村上 明

Phytochemicals are generally defined as secondary metabolites in plants that play crucial roles in their adaptation to a variety of environmental stressors. There is a great body of compelling evidence showing that these metabolites have pronounced potentials for regulating and modulating human health and disease onset, as shown by both experimental and epidemiological approaches. Concurrently, enormous efforts have been made to elucidate the mechanism of actions underlying their biological and physiological functions. For example, the pioneering work of Tachibana et al. uncovered the receptor for (-)-epigallocatechin-3-gallate (EGCg) as the 67 kDa laminin receptor, which was shown to partially mediate the functions of EGCg, such as anti-inflammatory, anti-allergic, and anti-proliferative activities. Thereafter, several protein kinases were identified as binding proteins of flavonoids, including myricetin, quercetin, and kaempferol. Isothiocyanates, sulfur-containing phytochemicals\ present in cruciferous plants, are well known to target Keap1 for activating the transcription factor Nrf2 for inducing self-defensive and anti-oxidative gene expression. In addition, we recently identified CD36 as a cell surface receptor for ursolic acid, a triterpenoid ubiquitously occurring in plants. Importantly, the above mentioned target proteins are indispensable for phytochemicals to exhibit, at least in part, their bioactivities. Nevertheless, it is reasonable to assume that some of the activities and potential toxicities of metabolites are exerted via their interactions with unidentified, off-target proteins. This notion may be supported by the fact that even rationally designed drugs occasionally display off-target effects and induce unexpected outcomes, including toxicity. Here we update the current status and future directions of research related to target molecules of food phytochemicals.

Six bryostatins were isolated from Japanese bryozoan by evaluating their binding to the C1B domain of protein kinase C delta (PKC delta). Structure-activity studies of bryostatins 4, 10, and 14 suggested that the ester group at C20 was not necessary for binding to and activating PKC delta. These bryostatins showed significant anti-tumor-promoting activity in induction tests with the Epstein-Barr virus early antigen.

Delphinidin-3-O-galactoside (D3G) is a water-soluble anthocyanin with antioxidant activity. (-)-Epigallocatechin-3-gallate (EGCG) is also known as a powerful antioxidant but concomitantly possesses a prooxidative property. We hypothesized that D3G is capable of protecting the EGCG-induced cytotoxicity and endoplasmic reticulum (ER) stress via inducing self-protective proteins and antioxidant enzymes. (-)-Epigallocatechin-3-gallate (200-500 mu M) dose dependently decreased the viability of hepa1c1c-7 mouse hepatocytes, whereas D3G (50-500 mu M) did not change it. Pretreatment with D3G significantly suppressed EGCG-induced cytotoxicity in a time-dependent manner (0, 6, and 24 hours). (-)-Epigallocatechin-3-gallate drastically decreased heme oxygenase-1 and heat shock protein 70 messenger RNA (mRNA) levels, whereas, pretreatment with D3G markedly attenuated their down-regulations. Delphinidin-3-O-galactoside remarkably decreased EGCG-induced ER stress responses such as C/EBP-homologus protein mRNA expression and X-box-binding protein-1 mRNA splicing. Taken together, our data suggest that D3G is capable of masking the EGCG-induced cytotoxicity and ER stress, presumably through up-regulation of antioxidant enzymes and heat shock proteins. (C) 2012 Elsevier Inc. All rights reserved.

Most physiologically functional food factors are derived from plants. Although those chemicals exhibit versatile bioactivities, their mechanisms of action are not fully understood. It is important to indicate that phytochemicals, but not plant nutrients, xenobiotics in humans and thus induce expressions of self-defense molecules, including anti-oxidative and xenobiotic function as metabolizing enzymes via the Keap1/Nrf2 system. In addition, recent reports have shown that several phytochemicals are capable of up-regulating the activities of molecular chaperones such as heat shock proteins (HSPs) and proteasomes. For example, sulforaphane, a sulfur-containing phytochemical present in cruciferous plants, was recently reported to induce HSP27 expression and increase proteasome activity. On the other hand, accumulating evidence shows that some toxins exert health beneficial effects under non-toxic doses, a phenomenon termed hormesis. Importantly, those beneficial effects are abrogated or disappear when given in high doses. Collectively, chronic exposure to phytochemicals, i.e., 'chemical training', may increase self-defense mechanisms, thereby contributing to health promotion and disease prevention.

Plants synthesize various secondary metabolites which play crucial roles in their adaptation to environmental stresses. These phytochemicals have been clarified to possess potentials for regulating mammalian physiological functions. Recently, their target molecules in mammals have been intensively studied. Zerumbone, a sesquiterpene occurring in Zingiber zerumbet Smith, is a promising chemopreventive agent which has various bioactivities including anti-inflammatory and detoxifying effects. This phytochemical suppresses the expression of cyclooxygenase-2 via a post-transcriptional mechanism and induces phase 2 detoxification enzymes. Our in vitro study with zerumbone-bound Sepharose revealed that it covalently binds to AU-rich element-binding proteins and Kelch-like ECH-associated protein 1 which are essential for those bioactivities. This review article describes versatile bioactivities of zerumbone, and highlights its target molecules.

