Two Prenylated Chalcones, 4-Hydroxyderricin, and Xanthoangelol Prevent Postprandial Hyperglycemia by Promoting GLUT4 Translocation via the LKB1/AMPK Signaling Pathway in Skeletal Muscle Cells.

SCOPE: Stimulation of glucose uptake in the skeletal muscle is crucial for the prevention of postprandial hyperglycemia. Insulin and certain polyphenols enhance glucose uptake through the translocation of glucose transporter 4 (GLUT4) in the skeletal muscle. The previous study reports that prenylated chalcones, 4-hydroxyderricin (4-HD), and xanthoangelol (XAG) promote glucose uptake and GLUT4 translocation in L6 myotubes, but their underlying molecular mechanism remains unclear. This study investigates the mechanism in L6 myotubes and confirms antihyperglycemia by 4-HD and XAG. METHODS AND RESULTS: In L6 myotubes, 4-HD and XAG promote glucose uptake and GLUT4 translocation through the activation of adenosine monophosphate-activated protein kinase (AMPK) and liver kinase B1 (LKB1) signaling pathway without activating phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) and Janus kinases (JAKs)/signal transducers and activators of transcriptions (STATs) pathways. Moreover, Compound C, an AMPK-specific inhibitor, as well as siRNA targeting AMPK and LKB1 completely canceled 4-HD and XAG-increased glucose uptake. Consistently, oral administration of 4-HD and XAG to male ICR mice suppresses acute hyperglycemia in an oral glucose tolerance test. CONCLUSION: In conclusion, LKB1/AMPK pathway and subsequent GLUT4 translocation in skeletal muscle cells are involved in Ashitaba chalcone-suppressed acute hyperglycemia.

Role of Intestinal Microbiota in the Bioavailability and Physiological Functions of Dietary Polyphenols.

Polyphenols are categorized as plant secondary metabolites, and they have attracted much attention in relation to human health and the prevention of chronic diseases. In recent years, a considerable number of studies have been published concerning their physiological function in the digestive tract, such as their prebiotic properties and their modification of intestinal microbiota. It has also been suggested that several hydrolyzed and/or fission products, derived from the catabolism of polyphenols by intestinal bacteria, exert their physiological functions in target sites after transportation into the body. Thus, this review article focuses on the role of intestinal microbiota in the bioavailability and physiological function of dietary polyphenols. Monomeric polyphenols, such as flavonoids and oligomeric polyphenols, such as proanthocyanidins, are usually catabolized to chain fission products by intestinal bacteria in the colon. Gallic acid and ellagic acid derived from the hydrolysis of gallotannin, and ellagitannin are also subjected to intestinal catabolism. These catabolites may play a large role in the physiological functions of dietary polyphenols. They may also affect the microbiome, resulting in health promotion by the activation of short chain fatty acids (SCFA) excretion and intestinal immune function. The intestinal microbiota is a key factor in mediating the physiological functions of dietary polyphenols.

Functional properties of anti-inflammatory substances from quercetin-treated Bifidobacterium adolescentis.

The genus Bifidobacterium is well known to have beneficial health effects. We discovered that quercetin and related polyphenols enhanced the secretion of anti-inflammatory substances by Bifidobacterium adolescentis. This study investigated characteristics of the anti-inflammatory substances secreted by B. adolescentis. The culture supernatant of B. adolescentis with quercetin reduced the levels of inflammatory mediators in activated macrophages. Spontaneous quercetin degradant failed to increase anti-inflammatory activity, while the enhancement of anti-inflammatory activity by quercetin was sustained after washout of quercetin. Physicochemical treatment of the culture supernatant indicated that its bioactive substances may be heat-stable, non-phenolic, and acidic biomolecules with molecular weights less than 3 kDa. Acetate and lactate have little or no effect on nitric oxide production. Taken together, the anti-inflammatory substances secreted by B. adolescentis may be small molecules but not short chain fatty acids. In agreement with these findings, stearic acid was tentatively identified as a bioactive candidate compound.

Triterpenoids Isolated from Ziziphus jujuba Enhance Glucose Uptake Activity in Skeletal Muscle Cells.

