村上 明

Overexpression of matrix metalloproteinases (MMPs) is associated with cancer metastasis. We assessed mRNA expression of MMPs in six human colorectal cancer cell lines and found a considerable level of MMP-7 expression in HT-29 cells. Next, we searched for natural and synthetic compounds that cause a reduction in the production of proMMP-7 protein, and found that nobiletin (NOB), quercetin, valeryl salicylate, and sulindac sulfone demonstrated marked inhibition. Importantly, NOB attenuated proMMP-7 protein and its mRNA expression both concentration- and time-dependently via a reduction of activator protein-1 (AP-1) DNA binding activity, suggesting it as a promising agent for suppression of cancer cell invasion and metastasis through MMP-7 gene repression.

There is increasing interest in the use of herbs for the treatment of human diseases including cancer. Therefore, the purpose of this study was to determine whether crude extracts obtained from 44 herbal plants in the Ryukyu Islands might contain components capable of inhibiting the growth of a variety of human colon carcinoma cell lines. Leaves, roots and other parts of the plants were extracted with chloroform, and the crude extracts were dissolved in dimethylsulfoxide and used for the experiments. Extracts of Hemerocallis fulva, Ipomoea batatas, Curcuma longa, and Nasturium officinale caused marked dose-dependent growth inhibition, with IC50 values in the range of 10-80 μ g/ml. With the HCT116 cell line, the extracts of Hemerocallis fulva and Ipomoea batatas induced G1 cell cycle arrest after 48 h of treatment. In addition, we found that extracts of Curcuma longa, and Nasturium officinale induced apoptosis in these cells after 48 h of treatment. The present studies are the first systematic examination of the growth inhibitory effects of crude extracts obtained from herbal plants in the Ryukyu Islands. The findings provide evidence that several plants in the Ryukyu Islands contain components that may have anticancer activity.

Okinawa prefecture in Japan is a distinct area characterized by unique traditional food habits and longevity. Prolonged exposure to activated leukocytes, playing pivotal roles in chronic inflammation-associated carcinogenesis, is known to lead to oxidative and nitrosative damage to macromolecules in the body since they are primary sources of free radicals, such as superoxide anion (O2 -) and nitric oxide (NO). In this study, we estimated anti-oxidative and anti-nitrosative activities of Okinawan food items by employing two cellular experimental systems: (1) phorbol ester-induced O2 - generation from differentiated HL-60 human promyelocytic leukemia cells and (2) lipopolysaccharide (LPS)-induced NO generation in RAW264.7 murine macrophages. A total of 138 food items, consisting of 42 samples unique to Okinawa and 96 common in the Japanese main island, were purchased at local markets in Okinawa and extracted with chloroform. When tested at a concentration of 100 μg/ml, 38% (16/42) of the former showed 70% or more inhibition of O2 - generation while 21% (20/96) of the latter did so. In parallel, 64%(27/42) of the former showed significant NO generation suppression in contrast to 48% (46/96) of the latter Twentyone active species were further tested at a concentration of 20 μg/ml, and eleven species, including sugar cane, wild turmeric, and zedoary, were indicated to be most promising items with anti-oxidative and anti-nitrosative properties. In addition, some of the active constituents (chebulagic acid, a resveratrol derivative, and sesquiterpenoids) wereidentified. Our results suggest that food items typical in the Okinawa area have higher cancer preventive potential than those common in Japan.

Zerumbone (ZER), a sesquiterpene compound occurring in tropical ginger Zingiber zerumbet Smith, has been implicated as one of the promising chemopreventive agents against colon and skin cancer. In the present study, we investigated the phase II detoxification enzymes induction of ZER using a cultured rat normal liver epithelial cell line. Exposure of RL34 cells to ZER resulted in the significant induction of glutathione S-transferase, while the reduced analogues of ZER (alpha-humulene and 8-hydroxy-alpha-humulene) did not show any inducing effect. Therefore, the electrophilic property, characterized by the reactivity with intracellular nucleophiles including protein sulfhydryls as well as low molecular weight thiols, at the 8-position alpha, beta-unsaturated carbonyl group plays an important role in the induction of phase II enzymes. ZER induced nuclear localization of the transcription factor Nrf2 that binds to antioxidant response element (ARE) of the phase II enzyme genes, suggesting that ZER is a potential activator of the Nrf2/ARE-dependent detoxification pathway. This is consistent with the observation that ZER potentiated the gene expression of several Nrf2/ARE-dependent phase II enzyme genes, including gamma-glutamylcysteine synthetase, glutathione peroxidase, and hemeoxygenase-1. The present study also implied the antioxidant role of this detoxification system activation by ZER in the neutralization of lipid peroxidation in hepatocytes, providing a new insight for cancer prevention. (C) 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