Protein kinase C (PKC) isozymes are promising targets for anticancer therapy. Bryostatin-1 (bryo-1), a unique PKC activator with little tumor-promoting activity, is currently in clinical trials for the treatment of cancer. However, its limited availability from natural sources and its synthetic complexity have hampered studies of its mode of action and structural optimization as a therapeutic agent. The development of synthetically more accessible compounds with bryo-1-like activities is thus needed. Recently, we developed a simple and less lipophilic analogue of tumor-promoting aplysiatoxin (ATX) (aplog-1) as a promising lead for bryo-1-like anticancer drugs. Structure-activity studies suggested that local hydrophobicity around the spiroketal moiety of aplog-1 is a crucial determinant of its antiproliferative activity. The hydrophobic analogue (12,12-dimethyl-aplog-1) displayed more potent antiproliferative activity. Moreover, it showed little tumor-promoting activity and even suppressed the tumor promotion by 12-O-tetradecanoylphorbol 13-acetate (TPA) in vivo and in vitro. Aplog-1 and bryo-1 bound selectively to novel PKC isozymes (delta, eta, and theta) while tumor promoters bound to both conventional and novel PKC isozymes. These results suggest that the unique biological activities of aplog-1 and bryo-1 are ascribable in part to the ability to bind to PKC delta, but weak binding to conventional PKC isozymes might also be important.

Previously, we reported that oral feeding of 1% green tea polyphenols (GTPs) aggravated the dextran sulfate sodium (DSS)-induced colitis in mice. In the present study, we assessed the toxicity of 1% GTPs in several organs from normal and DSS-exposed mice. Sixty-two male ICR mice were initially divided into four groups. Non-treated group (group 1, n = 15) was given standard diet and water, GTPs (group 2, n = 15) received 1% GTPs in diet and water, DSS (group 3, n = 15) received diet and 5% DSS in water, and GTPs + DSS group (group 4, n = 17) received 1% GTPs in diet and 5% DSS in water. We found that group 4 significantly increased (P < 0.05) kidney weight, the levels of serum creatinine and thiobarbituric acid-reactive substances in both kidney and liver, as compared with those in group 3. The mRNA expression levels of antioxidant enzymes and heat-shock proteins (HSPs) in group 4 were lower than those of group 3. For instance, heme oxygenase-1 (HO-1), HSP27, and 90 mRNA in the kidney of group 4 were dramatically down-regulated as compared with those of group 3. Furthermore, 1% GTPs diet decreased the expression of HO-1, NAD(P) H:quinone oxidoreductase 1 (NQO1) and HSP90 in kidney and liver of non-treated mice. Taken together, our results indicate that high-dose GTPs diet disrupts kidney functions through the reduction of antioxidant enzymes and heat-shock protein expressions in not only colitis but also non-treated ICR mice.

Previously, we reported that oral feeding of 1% green tea polyphenols (GTPs) aggravated the dextran sulfate sodium (DSS)-induced colitis in mice. In the present study, we assessed the toxicity of 1% GTPs in several organs from normal and DSS-exposed mice. Sixty-two male ICR mice were initially divided into four groups. Non-treated group (group 1, n = 15) was given standard diet and water, GTPs (group 2, n = 15) received 1% GTPs in diet and water, DSS (group 3, n = 15) received diet and 5% DSS in water, and GTPs + DSS group (group 4, n = 17) received 1% GTPs in diet and 5% DSS in water. We found that group 4 significantly increased (P < 0.05) kidney weight, the levels of serum creatinine and thiobarbituric acid-reactive substances in both kidney and liver, as compared with those in group 3. The mRNA expression levels of antioxidant enzymes and heat-shock proteins (HSPs) in group 4 were lower than those of group 3. For instance, heme oxygenase-1 (HO-1), HSP27, and 90 mRNA in the kidney of group 4 were dramatically down-regulated as compared with those of group 3. Furthermore, 1% GTPs diet decreased the expression of HO-1, NAD(P) H:quinone oxidoreductase 1 (NQO1) and HSP90 in kidney and liver of non-treated mice. Taken together, our results indicate that high-dose GTPs diet disrupts kidney functions through the reduction of antioxidant enzymes and heat-shock protein expressions in not only colitis but also non-treated ICR mice.

Glucose transporter-4 (GLUT4) is a transmembrane protein that plays a major role in insulin-mediated glucose transport in muscle and adipocytes. For glucose transport to occur, the GLUT4 protein needs to be translocated from the intracellular pool to the plasma membrane, and certain compounds may enhance this process. The present study investigated the promotion of glucose uptake in differentiated L6 myotubes by cardamonin, isolated from Alpinia katsumadai. Cardamonin increased translocation of GLUT4 to the plasma membrane in L6 cells, but did not activate protein kinase C zeta/lambda, Akt, or AMP-activated protein-kinase, all of which are known to regulate GLUT4 translocation. The glucose-uptake-promoting activity of cardamonin was not lowered by treatment with a phosphatidylinositol 3'-kinase inhibitor. These results suggest that cardamonin is a promising active compound for maintaining glucose homeostasis, and that it acts via an unknown mechanism that does not involve activation of the downstream insulin signal and AMP-activated protein kinase. Copyright (C) 2011 John Wiley & Sons, Ltd.