Jujube (Ziziphus jujuba Mill.), a traditional folk medicine and functional food in China and South Korea, is known for its beneficial properties, which include anti-cancer, anti-oxidative, and anti-obesity effects. To assess the anti-hyperglycemic effect of jujube in this study, we investigated the glucose uptake-promoting activity of jujube in rat L6 myotubes. After determining that the jujube extract induces muscle glucose uptake, we identified the following active compounds by bioassay-guided fractionation: betulonic acid, betulinic acid, and oleanonic acid. Ursonic acid, known to be present in jujube, was semi-synthesized from ursolic acid and also observed to enhance glucose uptake. These four triterpenic acids induced glucose uptake in a glucose transporter 4-dependent manner. Comparison experiments of jujube fruits from three countries, namely, China, South Korea, and Japan, revealed that Japanese jujube has a higher content of active triterpenoids and is the most potent enhancer of glucose uptake.

Measurement of Glucose Uptake in Cultured Cells.

Facilitative glucose uptake transport systems are ubiquitous in animal cells and are responsible for transporting glucose across cell surface membranes. Evaluation of glucose uptake is crucial in the study of numerous diseases and metabolic disorders such as myocardial ischemia, diabetes mellitus, and cancer. Detailed in this unit are laboratory methods for assessing glucose uptake into mammalian cells. The unit is divided into five sections: (1) a brief overview of glucose uptake assays in cultured cells; (2) a method for measuring glucose uptake using radiolabeled 3-O-methylglucose; (3) a method for measuring glucose uptake using radiolabeled 2-deoxyglucose (2DG); (4) a microplate method for measuring 2DG-uptake using an enzymatic, fluorometric assay; and (5) a microplate-based method using a fluorescent analog of 2DG.

Quercetin and related polyphenols: new insights and implications for their bioactivity and bioavailability.

The physiological functions and bioavailability of flavonoids have been widely investigated since their bioactivities were identified about 80 years ago. Quercetin is a typical flavonoid ubiquitously contained in vegetables and fruits with several biological effects demonstrated in vitro and in vivo including antioxidative, anti-inflammatory, anticancer, and antidiabetic activities. After the ingestion of vegetables and fruits, quercetin glycosides are metabolized, absorbed, and circulated as types of conjugates in the blood. Thereafter, quercetin-3-O-ß-D-glucuronide (Q3GA), a major metabolite of quercetin, is distributed throughout the body where it may exert beneficial functions in target tissues. Hydrophilic Q3GA has been found to be deconjugated into hydrophobic quercetin aglycone at injured sites which, in turn, may improve the pathological conditions. This review presents updated information on the biological aspects and mechanisms of action of quercetin and its related polyphenols. In particular, new insights into their beneficial health effects on the brain, blood vessels, muscle, and intestine will be discussed.

Effects of phytochemicals on in vitro anti-inflammatory activity of Bifidobacterium adolescentis.

Probiotics have been shown to improve the condition of not only the human gastrointestinal tract but also the entire body. We found that quercetin enhances the anti-inflammatory activity of Bifidobacterium adolescentis, which is abundant in human intestines. Here, we assessed whether certain phytochemicals could enhance the anti-inflammatory activity of B. adolescentis. Bifidobacteria were anaerobically cultured with phytochemicals for 3 h, and the anti-inflammatory activity of the supernatants was estimated by testing their ability to inhibit nitric oxide (NO) production by lipopolysaccharide-stimulated RAW264 macrophages. Of the 55 phytochemicals tested, phloretin, (+)-taxifolin, and (-)-epigallocatechin gallate as well as quercetin-3-O-glucoside and quercetin-4'-O-glucoside were similar to quercetin in promoting NO suppression by B. adolescentis. In addition, the phytochemicals excluding quercetin increased the concentrations of lactic and acetic acids in the co-culture supernatants. These results suggest that some phytochemicals may activate the anti-inflammatory function of B. adolescentis.

Quercetin-3-O-glucuronide inhibits noradrenaline-promoted invasion of MDA-MB-231 human breast cancer cells by blocking ß2-adrenergic signaling.