We recently showed that zerumbone, a sesquiterpene found in subtropical ginger, suppresses colonic tumor marker formation in rats and induces apoptosis in colon cancer cell lines. In our present study, the anti-tumor initiating and promoting activities of zerumbone in mouse skin were evaluated using a conventional 2-stage carcinogenesis model. A single topical pretreatment to mouse skin (2 mumol) 24 hr before application of dimethylbenz[a]anthracene (0.2 mumol) markedly suppressed tumor incidence by 60% and the number of tumors by 80% per mouse. Repeated pretreatment (16 nmol) twice weekly during the post-initiation phase reduced the number of 12-O-tetradecanoylphorbol-13-acetate (TPA, 1.6 nmol)-induced tumors by 83% as well as their diameter by 57%. Multiple reverse transcriptase (RT) PCR experiments revealed that zerumbone (2 mumol) enhanced the mRNA expression level of manganese superoxide dismutase, glutathione peroxidase-1, glutathione S-transferase-P1 and NAD(P)H quinone oxidoreductase in the epidermis, but not that of cytochrome P450 1A1 or 1B1. Further, it diminished TPA-induced cyclooxygenase-2 protein expression and phosphorylation of extracellular signal-regulated kinase 1/2, while pretreatment(s), in either the priming or activation stage or both, reduced double TPA application-induced hydrogen peroxide formation and edema induction by 29% to 86%, respectively. Histologic examination revealed that pretreatment(s) with zerumbone suppressed leukocyte infiltration and reduced proliferating cell nuclear antigen-labeling indices. Together, our results indicate that zerumbone is a promising agent for the prevention of both tumor initiating and promoting processes, through induction of anti-oxidative and phase II drug metabolizing enzymes as well as attenuation of proinflammatory signaling pathways. (C) 2004 Wiley-Liss, Inc.

Interleukin (IL)-1beta, an anti-apoptotic and pro-inflammatory cytokine, plays an important role in the onset of inflammation-associated disease. We examined the suppressive effects of a total of 39 synthetic or natural compounds on dextran sulfate sodium-induced IL-1beta production in murine peritoneal macrophages. Several compounds, including alpha-tocopherol, gallic acid, (-)-catechin and rutin, were found to be highly effective for attenuating IL-1beta production, suggesting that they would be useful for anti-inflammatory application.

We recently established a novel co-culture assay system using activated inflammatory cells and AS52 Chinese hamster ovary cells, and demonstrated that reactive oxygen and nitrogen species (RONS) generated from activated inflammatory leukocytes induce mutations in the gpt recorder gene in AS52 cells. In this study, we examined the inhibitory effects of 19 agents with antioxidative properties on RONS generation in cultured inflammatory cells and on mutagenesis in AS52 cells co-cultured with activated inflammatory cells. The results demonstrate that there is a linear correlation between the ability of these agents to suppress RONS production in activated inflammatory cells and to inhibit mutation in AS52 cells.

Cancer prevention strategies making use of combined agents with distinct molecular mechanisms, rather than individual agents. are considered promising for higher efficacy and lower toxicity. Although there is increasing understanding of the synergistic combinations of synthetic agents, our knowledge regarding such combinations of food factors remains limited. We recently found that free radical generation suppressants from food items in combination with their scavengers at low concentrations exhibited notable synergistic effects in activated leukocytes, whereas combinations of agents with similar modes of action showed additive or antagonistic effects. For example, (-)-epigallocatechin gallate (EGCG) from green tea has been shown to increase the endotoxin-induced production of proinflammatory mediators such as prostaglandin E-2 and tumor necrosis factor-a in RAW264.7 macrophages, whereas the soybean isoflavonoid genistein compensated for these inverse properties of EGCG, leading to marked suppression in combination. The present review briefly highlights the potential effectiveness of combinations of several agents with anti-oxidative and anti-inflammatory properties for cancer preventive strategies.

The inhibitory effects of dietary feeding of citrus nobiletin on azoxymethane (AOM)-induced rat colon carcinogenesis using a long-term bioassay were investigated. Five-week old male F344 rats were initiated with two weekly subcutaneous injections of AOM (20 rng/kg bw) to induce Colonic tumors. They were also given the diets containing 0.01% or 0.05% nobiletin for 34 weeks. starting one week after the last dosing of AOM. At the end of the study, the incidence of colonic adenocarcinoma were 67% in the AOM alone group, 55% in the AOM 0.01% nobiletin group, 35% (P < 0.05) in the AOM-0.05% nobiletin group. Also, nobiletin feeding reduced the cell-proliferation activity, increased the apoptotic index, and decreased the prostaglandin E-2 content in colonic adenocarcinoma and/or colonic mucosa. These findings might suggest that citrus nobiletin has chemopreventive ability against AOM-induced rat colon carcinogenesis.

Oxidative stress has been shown to play pivotal roles in the onset of inflammatory bowel disease. We evaluated the effects of a dietary anti-oxidant, Antioxidant Biofactor (AOB(R)), a processed grain food, on dextran sulfate sodium (DSS)induced colitis in mice. Female ICR mice were fed a diet containing 0.1% or 1% AOB for 2 weeks, during which they were given 5% DSS in drinking water for the latter I week to induce colitis. A diet containing 1% AOB, but not that with 0.1% AOB, attenuated DSS-induced body weight loss and colon shortening (each, P < 0.05), and dramatically improved colitis histologic scores. In addition, DSS-induced increases in colonic mucosal IL-1β, but not TNF-α, protein levels were significantly abrogated in 1% AOB-fed mice (P < 0.05). Further, 1% dietary AOB abolished the expression of IL-1beta mRNA levels in colonic mucosa (P < 0.01). Our results suggest that AOB is effective for the prevention of DSS-induced colitis in mice.