Reducing cancer incidence and mortality by use of cancer-chemopreventive agents is an important goal. We have established an in vitro bioassay that is able to screen large numbers of candidate chemicals that are positive for prevention of inflammation-related carcinogenesis. To accomplish this we have added candidate chemicals or vehicles and freshly isolated, fluorescent dye-labeled inflammatory cells that were overlaid on TNF-alpha-stimulated mouse endothelial cells in a 96-well plate. Inhibition of inflammatory cell attachment to the endothelial cells by the chemicals was quantified by the intensity of fluorescence from the adherent inflammatory cells after removing unattached cells. Using this assay, we selected two chemicals, auraptene and turmerones, for further study. As an in vivo test, diets containing these test chemicals were administered to mice with a piece of foreign body, gelatin sponge, that had been implanted to cause inflammation, and we found that the number of inflammatory cells that infiltrated into the subcutaneously implanted gelatin sponge was reduced compared to that found in the mice fed with a control diet. Moreover, diets containing either of the two chemicals prevented inflammation-based carcinogenesis in a mouse model. We found that the compounds reduced not only the number of infiltrating cells but also the expression of inducible nitric oxide synthase (iNOS) or formation of 8-hydroxy-2'-deoxyguanine (8-OHdG) in the infiltrated cells. Moreover, both compounds but not controls sustained the reducing activity in the inflammatory lesion, and this finding was confirmed by using non-invasive in vivo electron spin resonance. The newly established in vitro screening assay will be useful for finding biologically effective chemopreventive agents against inflammation-related carcinogenesis. (C) 2011 Elsevier Inc. All rights reserved.

Reducing cancer incidence and mortality by use of cancer-chemopreventive agents is an important goal. We have established an in vitro bioassay that is able to screen large numbers of candidate chemicals that are positive for prevention of inflammation-related carcinogenesis. To accomplish this we have added candidate chemicals or vehicles and freshly isolated, fluorescent dye-labeled inflammatory cells that were overlaid on TNF-alpha-stimulated mouse endothelial cells in a 96-well plate. Inhibition of inflammatory cell attachment to the endothelial cells by the chemicals was quantified by the intensity of fluorescence from the adherent inflammatory cells after removing unattached cells. Using this assay, we selected two chemicals, auraptene and turmerones, for further study. As an in vivo test, diets containing these test chemicals were administered to mice with a piece of foreign body, gelatin sponge, that had been implanted to cause inflammation, and we found that the number of inflammatory cells that infiltrated into the subcutaneously implanted gelatin sponge was reduced compared to that found in the mice fed with a control diet. Moreover, diets containing either of the two chemicals prevented inflammation-based carcinogenesis in a mouse model. We found that the compounds reduced not only the number of infiltrating cells but also the expression of inducible nitric oxide synthase (iNOS) or formation of 8-hydroxy-2'-deoxyguanine (8-OHdG) in the infiltrated cells. Moreover, both compounds but not controls sustained the reducing activity in the inflammatory lesion, and this finding was confirmed by using non-invasive in vivo electron spin resonance. The newly established in vitro screening assay will be useful for finding biologically effective chemopreventive agents against inflammation-related carcinogenesis. (C) 2011 Elsevier Inc. All rights reserved.

Aplog-1 is a unique analog of tumor-promoting aplysiatoxin that inhibits tumor-promotion by phorbol diesters and proliferation of tumor cells. While the structural features relevant to the biological activities of Aplog-1 remain to be identified, recent studies by us have suggested that local hydrophobicity around the spiroketal moiety of Aplog-1 is a crucial determinant of its anti-proliferative activity. This hypothesis led us to design 12,12-dimethyl-Aplog-1 (3), in which a hydrophobic geminal dimethyl group is installed proximal to the spiroketal moiety to improve biological potency. As expected, 3 was more effective than Aplog-1 in inhibiting cancer cell growth and binding to protein kinase C delta, a putative receptor responsible for the biological response of Aplog-1. Moreover, an induction test on Epstein-Barr virus early antigen demonstrated 3 to be a better anti-tumor promoter than Aplog-1. These results indicate that 3 is a superior derivative of Aplog-1, and thus a more promising lead for anti-cancer drugs.

Zerumbone, a sesquiterpene derived from tropical ginger, contains an electrophilic alpha,beta-unsaturated carbonyl moiety and was found to suppress chemically induced papilloma formation in mouse skin. Here, we report that topical application of zerumbone onto dorsal skin of hairless mice induces activation of NF-E2-related factor 2 (Nrf2) and expression of heme oxygenase-1 (HO-1). We compared the levels of HO-1 protein in the skin of zerumbone-treated Nrf2 wild-type and Nrf2 knockout mice, and nrf2-deficient mice expressed HO-1 protein to a much lesser extent than the wild-type animals following topical application of zerumbone. Treatment of mouse epidermal JB6 cells with zerumbone caused a marked increase of Nrf2 nuclear translocation followed by the promoter activity of HO-1, and also enhanced direct binding of Nrf2 to the antioxidant response element. Moreover, knockdown of Nrf2 in JB6 cells diminished the zerumbone-induced upregulation of HO-1. Notably, alpha-humulene and 8-hydroxy-alpha-humulene, the structural analogues of zerumbone that lack the alpha,beta-unsaturated carbonyl group, failed to activate Nrf2 and were unable to increase HO-1 expression. Unlike zerumbone, these nonelectrophilic analogues could not suppress the 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced JB6 cell transformation and the intracellular accumulation of reactive oxygen species (ROS). Interestingly, when JB6 cells were treated with carbon monoxide-releasing molecule that mimics the HO-1 activity, the TPA-induced ROS production was markedly reduced. Taken together, these findings suggest that upregulation of HO-1 expression by zerumbone is mediated through activation of Nrf2 signaling, which provides a mechanistic basis for the chemopreventive effects of this sesquiterpene on mouse skin carcinogenesis. Cancer Prev Res; 4(6); 860-70. (C) 2011 AACR.