Endogenous catecholamines such as adrenaline (A) and noradrenaline (NA) are released from the adrenal gland and sympathetic nervous system during exposure to stress. The adrenergic system plays a central role in stress signaling, and excessive stress was found to be associated with increased production of reactive oxygen species (ROS). Overproduction of ROS induces oxidative damage in tissues and causes the development of diseases such as cancer. In this study, we investigated the effects of quercetin-3-O-glucuronide (Q3G), a circulating metabolite of quercetin, which is a type of natural flavonoid, on the catecholamine-induced ß2-adrenergic receptor (ß2-AR)-mediated response in MDA-MB-231 human breast cancer cells expressing ß2-AR. Treatment with A or NA at concentrations above 1µM generated significant levels of ROS, and NA treatment induced the gene expression of heme oxygenase-1 (HMOX1), and matrix metalloproteinase-2 (MMP-2) and -9 (MMP9). Inhibitors of p38 MAP kinase (SB203580), cAMP-dependent protein kinase (PKA) (H-89), activator protein-1 (AP-1) transcription factor (SR11302), and NF-κB and AP-1 (Tanshinone IIA) decreased MMP2 and MMP9 gene expression. NA also enhanced cAMP induction, RAS activation and phosphorylation of ERK1/2. These results suggested that the cAMP-PKA, MAPK, and ROS-NF-κB pathways are involved in ß2-AR signaling. Treatment with 0.1µM Q3G suppressed ROS generation, cAMP and RAS activation, phosphorylation of ERK1/2 and the expression of HMOX1, MMP2, and MMP9 genes. Furthermore, Q3G (0.1µM) suppressed invasion of MDA-MB-231 breast cancer cells and MMP-9 induction, and inhibited the binding of [(3)H]-NA to ß2-AR. These results suggest that Q3G may function to suppress invasion of breast cancer cells by controlling ß2-adrenergic signaling, and may be a dietary chemopreventive factor for stress-related breast cancer.

Inhibitory effects of 4-hydroxyderricin and xanthoangelol on lipopolysaccharide-induced inflammatory responses in RAW264 macrophages.

The Japanese herb, Ashitaba (Angelica keiskei Koidzumi), contains two prenylated chalcones, 4-hydroxyderricin and xanthoangelol, which are considered to be the major active compounds of Ashitaba. However, their effects on inflammatory responses are poorly understood. In the present study, we investigated the effects and underlying molecular mechanisms of 4-hydroxyderricin and xanthoangelol on lipopolysaccharide (LPS)-induced inflammatory responses in RAW264 mouse macrophages. LPS-mediated production of nitric oxide (NO) was markedly reduced by 4-hydroxyderricin (10 µM) and xanthoangelol (5 µM) compared with their parent compound, chalcone (25 µM). They also inhibited LPS-induced secretion of tumor necrosis factor-alpha (TNF-α) and expression of inducible NO synthase (iNOS) and cyclooxygenase-2 (COX-2). Although chalcone decreased the DNA-binding activity of both activator protein-1 (AP-1) and nuclear factor-kappa B (NF-κB), 4-hydroxyderricin and xanthoangelol suppressed only AP-1 and had no effect on NF-κB. On the other hand, all of the tested chalcones reduced the phosphorylation (at serine 536) level of the p65 subunit of NF-κB. 4-Hydroxyderricin and xanthoangelol may be promising for the prevention of inflammatory diseases.

Mitochondrial dysfunction leads to deconjugation of quercetin glucuronides in inflammatory macrophages.

Dietary flavonoids, such as quercetin, have long been recognized to protect blood vessels from atherogenic inflammation by yet unknown mechanisms. We have previously discovered the specific localization of quercetin-3-O-glucuronide (Q3GA), a phase II metabolite of quercetin, in macrophage cells in the human atherosclerotic lesions, but the biological significance is poorly understood. We have now demonstrated the molecular basis of the interaction between quercetin glucuronides and macrophages, leading to deconjugation of the glucuronides into the active aglycone. In vitro experiments showed that Q3GA was bound to the cell surface proteins of macrophages through anion binding and was readily deconjugated into the aglycone. It is of interest that the macrophage-mediated deconjugation of Q3GA was significantly enhanced upon inflammatory activation by lipopolysaccharide (LPS). Zymography and immunoblotting analysis revealed that ß-glucuronidase is the major enzyme responsible for the deglucuronidation, whereas the secretion rate was not affected after LPS treatment. We found that extracellular acidification, which is required for the activity of ß-glucuronidase, was significantly induced upon LPS treatment and was due to the increased lactate secretion associated with mitochondrial dysfunction. In addition, the ß-glucuronidase secretion, which is triggered by intracellular calcium ions, was also induced by mitochondria dysfunction characterized using antimycin-A (a mitochondrial inhibitor) and siRNA-knockdown of Atg7 (an essential gene for autophagy). The deconjugated aglycone, quercetin, acts as an anti-inflammatory agent in the stimulated macrophages by inhibiting the c-Jun N-terminal kinase activation, whereas Q3GA acts only in the presence of extracellular ß-glucuronidase activity. Finally, we demonstrated the deconjugation of quercetin glucuronides including the sulfoglucuronides in vivo in the spleen of mice challenged with LPS. These results showed that mitochondrial dysfunction plays a crucial role in the deconjugation of quercetin glucuronides in macrophages. Collectively, this study contributes to clarifying the mechanism responsible for the anti-inflammatory activity of dietary flavonoids within the inflammation sites.