Zerumbone, a sesquiterpene occurring in zingiberaceous plants in Southeast Asian countries, has been shown to have anti-inflammatory effects in several independent experimental studies. We examined its effect on the expression of proinflammatory genes in human colon adenocarcinoma cell lines, Caco-2, Colo320DM, and HT-29, using reverse transcription-polymerase chain reaction (RT-PCR) assays. Surprisingly, zerumbone markedly induced the expression of interleukin (IL)-Ialpha, IL-Ibeta, IL-6, and tumor necrosis factor (TNF)-alpha in each cell line in concentration- and time-dependent manners. Results of a previous pharmacological approach using specific inhibitors of mitogen-activated protein kinases (MAPKs) suggested that the activation of both c-Jun N-terminal kinase and extracellular signal-regulated protein kinase, however, not that of p38 MAPK, may be involved in zerumbone-induced IL-1beta expression pathways in Caco-2 cells. The present results imply that zerumbone increases the production of proinflammatory cytokines in cancerous tissues in the colon and that this biochemical property may cause side-effects.

Objectives: We have previously reported that an antioxidant, auraptene (AUR), isolated from citrus fruit effectively inhibits chemically induced carcinogenesis in digestive tracts, such as the oral cavity, esophagus and large bowel. In this study, we investigated the modifying effects of dietary supplementation with AUR on N,N-diethylnitrosamine (DEN)-initiated hepatocarcinogenesis in male F344 rats in two different experiments to determine whether the compound exerts a cancer-chemo-preventive action in other organs. Methods: In the first experiment, animals were fed diets containing AUR at dose levels of 100 and 500 ppm for 7 weeks 1 week before, during, and 1 week after the start of liver carcinogenesis induced by DEN (40 ppm in drinking water for 5 weeks) to predict the modulatory effect on hepatocarcinogenesis. After 7 weeks, the numbers of hepatocellular enzyme-altered foci (EAF; cm(2)) which stained positive for the placental form of glutathione S-transferase (GST-P) and transforming growth factor (TGF)-alpha were determined on immunohistochemically stained sections. In the second experiment conducted to confirm the findings, animals subjected to DEN treatment were fed AUR-containing diets (100 and 500 ppm) during either the initiation stage ('initiation' feeding for 7 weeks) or post-initiation phase ('post-initiation' feeding for 25 weeks) of DEN-induced hepatocarcinogenesis. Results: In the first experiment, feeding with AUR at both doses during DEN exposure decreased the mean numbers of GST-P-positive and TGF-alpha-positive EAF/cm2, and the reduction in the number of TGF-alpha-positive EAF by feeding 500 ppm AUR was statistically significant (p < 0.005). In the second experiment, the 'initiation' feeding with 500 ppm AUR significantly inhibited the incidence (33 vs. 83%, p = 0.000511) and multiplicity (0.67 ± 1.09 vs. 1.96 ± 1.85, p < 0.005) of liver cell carcinoma. Also, the 'post-initiation' feeding with AUR at both doses significantly reduced the development of hepatocellular carcinoma (100 ppm: incidence, 15%, p = 0.000006; multiplicity: 0.25 +/- 0.64, p < 0.001; 500 ppm: incidence, 11%, p = 0.000002; multiplicity, 0.26 ± 0.81, p < 0.001). In addition, AUR feeding reduced cell proliferation and the apoptotic index in liver cell neoplasms. Conclusions: The results suggest that the citrus antioxidant AUR is a potential chemo-preventive agent against DEN-induced hepatocarcino-genesis in rats. Copyright (C) 2004 S. Karger AG, Basel.

We recently established a novel co-culture assay system using activated inflammatory cells and AS52 Chinese hamster ovary cells, and demonstrated that reactive oxygen and nitrogen species (RONS) generated from activated inflammatory leukocytes induce mutations in the gpt recorder gene in AS52 cells. In this study, we examined the inhibitory effects of 19 agents with antioxidative properties on RONS generation in cultured inflammatory cells and on mutagenesis in AS52 cells co-cultured with activated inflammatory cells. The results demonstrate that there is a linear correlation between the ability of these agents to suppress RONS production in activated inflammatory cells and to inhibit mutation in AS52 cells.

Ulcerative colitis (UC) and Crohn's disease are inflammatory disorders of unknown cause and difficult to treat, though some synthetic chemicals, including ligands for peroxisome proliferator-activated receptors (PPARs), are anticipated to be useful drugs. In contrast, few food phytochemicals have been reported to suppress colitis in animal models. The present study was undertaken to explore the suppressive efficacy of zerumbone (ZER), a sesquiterpenoid present in the rhizome of Zingiber zerumbet Smith that is used as a condiment in Southeast Asian countries and known to be a potent suppressant of cyclooxygenase (COX)-2 and inducible nitric oxide synthase expression in cell culture systems. Acute colitis was induced by exposing female ICR mice to 5% DSS in drinking water for 1 week. One week prior to DSS administration, the experimental mice were fed ZER alone, nimesulide (NIM, a selective COX-2 inhibitor) alone, or both in combination (1000 ppm each) for a total of 2 weeks. Inflammatory biomarkers, i.e. interleukin (IL)-1alpha and IL-1beta, tumor necrosis factor (TNF)-alpha, and prostaglandin (PG)E-2 and PGF(2alpha) in colonic mucosa were quantified by an enzyme-linked immunosorbent assay in conjunction with histological alterations. Oral feeding of ZER significantly lowered the levels of IL-1beta [inhibitory rate (IR) = 34%], TNF-alpha (IR = 29%), and PGE(2) (IR = 73%) and suppressed DSS-induced colitis, whereas NIM suppressed the histological changes induced by DSS without affecting inflammatory biomarkers. However, their treatment in combination was most effective for suppressing these biomarkers. Our results suggest that ZER is a novel food factor for mitigating experimental UC and that use of a combination of agents, with different modes of actions, may be an effective anti-inflammatory strategy. (C) 2003 Elsevier Inc. All rights reserved.