To assess the effectiveness of selected food phytochemicals in reducing the toxic effects of the environmental toxicants, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and p,p'-DDT (DDT), we tested the potencies of auraptene, nobiletin, zerumbone, and (+/-)-13-hydroxy-10-oxo-trans-11-octadecenoic acid (13-HOA) in reversing the inflammatory action of these toxicants in U937 human macrophages. Using quantitative RT-PCR as the initial screening assay, we identified antagonistic actions of zerumbone and auraptene against the action of TCDD and DDT in up-regulating the mRNA expressions of COX-2 and VEGF. The functional significance of the inhibitory action of zerumbone on COX-2 expression was confirmed by demonstrating its suppression of TCDD-induced activation of COX-2 gene expression in mouse MMDD1 cells. We tested auraptene on DDT-induced reactive oxygen species (ROS) formation in U937 macrophages and found that auraptene is a powerful agent antagonizing this action of DDT. To confirm the significance of these actions of zerumbone and auraptene at the cellular level, we assessed their influence on TCDD-induced apoptosis resistance in intact U937 macrophages and found that they are capable of reversing this action of TCDD. In conclusion, zerumbone and auraptene were identified to be the most effective agents in protecting U937 macrophages from developing these cell toxic effects of TCDD and DDT.

The 18-deoxy derivative (3) of a simplified analogue (1) of aplysiatoxin with antiproliferative activity was synthesized to examine the role of the phenolic hydroxyl group at position 18 in the biological activities of 1. Compound 3 as well as 1 showed significant affinity for protein kinase C delta (PKC delta), and the antiproliferative activity of 3 was slightly higher than that of 1. However, the anti-tumor-promoting activity of 3 was less than that of 1 in vitro, suggesting that the phenolic hydroxyl group of 1 is necessary for the anti-tumor-promoting activity but not for the binding of PKCd and antiproliferative activity. Moreover, PKC isozyme selectivity of 3 was similar to that of 1, suggesting non-PKC receptors for these compounds to play some roles in the anti-tumor-promoting activity of 1. (c) 2010 Elsevier Ltd. All rights reserved.

The 18-deoxy derivative (3) of a simplified analogue (1) of aplysiatoxin with antiproliferative activity was synthesized to examine the role of the phenolic hydroxyl group at position 18 in the biological activities of 1. Compound 3 as well as 1 showed significant affinity for protein kinase C delta (PKC delta), and the antiproliferative activity of 3 was slightly higher than that of 1. However, the anti-tumor-promoting activity of 3 was less than that of 1 in vitro, suggesting that the phenolic hydroxyl group of 1 is necessary for the anti-tumor-promoting activity but not for the binding of PKCd and antiproliferative activity. Moreover, PKC isozyme selectivity of 3 was similar to that of 1, suggesting non-PKC receptors for these compounds to play some roles in the anti-tumor-promoting activity of 1. (c) 2010 Elsevier Ltd. All rights reserved.

Phenethyl isothiocyanate (PEITC), a constituent of many cruciferous vegetables, is well known to have versatile physiological activities, including chemopreventive effects. On the other hand, its anti-inflammatory effects are poorly reported. Nitric oxide (NO) is associated with a wide variety of inflammatory diseases. In this study, we investigated the effects of PEITC on NO production in LPS-activated peritoneal macrophages from ICR mice. The signaling pathway of LPS-induced NO production was examined using neutralizing antibodies [anti-interferon (IFN)-gamma and anti-interleukin (IL-12)] and specific protein kinase inhibitors, as well as others. The activity of PEITC toward NOx production was assessed in mice that received LPS via intraperitoneal administration. The neutralizing antibody of anti-IFN-gamma, but not anti-IL-12, suppressed LPS-induced NO production by 90%. LY294002, a specific inhibitor of phosphoinositide-3-kinase, suppressed Akt and IFN-gamma mRNA expression up-regulated by LPS, whereas PEITC exhibited a similar inhibition profile. Furthermore, oral administration of PEITC significantly suppressed the serum concentration of NOx in ICR mice. Our results suggest that PEITC suppresses LPS-induced NO production via inhibition of Akt activation and the resultant decrease in expression of IFN-gamma. This is one of the first reports to demonstrate a marked anti-inflammatory effect of PEITC following its oral administration.

