounds on the invasion of hepatoma cells in culture

Actions of chlorogenic acid, a major component of coffee, andits constituents, caffeic and quinic acids, on theproliferation and invasion of AH109A, a rat ascites hepatomacell line, were investigated using in vitro assay systems. Allthree components suppressed the AH109A invasion atconcentrations of 5–40 M without altering the cellproliferation. At the concentration of 10 M, chlorogenic,caffeic and quinic acids significantly (P < 0.05) suppressedthe invasion by 68%, 36% and 31%, respectively, implying thatthe suppressive effect of chlorogenic acid on the AH109Ainvasion might result from the additive effects of itsconstituents, caffeic and quinic acids. At the concentrationof 10 M, cinnamic acid and p-coumaric acid (4-hydroxycinnamicacid) exerted no or little influence on the invasion, whereascaffeic acid (3,4-dihydroxycinnamic acid) significantly (P <0.05) suppressed it, suggesting the possible involvement ofthe 3,4-dihydroxy group of caffeic acid in the suppression.Chlorogenic acid was thus demonstrated to be one of thechemical entities in coffee suppressing the hepatoma invasionin vitro, and both of its constituents, caffeic and quinicacids, to be responsible for the anti-invasive activity. Theseresults suggest the existence of nutritionally andpharmacologically important substances in coffee which controltumor cell invasion.     

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.     

白化和黄化茶树品种绿茶游离氨基酸、儿茶素类及咖啡碱差异分析

为探究以白化和黄化茶树品种鲜叶为原料制成的绿茶滋味品质和代谢物差异,对广德市6个白化品种绿茶(奶白茶)和14个黄化品种绿茶(黄金芽茶)进行感官审评和代谢物分析。结果表明,奶白茶滋味鲜爽而收敛性略弱;黄金芽茶滋味收敛性强而鲜度低于奶白茶。游离氨基酸总量以及呈现鲜味、甜味的游离氨基酸在奶白茶中的含量显著高于黄金芽茶,而贡献收敛性的儿茶素类化合物和没食子酸含量以及呈现苦味的咖啡碱含量在奶白茶中显著低于黄金芽茶。偏最小二乘法判别分析(PLS-DA)表明导致两种绿茶滋味差异的标志性化合物有7种,分别是茶氨酸、精氨酸、谷氨酸、天冬氨酸、表没食子儿茶素没食子酸酯(EGCG)、表儿茶素没食子酸酯和咖啡碱。味觉活性值(Dot)最高的EGCG在黄金芽茶中的呈味贡献显著高于奶白茶。因此,游离氨基酸、儿茶素类化合物、没食子酸和咖啡碱含量差异导致白化和黄化茶树品种绿茶滋味不同。     

Quantitative comparison of cancer and normal cell adhesion using organosilane monolayer templates: an experimental study on the anti-adhesion effect of green-tea catechins

The main constituent of green tea, (?)-Epigallocatechin-3-O-gallate (EGCG), is known to have cancer-specific chemopreventive effects. In the present work, we investigated how EGCG suppresses cell adhesion by comparing the adhesion of human pancreatic cancer cells (AsPC-1 and BxPC-3) and their counterpart, normal human embryonic pancreas-derived cells (1C3D3), in catechin-containing media using organosilane monolayer templates (OMTs). The purpose of this work is (1) to evaluate the quantitativeness in the measurement of cell adhesion with the OMT and (2) to show how green-tea catechins suppress cell adhesion in a cancer-specific manner. For the first purpose, the adhesion of cancer and normal cells was compared using the OMT. The cell adhesion in different type of catechins such as EGCG, (?)-Epicatechin-3-O-gallate (ECG) and (?)-Epicatechin (EC) was also evaluated. The measurements revealed that the anti-adhesion effect of green-tea catechins is cancer-specific, and the order is EGCG?ECG>EC. The results agree well with the data reported to date, showing the quantitativeness of the new method. For the second purpose, the contact area of cells on the OMT was measured by reflection interference contrast microscopy. The cell-OMT contact area of cancer cells decreases with increasing EGCG concentration, whereas that of normal cells remains constant. The results reveal a twofold action of EGCG on cancer cell adhesion—suppressing cell attachment to a candidate adhesion site and decreasing the contact area of the cells—and validates the use of OMT as a tool for screening cancer cell adhesion.     

Green tea (-)-epigallocatechin-3-gallate inhibits HGF-induced progression in oral cavity cancer through suppression of HGF/c-Met

Hepatocyte growth factor (HGF) and c-Met have recently attracted a great deal of attention as prognostic indicators of patient outcome, and they are important in the control of tumor growth and invasion. Epigallocatechin-3-gallate (EGCG) has been shown to modulate multiple signal pathways in a manner that controls the unwanted proliferation and invasion of cells, thereby imparting cancer chemopreventive and therapeutic effects. In this study, we investigated the effects of EGCG in inhibiting HGF-induced tumor growth and invasion of oral cancer in vitro and in vivo. We examined the effects of EGCG on HGF-induced cell proliferation, migration, invasion, induction of apoptosis and modulation of HGF/c-Met signaling pathway in the KB oral cancer cell line. We investigated the antitumor effect and inhibition of c-Met expression by EGCG in a syngeneic mouse model (C3H/HeJ mice, SCC VII/SF cell line). HGF promoted cell proliferation, migration, invasion and induction of MMP (matrix metalloproteinase)-2 and MMP-9 in KB cells. EGCG significantly inhibited HGF-induced phosphorylation of Met and cell growth, invasion and expression of MMP-2 and MMP-9. EGCG blocked HGF-induced phosphorylation of c-Met and that of the downstream kinases AKT and ERK, and inhibition of p-AKT and p-ERK by EGCG was associated with marked increases in the phosphorylation of p38, JNK, cleaved caspase-3 and poly-ADP-ribose polymerase. In C3H/HeJ syngeneic mice, as an in vivo model, tumor growth was suppressed and apoptosis was increased by EGCG. Our results suggest that EGCG may be a potential therapeutic agent to inhibit HGF-induced tumor growth and invasion in oral cancer.     

Inhibition of melanoma growth and metastasis by combination with (-)-epigallocatechin-3-gallate and dacarbazine in mice.

(-)-Epigallocatechin-3-gallate (EGCG), a major polyphenol in green tea, was shown to have cancer chemopreventive activity. In this study, we examined the antimetastatic effects of EGCG or the combination of EGCG and dacarbazine on B16-F3m melanoma cells in vitro and in vivo. First, the antimetastatic potentials of five green tea catechins were examined by soft agar colony formation assay, and the results show that EGCG was more effective than the other catechins in inhibiting soft agar colony formation. Second, EGCG dose-dependently inhibited B16-F3m cell migration and invasion by in vitro Transwell assay. Third, EGCG significantly inhibited the spread of B16-F3m cells on fibronectin, laminin, collagen, and Matrigel in a dose-dependent manner. In addition, EGCG significantly inhibited the tyrosine phosphorylation of focal adhesion kinase (FAK) and the activity of matrix metalloproteinase-9 (MMP-9). In animal experiments, EGCG alone reduced lung metastases in mice bearing B16-F3m melanomas. However, a combination of EGCG and dacarbazine was more effective than EGCG alone in reducing the number of pulmonary metastases and primary tumor growths, and increased the survival rate of melanoma-bearing mice. These results demonstrate that combination treatment with EGCG and dacarbazine strongly inhibits melanoma growth and metastasis, and the action mechanisms of EGCG are associated with the inhibition of cell spreading, cell-extracellular matrix and cell-cell interactions, MMP-9 and FAK activities.