Combinatorial chemopreventive strategies, in contrast to those with individual agents, show potential in terms of potentially lower toxicity and higher efficacy. In this study, we combined several agents and examined their suppressive effects on the combined lipopolysaccharide (LPS)- and interferon(IFN)-gamma-induced formation of proinflammatory mediators, including prostaglandin (PG) E-2 and tumor necrosis factor (TNF)-alpha, in RAW264.7 murine macrophages. The combinatorial effects of indomethacin/genistein (GEN) and aspirin/GEN were found to be synergistic for PGE(2) suppression, while the nimesulide/GEN combination was antagonistic. Further, while (-)-epigallocatechin gallate (EGCG) alone increased LPS/IFM-gamma-induced production of PGE(2) and TNF-alpha as well as cyclooxygenase-2 expression, the EGCG/GEN combination markedly suppressed these parameters. Our results suggest that certain chemopreventive agents act complexly and that, when used in combination, they affect the intracellular signaling pathways of the paired agents to exert additive, synergistic, or antagonistic effects.

We have developed a simple system for the sensitive detection and measurement of glutathione S-transferase (GST) activity that detoxifies polycyclic aromatic hydrocarbons using the cultured rat normal liver epithelial cell line, RL34 cells. Citral (3,7-dimethyl-2,6-octadienal) was isolated from the methanol extract of lemongrass (Cymbopogon citratus) and identified as a novel inducer of GST. Citral, a mixture of the two stereoisomers geranial and neral, dose- and time-dependently induced the total and pi-class-specific activities of GST. The structure-activity relationship study revealed that geranial, an E-isomer, was mainly responsible for the inducing activity of citral mixture and the aldehyde group conjugated with a trans-double bond is an essential structural factor. The data were consistent with the in vitro observation that both glutathione (GSH) and protein thiol quickly and specifically reacted with the active isomer geranial, but not neral. Pretreatment of the cells with diethyl maleate significantly enhanced not only the basal activity but also the citral-stimulated activity of GST, while pretreatment with N-acetyl-cysteine inhibited it. Moreover, the treatment of RL 34 cells with geranial for 30 min significantly attenuated the intracellular GSH level, while application for 18 h enhanced it. These results strongly suggested that the electrophilic property characterized by the reactivity with intracellular nucleophiles including protein thiol or glutathione (GSH) plays an important role in the induction of GST. The present study also implied the antioxidant role of GST induction by citral in mouse skin, providing a new insight into skin cancer prevention. (C) 2003 Elsevier Science (USA). All rights reserved.

We have developed a simple system for the sensitive detection and measurement of glutathione S-transferase (GST) activity that detoxifies polycyclic aromatic hydrocarbons using the cultured rat normal liver epithelial cell line, RL34 cells. Citral (3,7-dimethyl-2,6-octadienal) was isolated from the methanol extract of lemongrass (Cymbopogon citratus) and identified as a novel inducer of GST. Citral, a mixture of the two stereoisomers geranial and neral, dose- and time-dependently induced the total and pi-class-specific activities of GST. The structure-activity relationship study revealed that geranial, an E-isomer, was mainly responsible for the inducing activity of citral mixture and the aldehyde group conjugated with a trans-double bond is an essential structural factor. The data were consistent with the in vitro observation that both glutathione (GSH) and protein thiol quickly and specifically reacted with the active isomer geranial, but not neral. Pretreatment of the cells with diethyl maleate significantly enhanced not only the basal activity but also the citral-stimulated activity of GST, while pretreatment with N-acetyl-cysteine inhibited it. Moreover, the treatment of RL 34 cells with geranial for 30 min significantly attenuated the intracellular GSH level, while application for 18 h enhanced it. These results strongly suggested that the electrophilic property characterized by the reactivity with intracellular nucleophiles including protein thiol or glutathione (GSH) plays an important role in the induction of GST. The present study also implied the antioxidant role of GST induction by citral in mouse skin, providing a new insight into skin cancer prevention. (C) 2003 Elsevier Science (USA). All rights reserved.

In contrast to chemopreventive strategies using individual agents, a combination of specified compounds may be effectual to achieve desirable results with higher efficacy and lower toxicity. In the present in vitro study, we examined combinations of agents and assessed which concentrations were appropriate to yield notable synergism. L-N-G-Monomethyl-L-arginine (L-NMMA), a synthetic inducible nitric oxide synthase (iNOS) inhibitor, and zerumbone, a natural sesquiterpene that suppresses iNOS de novo synthesis, were combined at various concentrations, with the aim to diminish combined lipopolysaccharide- and interferon-gamma-induced nitric oxide generation in a murine macrophage line, RAW264.7. Although the combinatorial effects (CEs) were antagonistic or additive at higher concentrations, significant synergism was obtained at lower concentrations where each agent alone did not cause significant inhibition. Similarly, the CEs were synergistic when (-)-epigallocatechin gallate (EGCG) and genistein were combined at lower concentrations, whereas those of two iNOS inhibitors, L-NMMA and L-N-G-aminoethyl-L-ornithine, were either additive or antagonistic at all concentrations tested, suggesting that a combination of given agents with different action mechanisms is a prerequisite for synergistic effects. For suppression of phorbol ester-induced superoxide anion radical (O-2(.-)) generation in differentiated HL-60 cells, the CEs of 1'-acetoxycahvicol acetate (ACA), a phenyl propanoid that suppresses O-2(.-) generation, and O-2(.-) dismutase were also synergistic, though only at lower concentrations. The CEs of ACA/EGCG were antagonistic or additive, even at low concentrations, suggesting that the signal transduction pathways triggered by these agents are antagonistic. The present findings suggest that individual food phytochemicals have complex interactions that can be antagonistic, additive, and/or synergistic in biological systems, depending upon certain environmental factors including concentrations. Further, these results support and emphasize the concept that combinations of different types of chemicals at low concentrations are one of the essential areas of study for chemopreventive strategies. (C) 2002 Elsevier Science B.V. All rights reserved.