Ample evidence has shown key roles of inflammation in tumor promotion and carcinogenesis, and tumor-associated macrophages are known to promote tumor growth and dissemination. Programmed cell death 4 (Pdcd4) is a novel tumor suppressor, and although various studies have revealed that the functions and expression mechanisms of Pdcd4 in tumor promotion, those in regard to inflammation remain unclear. In the present study, we examined whether inflammatory stimuli regulate Pdcd4 expression. 12-O-tetradecanoylphorbol 13-acetate (TPA) suppressed expression of pdcd4 mRNA in human monocytic cell lines (U937, THP-1). Similarly, the bacterial endotoxin lipopolysaccharide (LPS) downregulated pdcd4 level in mouse RAW264.7 and peritoneal macrophages. Furthermore, conditioned medium from LPS-stimulated RAW264.7 macrophages suppressed pdcd4 mRNA in RAW264.7 macrophages, and findings obtained with recombinant tumor necrosis factor-alpha (TNF-alpha) and TNF-alpha-specific siRNA suggested that TNF-alpha partly mediates [PS-triggered Pdcd4 downregulation via an autocrine mechanism. Specific inhibitors of phosphoinositide-3-kinase (PI3K) and c-jun N-terminus kinase (JNK) restored LPS-abolished pdcd4 mRNA. Consistently, in MCF7 mammary carcinoma cells, conditioned medium from TPA-differentiated/activated U937 cells suppressed pdcd4 mRNA. Additionally, knockdown of pdcd4 in RAW264.7 macrophages using siRNA significantly enhanced [PS-induced TNF-alpha protein production, and interferon-gamma, CC chemokine ligand (Ccl) 1, Ccl20, and interleukin-10 mRNA expression. These results suggest that Pdcd4 suppresses the induction of these inflammatory mediators. Taken together, loss of Pdcd4 in macrophages may be a critical step in establishing the inflammatory environment while that in tumor cells contributes to tumor progression. (C) 2010 Wiley-Liss, Inc.

Programmed cell death 4 (Pdcd4), a novel tumor suppressor, has recently emerged as an anti-inflammatory protein. We assessed in the present study the effects of 18 different food factors with anti-inflammatory activity on the lipopolysaccharide-induced down-regulation of pdcd4 mRNA expression in mouse RAW 264.7 macrophages. Genistein, a soy isoflavone, significantly inhibited pdcd4 down-regulation.

Helicobacter pylori (H. pylori) is a major human pathogen and plays a central role in chronic gastritis and gastric cancer. Since the adhesion of H. pylori to the human gastric epithelium is the initial and critical step of its infection, anti-H. pylori adhesion agents may be effective for the prevention and therapy of H. pylori-associated diseases. CD74 has recently been identified as a new receptor for H. pylori urease, and we have previously reported that several citrus components strongly suppressed CD74 expression in NCI-N87 gastric carcinoma cells. We found in this present study that auraptene (citrus coumarin) disrupted serum starvation-induced extracellular signaling-regulated kinase (ERK) 1/2 activation and attenuated H. pylori adhesion and IL-8 production in a co-culture system. In addition, the knockdown of CD74 expression led to a significant decrease of H. pylori adhesion, but unexpectedly increased IL-8 production. However, PD98059 (a MEK1/2 inhibitor) dramatically down-regulated this cytokine, suggesting MEK/ERK-dependent IL-8 production. Our results suggest that auraptene suppressed H. pylori adhesion and resulting chemokine production by disrupting ERK1/2 activation.

Programmed cell death 4 (Pdcd4), a novel tumor suppressor, has recently emerged as an anti-inflammatory protein. We assessed in the present study the effects of 18 different food factors with anti-inflammatory activity on the lipopolysaccharide-induced down-regulation of pdcd4 mRNA expression in mouse RAW 264.7 macrophages. Genistein, a soy isoflavone, significantly inhibited pdcd4 down-regulation.

Obesity is known to be a risk factor for colon carcinogenesis. Although there are several reports on the chemopreventive abilities of dietary flavonoids in chemically induced colon carcinogenesis, those have not been addressed in an obesity-associated carcinogenesis model. In the present study, the effects of 3 flavonoids (chrysin, quercetin and nobiletin) on modulation of the occurrence of putative preneoplastic lesions, aberrant crypt foci (ACF), and beta-catenin-accumulated crypts (BCACs) in the development of colon cancer were determined in male db/db mice with obesity and diabetic phenotypes. Male db/db mice were given 3 weekly intraperitoneal injections of azoxymethane (AOM) to induce the ACF and BCAC. Each flavonoid (100 ppm), given in the diet throughout the experimental period, significantly reduced the numbers of ACF by 68-91 % and BCAC by 64-71 %, as well as proliferation activity in the lesions. Clinical chemistry results revealed that the serum levels of leptin and insulin in mice treated with AOM were greater than those in the untreated group. Interestingly, the most pronounced suppression of development of preneoplastic lesions and their proliferation were observed in the quercetin-fed group, in which the serum leptin level was lowered. Furthermore, quercetin-feeding decreased leptin mRNA expression and secretion in differentiated 3T3-L1 mouse adipocytes. These results suggest that the present dietary flavonoids are able to suppress the early phase of colon carcinogenesis in obese mice, partly through inhibition of proliferation activity caused by serum growth factors. Furthermore, they indicate that certain flavonoids may be useful for prevention of colon carcinogenesis in obese humans. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Leptin, a pleiotropic hormone regulating food intake and metabolism, plays an important role in the regulation of inflammation and immunity. We previously demonstrated that serum leptin levels are profoundly increased in mice which received azoxymethane (AOM) and dextran sulfate sodium (DSS) as tumor-initiator and -promoter, respectively, in a colon carcinogenesis model. In this study, we attempted to address underlying mechanism whereby leptin is up-regulated in this rodent model. Five-week-old male ICR mice were given a single intraperitoneal injection of AOM (week 0), followed by 1% DSS in drinking water for 7 days. Thereafter, the weights of visceral fats and the serum concentration of leptin were determined at week 20. Of interest, the relative epididymal fat pad and mesenteric fat weights, together with serum leptin levels in the AOM and/or DSS-treated mice were markedly increased compared to that in untreated mice. In addition, leptin protein production in epididymal fat pad with AOM/DSS-treated mice was 4.7-fold higher than that of control. Further, insulin signaling molecules, such as protein kinase B (Akt), S6, mitogen-activate protein kinase/extracellular signaling-regulated kinase 1/2, and extracellular signaling-regulated kinase 1/2, were concomitantly activated in epididymal fat of AOM/DSS-treated mice. This treatment also increased the serum insulin and IGF-1 levels. Taken together, our results suggest that higher levels of serum insulin and lGF-1 promote the insulin signaling in epididymal fat and thereby increasing serum leptin, which may play an crucial role in, not only obesity-related, but also -independent colon carcinogenesis. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Reactive oxygen species (ROS) have been implicated as mediators of intestinal inflammation and carcinogenesis. Although green tea polyphenols (GTP) have anticancer property as antioxidants they also generate ROS in vitro. In this study, we investigated the modifying effects of GTP on dextran sulfate sodium (DSS)-induced acute colitis and on 1,2-dimethylhydrazine (DMH) and DSS-induced colon carcinogenesis in male ICR mice. At sacrifice after 6 days, the colon shortening induced by 2% DSS was unchanged by 0.1% and 0.25% GTP, but increased by 0.5% and 1% GTP-containing diet. The expression of interleukin-1 beta and macrophage-migration inhibitory factor in the DSS + 0.1% GTP group were lower than the DSS alone group, whereas the expression levels were increased in the DSS + 0.5% GTP and DSS + 1% GTP groups when compared with the DSS alone group. In a subsequent experiment to determine the effects of 0.01-1% GTP on inflammation-associated colon carcinogenesis induced by DMH/DSS, 0.5 and 1% doses of GTP failed to prevent the development of multiple colon tumors, rather, they tended to increase it. Our results thus indicate that the modifying effects of GTP on DSS-induced acute colitis and DMH/DSS-induced colon carcinogenesis depends upon its dosage and the expression of proinflammatory cytokines. (C) 2010 International Union of Biochemistry and Molecular Biology, Inc