Effect of quercetin and its glucuronide metabolite upon 6-hydroxydopamine-induced oxidative damage in Neuro-2a cells.

Quercetin is ubiquitously distributed in plant foods. This antioxidative polyphenol is mostly converted to conjugated metabolites in the body. Parkinson disease (PD) has been suggested to be related to oxidative stress derived from abnormal dopaminergic activity. We evaluated if dietary quercetin contributes to the antioxidant network in the central nervous system from the viewpoint of PD prevention. A neurotoxin, 6-hydroxydopamine (6-OHDA), was used as a model of PD. 6-OHDA-induced H2O2 production and cell death in mouse neuroblastoma, Neuro-2a. Quercetin aglycone suppressed 6-OHDA-induced H2O2 production and cell death, although aglycone itself reduced cell viability at higher concentration. Quercetin 3-O-ß-D-glucuronide (Q3GA), which is an antioxidative metabolite of dietary quercetin, was little incorporated into the cell resulting in neither suppression of 6-OHDA-induced cell death nor reduction of cell viability. Q3GA was found to be deconjugated to quercetin by microglial MG-6 cells. These results indicate that quercetin metabolites should be converted to their aglycone to exert preventive effect on damage to neuronal cells.

Measurement of glucose uptake in cultured cells.

Facilitative glucose uptake transport systems are ubiquitous in animal cells and responsible for transporting glucose across the cell surface membrane. Evaluation of glucose uptake is crucial in the study of numerous diseases and metabolic disorders, such as myocardial ischemia, diabetes mellitus, and cancer. Methods for assessing glucose uptake into mammalian cells are detailed in this unit. The work is divided into four sections: (1) a brief overview of glucose uptake assays in cultured cells; (2) a method for measuring glucose uptake using radiolabeled 3-O-methylglucose; (3) a method for measuring glucose uptake using radiolabeled 2-deoxyglucose (2DG); and (4) an improved method for measuring 2DG-uptake using an enzymatic, fluorometric assay, eliminating the need for radiolabeled glucose analogs.

Dietary flavonoids as cancer-preventive and therapeutic biofactors.

Flavonoids are present in many plants, and hence, in foods and ingredients derived from them. These polyphenolic compounds have attracted renewed attention as potential anticarcinogens, and the molecular mechanisms of their anticarcinogenic effects and their bioavailability have been extensively explored. In this review, we focus on the major dietary flavonoids; flavones, flavonols, and flavan-3-ols (catechins), and evaluate their roles in cancer prevention. After absorption with or without metabolic conjugation, flavonoids are transported to target organs where they exert their anticarcinogenic activity. The molecular mechanisms of the anticarcinogenic effects of flavonoids include their antagonistic effect on the aryl hydrocarbon receptor (AhR), and regulation of phase I and II drug metabolizing enzymes and phase III transporters. Experimental evidence suggests that flavonoids modulate signal transduction pathways at each stage of carcinogenesis. The interactions between flavonoids and biomolecules in vivo must be investigated in detail to identify specific targets. In addition, the potential side effects should be considered when flavonoid supplements are used for cancer prevention. Therefore, the use of flavonoids as chemopreventive agents should be further investigated to establish safe levels of flavonoid intake.

Prenylated chalcones 4-hydroxyderricin and xanthoangelol stimulate glucose uptake in skeletal muscle cells by inducing GLUT4 translocation.