Activated inflammatory leukocytes generate a variety of reactive oxygen and nitrogen species (RONS) that may have roles in mutagenesis and carcinogenesis. The purpose of the present study was to explore the relationship between inflammatory leukocyte activation and mutagenesis using co-culture systems. We investigated the mutagenic potentials of 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated differentiated HL-60 (human promyelocytic leukemia cells), and RAW 264.7 cells (murine macrophages) stimulated with lipopolysaccharide (LPS) and interferon (IFN)-gamma by co-culturing each cell line with AS52 cells, a transgenic Chinese hamster ovary cell line. HL-60 cells rapidly generated superoxide (O-2(-)) 15 min to 1 h (peak at 30 min) following TPA stimulation. RAW 264.7 cells stimulated with LPS and IFN-gamma produced O-2(-), nitric oxide (NO) and peroxynitrite (ONOO-) continuously for 5-25 h. There was a 2.0-fold increase in the mutation frequency of the gpt gene in AS52 cells co-cultured with TPA stimulated HL-60 cells, when compared with non-treated cells. Importantly, this increase in mutation frequency was significantly suppressed by antioxidants, such as superoxide dismutase (SOD) and diphenylene iodonium. (DPI), an NADPH oxidase inhibitor (inhibition rates: IRs = 18.2 and 35.1%, respectively). Similarly, co-culture of AS52 cells with LPS/IFN-gamma-stimulated RAW 264.7 cells also increased the mutation frequency of the gpt gene by 2.6-fold, and this increase in mutation frequency was suppressed by SOD, DPI and N-5-(1-iminoethyl)-L-ornithine dihydrochloride (L-NIO), an specific iNOS inhibitor (IRs = 58.3, 70.8 and 70.8%, respectively). In co-culture experiments, activated HL-60 and RAW 264.7 cells increased 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels in AS52 cells when compared with non-treated controls (1.7- and 1.6-fold, respectively). Treatment of AS52 cells with hydrogen peroxide (H2O2, 100 muM), ONOO- (100 muM) and SIN-1 (100 muM), a ONOO- generator, also increased the mutation frequency of the gpt gene (4.6-, 5.4- and 2.8-fold, respectively). Taken together, these results support the hypothesis that RONS, derived from activated inflammatory leukocytes, are mutagenic in the biological systems, and that RONS generation inhibitors are potentially anti-mutagenic, and thus may be useful in cancer preventive strategies.

In contrast to chemopreventive strategies using individual agents, a combination of specified compounds may be effectual to achieve desirable results with higher efficacy and lower toxicity. In the present in vitro study, we examined combinations of agents and assessed which concentrations were appropriate to yield notable synergism. L-N-G-Monomethyl-L-arginine (L-NMMA), a synthetic inducible nitric oxide synthase (iNOS) inhibitor, and zerumbone, a natural sesquiterpene that suppresses iNOS de novo synthesis, were combined at various concentrations, with the aim to diminish combined lipopolysaccharide- and interferon-gamma-induced nitric oxide generation in a murine macrophage line, RAW264.7. Although the combinatorial effects (CEs) were antagonistic or additive at higher concentrations, significant synergism was obtained at lower concentrations where each agent alone did not cause significant inhibition. Similarly, the CEs were synergistic when (-)-epigallocatechin gallate (EGCG) and genistein were combined at lower concentrations, whereas those of two iNOS inhibitors, L-NMMA and L-N-G-aminoethyl-L-ornithine, were either additive or antagonistic at all concentrations tested, suggesting that a combination of given agents with different action mechanisms is a prerequisite for synergistic effects. For suppression of phorbol ester-induced superoxide anion radical (O-2(.-)) generation in differentiated HL-60 cells, the CEs of 1'-acetoxycahvicol acetate (ACA), a phenyl propanoid that suppresses O-2(.-) generation, and O-2(.-) dismutase were also synergistic, though only at lower concentrations. The CEs of ACA/EGCG were antagonistic or additive, even at low concentrations, suggesting that the signal transduction pathways triggered by these agents are antagonistic. The present findings suggest that individual food phytochemicals have complex interactions that can be antagonistic, additive, and/or synergistic in biological systems, depending upon certain environmental factors including concentrations. Further, these results support and emphasize the concept that combinations of different types of chemicals at low concentrations are one of the essential areas of study for chemopreventive strategies. (C) 2002 Elsevier Science B.V. All rights reserved.