Leptin, a pleiotropic hormone regulating food intake and metabolism, plays an important role in the regulation of inflammation and immunity. We previously demonstrated that serum leptin levels are profoundly increased in mice which received azoxymethane (AOM) and dextran sulfate sodium (DSS) as tumor-initiator and -promoter, respectively, in a colon carcinogenesis model. In this study, we attempted to address underlying mechanism whereby leptin is up-regulated in this rodent model. Five-week-old male ICR mice were given a single intraperitoneal injection of AOM (week 0), followed by 1% DSS in drinking water for 7 days. Thereafter, the weights of visceral fats and the serum concentration of leptin were determined at week 20. Of interest, the relative epididymal fat pad and mesenteric fat weights, together with serum leptin levels in the AOM and/or DSS-treated mice were markedly increased compared to that in untreated mice. In addition, leptin protein production in epididymal fat pad with AOM/DSS-treated mice was 4.7-fold higher than that of control. Further, insulin signaling molecules, such as protein kinase B (Akt), S6, mitogen-activate protein kinase/extracellular signaling-regulated kinase 1/2, and extracellular signaling-regulated kinase 1/2, were concomitantly activated in epididymal fat of AOM/DSS-treated mice. This treatment also increased the serum insulin and IGF-1 levels. Taken together, our results suggest that higher levels of serum insulin and lGF-1 promote the insulin signaling in epididymal fat and thereby increasing serum leptin, which may play an crucial role in, not only obesity-related, but also -independent colon carcinogenesis. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Obesity is known to be a risk factor for colon carcinogenesis. Although there are several reports on the chemopreventive abilities of dietary flavonoids in chemically induced colon carcinogenesis, those have not been addressed in an obesity-associated carcinogenesis model. In the present study, the effects of 3 flavonoids (chrysin, quercetin and nobiletin) on modulation of the occurrence of putative preneoplastic lesions, aberrant crypt foci (ACF), and beta-catenin-accumulated crypts (BCACs) in the development of colon cancer were determined in male db/db mice with obesity and diabetic phenotypes. Male db/db mice were given 3 weekly intraperitoneal injections of azoxymethane (AOM) to induce the ACF and BCAC. Each flavonoid (100 ppm), given in the diet throughout the experimental period, significantly reduced the numbers of ACF by 68-91 % and BCAC by 64-71 %, as well as proliferation activity in the lesions. Clinical chemistry results revealed that the serum levels of leptin and insulin in mice treated with AOM were greater than those in the untreated group. Interestingly, the most pronounced suppression of development of preneoplastic lesions and their proliferation were observed in the quercetin-fed group, in which the serum leptin level was lowered. Furthermore, quercetin-feeding decreased leptin mRNA expression and secretion in differentiated 3T3-L1 mouse adipocytes. These results suggest that the present dietary flavonoids are able to suppress the early phase of colon carcinogenesis in obese mice, partly through inhibition of proliferation activity caused by serum growth factors. Furthermore, they indicate that certain flavonoids may be useful for prevention of colon carcinogenesis in obese humans. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Zerumbone (ZER), a sesquiterpene in Zingiber zerumbet Smith, has been implicated as a promising chemopreventive agent. Here we found that ZER suppressed expression of pro-inflammatory genes (COX-2 and iNOS) and induced detoxification genes (GSTP1 and NQO1) in RAW264.7 macrophages. Using the ZER-bound Sepharose gel, it appeared ZER was covalently bound to proteins, Keap1 and HuR, that play key roles in these molecular mechanisms.