Two antioxidant compounds were isolated from C sappan L by multiple steps of column chromatography and thin layer chromatography in succession with superoxide scavenging assay as activity monitor. Structures of the two compounds were determined by spectroscopic methods as 1',4'-dihydro-spiro[benzofuran-3(2H),3'-[3H-2]benzopyran]-1',6',6',7'-tetrol (compound 1) and 3-[[4,5-dihydroxy-2(hydroxymethyl) phenyl]-methyl]-2,3-dihydro-3,6-benzofurandiol (compound 2). Characterization of antioxidant properties of these two compounds was done by determining the inhibitory effect on xanthine oxidase activity as well as scavenging effect on superoxide anion and hydroxyl radicals. Our results indicated that compounds 1 and 2 inhibited xanthine oxidase activity and scavenged superoxide anion and hydroxyl radicals. Compounds 1 and 2 possessed similar radical scavenging activities as ascorbic acid, and they were more effective than other well-known antioxidants such as a-tocopherol, beta-carotene, and BHT. As inhibitors of free radical formation, compounds 1 and 2 were more effective than all the other antioxidants tested. In conclusion, compounds 1 and 2 can be regarded as primary antioxidants with radical-scavenging and chain-breaking activities as well as secondary antioxidants with inhibitory effect on radical generation.

Purpose: Recent preclinical assays using animal models have shown that naturally-occurring and synthetic chemicals such as auraptene (AUR), nobiletin (NOB), hesperidin (HE), diosmin (DIO), indole-3-carbinol (13C), F-acetoxychavicol acetate (ACA), 2,5-di-O-acetyl-D-1,4-glucaro-6,3-dilactone (ACE), D-glucuronic acid gamma-lactone (GL), chlorogenic acid (CGA), protocatechuic acid (PA), and sinigrin (SIN) are possible preventive agents against the development of cancer. However, the mode of action of such preventive agents remains to be elucidated. The current study, therefore, was conducted to analyze whether these agents induce apoptosis and/or inhibit DNA synthesis in human colorectal cancer cell lines. Methods: We performed an 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium assay to evaluate the modifying effects of the chemicals on cell viability as the first screening. Then, induction of apoptosis was detected by means of a DNA fragmentation assay, a quantitative enzyme immunoassay, and morphological analysis using 4-diamidino-2-phenylindole staining. In addition, the modulating effects of the compounds on DNA synthesis of the cells with fixed doses of the compounds were analyzed by scoring the 5-bromo-2'-deoxyuridine labeling index. Results: AUR, NOB, 13C, ACA, and ACE had apoptosis-inducing effects in a concentration- and time-dependent manner, some of which were followed by a reduction in replicating DNA synthesis. CGA, PA, SIN, GL, DIO, and HE had little modulating effect on cell viability, apoptosis, and DNA synthesis in this cell system. Conclusions: Our results suggest that AUR, 13C, ACA, NOB, and ACE might exert tumor-preventive action through apoptosis- and/or cell proliferation-dependent mechanisms and, on the other hand, CGA, PA, SIN, HE, DIO, and GL might be apoptosis- and cell proliferation-independent. These assays provided an initial tool for further mechanical studies of tumor-preventive agents and future applications to mechanism-based chemopreventive studies.

Some quassinoids (1-6) isolated previously as plant growth inhibitors from the leaves of Eurycoma longifolia Jack. (Simaroubaceae) were subjected to in vitro tests on anti-tumor promoting, antischistosomal and plasmodicidal activities. The most active compound for inhibition of tumor promoter-induced Epstein-Barr virus activation (anti-tumor promotion) was 14,15beta-dihydroxyklaineanone (5, IC50 = 5 muM). Longilactone (1) gave significant antischistosomal effect at a concentration of 200 mug/ml. 11-Dehydroklaineanone (3) and 15beta-O-acetyl-14-hydroxyklaineanone (6) showed potent plasmodicidal activity (IC50 = 2 mug/ml). Thus it was suggested that E longifolia possesses high medicinal values due to the occurrence of a variety of quassinoids. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

In our previous study. FA15 (2-methyl-I-butyl ferulic acid) was chemically synthesized as a novel ferulic acid (FA) analog, and found to notably suppress phorbol ester-induced Epstein-Barr virus activation and superoxide anion generation in vitro. In this report, we demonstrated that FA15 in contrast to FA, markedly Suppressed the combined lipopolysaccharide and interferon-gamma-induced protein expressions of inducible nitric oxide synthase and cyclooxygenase-2, and also inhibited the release of tumor necrosis factor-alpha accompanied by suppression of I-kappaB degradation in RAW264.7, a murine macrophage cell line. In ICR mouse skin, topical application of FA15 significantly attenuated phorbol ester-triggered hydrogen peroxide production and edema formation as well as papilloma development while that of FA did not. Our results suggest that FA15, derived front natural sources. is a novel chemopreventive agent, both structurally and functionally. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

We prepared 14 feruloyl-myo-inositol derivatives, and evaluated the relationships between their stereostructure and inhibitory activity toward the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced superoxide (O-2(-)) generation. And further, their suppressive effect on the TPA-induced Epstein-Barr virus (EBV) activation was examined in order to estimate their anti-carcinogenic potentials. Among the derivatives tested, 1,6-O-bis[3-(4'-hydroxy-3'-methoxyphenyl)-2-propenoyl]-myo-inositol (6b) showed an excellent suppressive activity on the O-2(-) generation at a concentration of 20 muM. For the suppressive effects on the EBV activation, 2,4,6-O-tris[3-(4'-hydroxy-3'-methoxyphenyl)-2-propenoyl]-myo-inositol 1,3,5-orthoformate (9b) showed the highest activity at a concentration of 100 muM anion g the derivatives tested. These results suggest that the inhibitory potencies of feruloyl-myo-inositol derivatives depend on the stereostructure of molecules rather than the hydrophobicity of molecules. (C) 2002 Elsevier Science Ltd. All rights reserved.

Aurapten (7-geranyloxycoumarin) has been reported to be an effective inhibitor of chemical carcinogenesis in some,rodent models. In the present study, a method for preparing an aurapten,enriched agricultural product has been established. Out of 17 Rutaceae varieties, the aurapten content in hassaku (Citrus hassaku Hort ex Y. Tanaka) fruit peel was marked, as well as that in natsumikan (C. natsudaidai) and grapefruit (C. paradisi). The aurapten content in hassaku peel was most abundant in April. Hassaku fruit peel oil, which was dissolved by heating precipitates including aurapten which had formed after freezing the peel oil at -20 degreesC, was used. After adsorbing aurapten from peel oil onto synthetic adsorbent SP70, the adsorbent was washed with 40% (v/v) ethanol in water to remove essential oils and pigments remaining on the adsorbent. Aurapten was then eluted with 80% (v/v) ethanol. In a-laboratory-scale test, the recovery rates of aurapten and total carotenoids from the eluates were 74.3 and 4.6%, respectively. In a pilot-scale test, the recovery rate of aurapten in the aurapten-enriched preparation from dissolved hassaku oil was 91.0%, and its concentration was 64.1% (w/w). When stored,for 180 days under sunlight, aurapten in powder form remained at 88.0-89.0% of the initial level, but only 31.3-43.8% in ethanol. The stability of aurapten in the aurapten-enriched preparation was higher than that of purified aurapten. These results suggest that aurapten is readily recovered from hassaku peel oil using SP70, and thus may be used as a food additive.

We prepared novel polyphenols which were esters composed of two naturally occurring products, ferulic and gallic acids, and investigated their inhibitory effects on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced Epstein-Barr virus (EBV) activation and superoxide (O-2(-)) generation. Most of these compounds exhibited significant EBV activation suppression at a concentration of 20 muM and in particular, the ester 5f having 2-metyl-1-butyl group showed high activity. The suppressive effects on O-2(-) generation were also observed in most of the esters. (C) 2002 Elsevier Science Ltd. All rights reserved.

Epithelial xanthine oxidase (XOD) is one of the major enzymes responsible for superoxide (O-2(-)) generation, which is involved in oxidative stress. However, there are few known reports of a convenient bioassay to detect Cellular XOD activity. We tested several cell lines, and found that AS52, from Chinese hamster ovary cells, produced a significant level of O-2(-) in 2 response to 12-O-tetradecanoylphorbol 13-acetate (TPA), and this activity was markedly inhibited by allopurinol, an XOD inhibitor. Using AS52 cells and differentiated HL-60 cells, we conducted screening tests of edible Japanese plant extracts for their inhibitory activities toward TPA-induced O-2(-) generation from both reduced nicotinamide adenine dinucleotide oxidase (HL-60) and XOD (AS52). Notably, the extracts from mioga ginger, rape, avocado, carrot, turnip, taro, and shiineji showed potent inhibition of O-2(-) generation in both cell lines. These results suggest that several edible Japanese plants carry a significant antioxidative and cancer preventive potential. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

Nobiletin (NOB), a polymethoxyflavonoid, is an effective anti-inflammatory and chemopreventive phytochemical found in citrus fruits. We compared the absorption and metabolism characteristics of NOB with those of luteolin (LT) in male SD rats. Each flavonoid (67.1 mumol/kg of body weight) was given separately by gastric intubation, and then concentrations were measured at 1, 4, and 24 hours after administration. In the digestive organs, NOB showed a notable tendency for localizing into the mucous membrane and muscularis from 1 to 4 hours, in contrast to LT, though both NOB and LT were completely excreted within 24 hours. Further, significant amounts of NOB were detected in the whole liver and kidney specimens, whereas LT accumulation was slight. Although serum concentrations of NOB from 1 to 4 hours were comparable to those of LT, urinary concentrations of LT were significantly higher from 4 to 24 hours. Following glucuronidase/sulfatase treatments of urinary materials, we detected 3 types of mono-demethylated NOB, including 3'-demethyl-NOB, and two di-demethylated types, as well as 3'-demethyl-NOB alone in serum samples using liquid chromatography-mass spectral analysis. Our results suggest that the metabolic properties of polymethoxyflavonoids are distinct from those of other general flavonoids, because of their wide distribution and accumulation in tissue.

Cancer chemoprevention with food phytochemicals is currently regarded as one of the noticeable scientific fields. We have screened more than 400 extracts from vegetables and fruits for the anti-tumor promoting activity using a cell culture system. The Zingiberaceae plants in Southeast Asian countries were indicated to be notable sources to identify effective cancer chemopreventive agents. In addition, F-acetoxychavicol acetate (ACA), a phenyl propanoid, isolated from the rhizomes of Languas galanga, was found to be a potent chemopreventive agent which markedly suppresses phorbol ester-induced superoxide (O-2(.)) generation and endotoxin-induced nitric oxide (NO) production.

Seven quassinoids including a now 12-epi-11-dehydroklaineanone were isolated from the leaves of Eurycoma longifolia (Simaroubaceae) as plant growth inhibitors or related compounds. The strongest activity was found in 14,15 beta -dihydroxyklaineanone. (C) 2001 Elsevier Science Ltd. All rights reserved.