Zerumbone (ZER), a sesquiterpene in Zingiber zerumbet Smith, has been implicated as a promising chemopreventive agent. Here we found that ZER suppressed expression of pro-inflammatory genes (COX-2 and iNOS) and induced detoxification genes (GSTP1 and NQO1) in RAW264.7 macrophages. Using the ZER-bound Sepharose gel, it appeared ZER was covalently bound to proteins, Keap1 and HuR, that play key roles in these molecular mechanisms.

(+/-)-13-Hydroxy-10-oxo-trans-11-octadecenoic acid (13-HOA) is one of the lipoxygenase metabolites of linoleic acid (LA) from corn germ. Recently, we reported that this metabolite suppressed the expression of lipopolysaccharide-induced proinflammatory genes in murine macrophages by disrupting mitogen-activated protein kinases and Akt pathways. In this study, we investigated the inhibitory effects of 13-HOA on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation in ears and skin, as well as tumor promotion in female ICR mice. Pretreatment with 13-HOA (1600 nmol) inhibited ear edema formation by 95% (P < 0.05) in an inflammation test and reduced tumor incidence and the number of tumors per mouse by 40 and 64% (P < 0.05 each), respectively, in a two-stage skin carcinogenesis model. Histological examinations revealed that it decreased epidermal thickness, the number of infiltrated leukocytes and cell proliferation index. Furthermore, 13-HOA (8-40 mu M) suppressed TPA-induced anchorage-independent growth of JB6 mouse epidermal cells by 70-100%, whereas LA was virtually inactive. 13-HOA (40 mu M) inhibited TPA-induced activator protein-1 transactivation but not extracellular signal-regulated kinase1/2 activation. Interestingly, 13-HOA (40 mu M and 1600 nmol in JB6 cells and mouse skin, respectively) induced expression of programmed cell death 4 (Pdcd4), a novel tumor suppressor protein. To our knowledge, this is the first report of a food factor that is able to induce Pdcd4 expression. Collectively, our results indicate that 13-HOA may be a novel anti-inflammatory and antitumor chemopreventive agent with a unique mode of action.

Zerumbone (ZER), present in subtropical ginger Zingiber zerumbet Smith, possesses anti-growth and anti-inflammatory properties in several human cancer cell lines. ZER also down-regulates the cyclooxygenase-2 and inducible nitric oxide synthase expression via modulation of nuclear factor (NF)-kappa B activation in cell culture systems. These findings led us to investigate whether ZER is able to inhibit carcinogenesis in the colon and lung, using 2 different preclinical mouse models. In Exp. 1, a total of 85 male ICR mice were initiated using a single intraperitoneal (i.p.) injection with azoxymethane (AOM, 10 mg/kg bw) and promoted by 1.5% dextran sulfate sodium (DSS) in drinking water for 7 days for rapid induction of colonic neoplasms. Animals were then fed the die containing 100, 250 or 500 ppm ZER for 17 weeks. In Exp. 2, a total of 50 female A/J mice were given a single i.p. injection of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (10 mu mol/mouse) to induce lung proliferative lesions. They were then fed the diet mixed with 100, 250 or 500 ppm ZER for 21 weeks. At the termination of the experiments (wk 20 of Exp. 1 and wk 22 of Exp. 2), all animals were subjected to complete necropsy examination to determine the pathological lesions in both tissues. Oral administration of ZER at 100, 250 and 500 ppm significantly inhibited the multiplicity of' colonic adenocarcinomas. The treatment also suppressed colonic inflammation. In the lung carcinogenesis, ZER feeding at 250 and 500 ppm significantly inhibited the multiplicity of lung adenomas in a dose-dependent manner. Feeding with ZER resulted in inhibition of proliferation, induction of apoptosis, and suppression of NF kappa B and heme oxygenase (HO)-1 expression in tumors developed in both tissues. Our findings suggest that dietary administration of ZER effectively suppresses mouse colon and lung carcinogenesis through multiple modulatory mechanisms of growth, apoptosis, inflammation and expression of NF kappa B and HO-1 that are involved in carcinogenesis in the colon and lung. (C) 2008 Wiley-Liss, Inc.

Quercetin is an anti-oxidative flavonoid widely distributed in the plant kingdom. Phenolic hydroxyl groups at the B-ring and the 3-position are responsible for its free radical-scavenging activity. Quercetin is commonly present as a glycoside and is converted to glucuronide/sulfate conjugates during intestinal absorption and only conjugated metabolites are therefore found in circulating blood. Although metabolic conversion attenuates its biological effects, active aglycone may be generated from the glucuronide conjugates by enhanced beta-glucuronidase activity during inflammation. With respect to its relationship with molecular targets relevant to cancer prevention, quercetin aglycone has been shown to interact with some receptors, particularly an aryl hydrocarbon receptor, which is involved in the development of cancers induced by certain chemicals. Quercetin aglycone has also been shown to modulate several signal transduction pathways involving MEK/ERK and Nrf2/keap 1, which are associated with the processes of inflammation and carcinogenesis. Rodent studies have demonstrated that dietary administration of this flavonol prevents chemically induced carcinogenesis, especially in the colon, whilst epidemiological studies have indicated that an intake of quercetin may be associated with the prevention of lung cancer. Dietary quercetin is, therefore, a promising agent for cancer prevention and further research is warranted. (C) 2008 Elsevier Ireland Ltd. All rights reserved.