The modifying effects of dietary feeding of zerumbone isolated from Zingiber zerumbet on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) were investigated in male F344 rats. Expression of cyclooxygenase (COX)-2 in colonic mucosa exposed to AOM and/or zerumbone was also assayed. In addition, we assessed the effects of zerumbone on cell proliferation activity of crypts by counting silver-stained nucleolar organizer regions protein (AgNORs) in colonic cryptal cell nuclei. To induce ACF rats were given three weekly subcutaneous injections of AOM (15 mg/kg body weight). They were also fed the experimental diet containing 0.01% or 0.05% zerumbone for 5 weeks, starting one week before the first dosing of AOM. AOM exposure produced 84 +/- 13 ACF/rat at the end of the study (week 5). Dietary administration of zerumbone caused reduction in the frequency of ACF: 72 +/- 17 (14% reduction) at a dose of 0.01% and 45 +/- 18 (46% reduction, p<0.001) at a dose of 0.05%. Feeding of zerumbone significantly reduced expression of COX-2 and prostaglandins in colonic mucosa. Zerumbone feeding significantly lowered the number of AgNORs in colonic crypt cell nuclei. These Endings might suggest possible chemopreventive ability of zerumbone, through suppression of COX-2 expression, cell proliferating activity of colonic mucosa, and induction of phase II detoxification enzymes in the development of carcinogen-induced ACE (C) 2001 Elsevier Science Inc. All rights reserved.

The modifying effects of dietary feeding of zerumbone isolated from Zingiber zerumbet on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) were investigated in male F344 rats. Expression of cyclooxygenase (COX)-2 in colonic mucosa exposed to AOM and/or zerumbone was also assayed. In addition, we assessed the effects of zerumbone on cell proliferation activity of crypts by counting silver-stained nucleolar organizer regions protein (AgNORs) in colonic cryptal cell nuclei. To induce ACF rats were given three weekly subcutaneous injections of AOM (15 mg/kg body weight). They were also fed the experimental diet containing 0.01% or 0.05% zerumbone for 5 weeks, starting one week before the first dosing of AOM. AOM exposure produced 84 +/- 13 ACF/rat at the end of the study (week 5). Dietary administration of zerumbone caused reduction in the frequency of ACF: 72 +/- 17 (14% reduction) at a dose of 0.01% and 45 +/- 18 (46% reduction, p<0.001) at a dose of 0.05%. Feeding of zerumbone significantly reduced expression of COX-2 and prostaglandins in colonic mucosa. Zerumbone feeding significantly lowered the number of AgNORs in colonic crypt cell nuclei. These Endings might suggest possible chemopreventive ability of zerumbone, through suppression of COX-2 expression, cell proliferating activity of colonic mucosa, and induction of phase II detoxification enzymes in the development of carcinogen-induced ACE (C) 2001 Elsevier Science Inc. All rights reserved.

The modifying effects of dietary feeding of a polymethoxyflavonoid nobiletin isolated from Citrus unshiu on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) were investigated in male F344 rats. We also assessed the effects of nobiletin on cell proliferation activity of ACF using a monoclonal antibody MIB-5. Rats were given subcutaneous injections of AOM (15 mg/kg body weight) once a week for 3 weeks to induce ACF. They also received the experimental diet containing 0.01% or 0.05% nobiletin for 5 weeks, starting one week before the first dosing of AOM, AOM exposure produced 139 +/- 35 ACF/rat at the end of the study (week 5). Dietary administration of nobiletin caused significant reduction in the frequency of ACF: 70 +/- 15 (50% reduction, p <0.001) at a dose of 0.01% and 63 +/- 10 (55% reduction, p <0.001) at a dose of 0.05%. Nobiletin feeding significantly lowered MIB-5-index in ACF, Also, dietary administration of nobiletin significantly reduced prostaglandin E(2) content in the colonic mucosa. These findings might suggest possible chemopreventive ability of nobiletin, through suppression of cell proliferating activity of ACF, in the development of ACF. (C) 2001 Elsevier Science Inc. All rights reserved.

Five bafadienolides (1-5) isolated from the leaves of Kalanchoe pinnata and K. daigremontiana x tubiflora (Crassulaceae) mere examined for their inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation in Raji cells induced by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate. All bufadienolides showed inhibitory activity, and bryophyllin A (1) exhibited the most marked inhibition (IC50 = 0.4 mum) among the tested compounds. Bryophyllin C (2), a reduction analogue of 1, and bersaldegenin-3-acetate (3) lacking the orthoacetate moiety mere less active. These results strongly suggest that bufadienolides are potential cancer chemopreventive agents.

The polymethoxyflavonoid (PMF), nobiletin (NOB), specifically occurs in citrus fruits, and is currently believed to be a promising anti-inflammatory and antitumor promoting agent, In the present study, we investigated the in vitro absorption and metabolism of NOB and compared them with those of the polyhydroxyflavonoid (PHF), luteolin (LT), NOB preferentially accumulated in a differentiated Caco-2 cell monolayer, which is a model for small intestinal epithelial cells, while LT did not, Treatment of NOB with a rat liver S-9 mixture led to the formation of 3'-demethyl-NOB, while that of LT did not, We thus suggest that PMFs including NOB have properties distinct from those of general flavonoids for absorption and metabolism in vitro.