To determine whether tobacco-derived carcinogens affect colon carcinogenesis, the effects of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) on colon carcinogenesis were examined using an azoxymethane (AOM)/dextran sulfate sodium (DSS) mouse model. NNK (10 mu mol) was administered to male A/J mice by a single intraperitoneal (i.p.) injection and then AOM (10 mg/kg body weight, i.p.) was given 1 week after NNK administration. One week later, the mice received 1.5% (w/v) DSS in their drinking water for 7 days. All animals were sacrificed at week 22 to examine the pathological lesions in the colon and lung. The incidence (80%, p<0.05) and multiplicity (4.0 +/- 3.6, p<0.05) of colonic tumors of the NNK+AOM+DSS group were significantly higher than that of the AOM+DSS group (incidence, 40%; and multiplicity, 1.2 +/- 1.7). The differences in incidence and multiplicity of lung tumors were insignificant between these two groups. Our findings may suggest that smoking increases the risk of inflammation-related colon cancer development.

Interleukin (IL)-1 beta is a proinflammatory cytokine responsible for the onset of a broad range of diseases, such as inflammatory bowel disease and rheumatoid arthritis. We have recently found that aggregated ursolic acid (UA), a triterpene carboxylic acid, is recognized by CD36 for generating reactive oxygen species (ROS) via NADPH oxidase (NOX) activation, thereby releasing IL-1 beta protein from murine peritoneal macrophages (pM phi) in female ICR mice. In the present study, we investigated the ability of UA for inducing IL-1 beta production in pM phi from 4 different strains of female mice (C57BL/6J, C3H/He, DDY, and ICR), as well as an established macrophage line (RAW264.7 cells). The levels of IL-1 beta released from UA-treated pM phi of C57BL/6J and DDY mice were significantly lower than from those of ICR mice, whereas IL-1 beta was not released from the pM phi of C3H/He mice or RAW264.7 cells. Of paramount importance, CD36 as well as the NOX components gp91(phox) and p47(phox) (C3H/He mice) and gp91(phox) (RAW264.7 cells) were scarcely detected. In addition, the different susceptibilities to UA-induced IL-1 beta release were suggested to be correlated with the amount of superoxide anion (O-2(-)) generated from the 5 different types of M phi. Notably, intracellular, but not extracellular, O-2(-) generation was indicated to play a major role in UA-induced IL-1 beta release. Together, our results indicate that the UA-incluced IL-1 beta release was strain-dependent, and the expression status of CD36 and gp91(phox) is strongly associated with inducibility. (C) 2008 Elsevier Inc. All rights reserved.

Adipocytokines are a group of adipocyte-secreted proteins that have significant effects on the metabolism of lipids and carbohydrates, as well as numerous other processes. A number of recent studies have indicated that some adipocytokines may significantly influence the proliferation of malignant cells in vitro, whereas it remains unclear whether they have similar roles in vivo. In this study, we determined serum levels of adipocytokines in mice with azoxymethane (AOM)- and dextran sulfate sodium (DSS)-induced colon carcinogenesis. Five-week-old ICR mice were given a single intraperitoneal injection of AOM followed by 1% DSS in drinking water for 7 days. Nobiletin (NOB), a citrus flavonoid, was given in the diet (100 p.p.m) for 17 weeks. Thereafter, the incidence and number of colon tumors and serum concentration of adipocytokines were determined at the end of week 20. The serum leptin level in AOM/DSS-treated mice was six times higher than that in untreated mice, whereas there were no significant differences in the levels of triglycerides, adiponectin and interleukin-6. Feeding with NOB abolished colonic malignancy and notably decreased the serum leptin level by 75%. Further, NOB suppressed the leptin-dependent, but not independent, proliferation of HT-29 colon cancer cells and decreased leptin secretion through inactivation of mitogen-activated protein kinase/extracellular signaling-regulated protein kinase, but not that of adiponectin in differentiated 3T3-L1 mouse adipocytes in a dose-dependent manner. Taken together, our results suggest that higher levels of leptin in serum promote colon carcinogenesis in mice, whereas NOB has chemopreventive effects against colon carcinogenesis, partly through regulation of leptin levels.

We previously reported that auraptene (7-geranyloxycoumarin; AUR), a coumarin that occurs widely in citrus fruit, has been shown to be a promising cancer-preventive agent in several rodent models. However, its bioavailability and metabolism have not been investigated. In this study, we compared the metabolism characteristics of AUR with those of 7-ethoxycoumarin (ETC) in male Sprague Dawley rats. Each (500 mu mol/kg body weight) was given separately by a single gastric intubation procedure, and digestive tract, liver, and kidney were removed at 1, 4, and 24 h after administration. The localization profiles of AUR and ETC in the gastrointestinal tract were similar. However, AUR, in contrast to ETC, showed significant localization in the liver from 1 to 4 h. Treatments of serum and urinary samples with glucuronidase/sulfatase led to the detection of significant amounts of umbelliferone (7-hydroxycoumarin; UMB), and serum and urinary concentrations of UMB following ETC administration were significantly higher than with AUR administration. Our results suggest that AUR, which bears a geranyloxyl side chain, has a longer life span than ETC, and this property may be associated with its previously reported chemopreventive and xenobiotics metabolizing activities.