SCOPE: Glucose uptake in skeletal muscle is crucial for glucose homeostasis. METHODS AND RESULTS: Insulin and muscle contraction increase glucose uptake accompanied by the translocation of glucose transporter (GLUT) 4. In a search for promising foods, which can increase glucose uptake in skeletal muscle, we screened for active polyphenols by assaying for uptake of 2-deoxyglucose (2DG) in rat L6 muscle cells. Among 37 compounds, 4-hydroxyderricin and xanthoangelol, prenylated chalcones abundant in Ashitaba (Angelica keiskei Koidzumi, family Apiaceae), significantly increased 2DG uptake in L6 cells by 1.9-fold at 10 µM, compared with the level in DMSO-treated control cells. Next, we investigated the effect of these chalcones on the translocation of GLUT4 and its underlying mechanisms. The chalcones increased the GLUT4 level in the plasma membrane of L6 cells, but activated neither protein kinase C ζ/λ, Akt, nor adenosine monophosphate-activated protein kinase, all of which regulate the GLUT4 translocation. Interestingly, the oral administration of a titrated chalcone-enriched Ashitaba extract containing 150.6 mg/g (dry base) of 4-hydroxyderricin and 146.0 mg/g (dry base) of xanthoangelol suppressed acute hyperglycemia in oral glucose tolerance tests of mice. CONCLUSIONS: Ashitaba is a promising functional food for the maintenance of the blood glucose level by inducing skeletal muscle-associated glucose uptake.

Artemisia princeps extract promoted glucose uptake in cultured L6 muscle cells via glucose transporter 4 translocation.

Artemisia princeps is a familiar plant as a food substance and medicinal herb. In this study, we evaluated the effects of an ethanol extract of A. princeps (APE) on glucose uptake in differentiated L6 muscle cells. Treatment with APE elevated deoxyglucose uptake, and translocation of the insulin-responsive glucose transporter (GLUT4) to the plasma membrane in L6 myotubes occurred. The PI3K inhibitor LY294002 attenuated glucose uptake induced by APE. Phosphorylation of the Ser(473) residue of Akt was not observed, but phosphorylation of PI3K, Akt (Thr(308)), and atypical PKC was. In addition, APE stimulated phosphorylation of AMP-activated protein kinase (AMPK) at a level similar to 5'-amino-5-imidazolecarboxamide-riboside (AICAR). These results indicate that APE stimulates glucose uptake by inducing GLUT4 translocation, which is in part mediated by combination of the PI3K-dependent atypical PKC pathway and AMPK pathways.

Suppressive effect of quercetin on acute stress-induced hypothalamic-pituitary-adrenal axis response in Wistar rats.

The flavonoid quercetin is considered to have beneficial effects on human health. We recently have shown that quercetin-enriched foods reduced the duration of immobility time in a rat forced swimming test, indicating that dietary quercetin is promising as an antidepressant-like factor, whereas its mechanism of action is poorly understood. The aim of this study is to investigate the effects of quercetin on water immersion-restraint (WIR), stress-induced hypothalamic-pituitary-adrenal (HPA) axis activation, which is a major component of stress response and plays an important role in the pathology of depression. Quercetin administration to rats significantly suppressed WIR stress-induced increase of plasma corticosterone and adrenocorticotropic hormone levels as well as the mRNA expression of corticotropin-releasing factor (CRF) in the hypothalamic region. In addition, quercetin modulated the DNA binding activities of glucocorticoid receptor and phosphorylated cyclic adenosine 3',5'-monophosphate (cAMP) response element binding protein as well as the phosphorylation of extracellular signal-regulated kinase 1/2 in the hypothalamic region, all of which are known to regulate the expression of CRF mRNA. Taken together, these results suggest that dietary quercetin attenuates the HPA axis activation by the suppression of the CRF mRNA expression.

Flavonoids as substrates and inhibitors of myeloperoxidase: molecular actions of aglycone and metabolites.

Myeloperoxidase (MPO), secreted by activated neutrophils and macrophages at the site of inflammation, may be implicated in the oxidation of protein/lipoprotein during the development of cardiovascular diseases. Flavonoids have been suggested to act as antioxidative and anti-inflammatory agents in vivo; however, their molecular actions have not yet been fully understood. In this study, we examined the molecular basis of the inhibitory effects of dietary flavonoids, such as quercetin, and their metabolites on the catalytic reaction of MPO using a combination of biological assays and theoretical calculation studies. Immunohistochemical staining showed that a quercetin metabolite was colocalized with macrophages, MPO, and dityrosine, an MPO-derived oxidation product of tyrosine, in human atherosclerotic aorta. Quercetin and the plasma metabolites inhibited the formation of dityrosine catalyzed by the MPO enzyme and HL-60 cells in a dose-dependent manner. Spectrometric analysis indicated that quercetin might act as a cosubstrate of MPO resulting in the formation of the oxidized quercetin. Quantitative structure-activity relationship studies showed that the inhibitory actions of flavonoids strongly depended not only on radical scavenging activity but also on hydrophobicity (log P). The requirement of a set of hydroxyl groups at the 3, 5, and 4'-positions and C2-C3 double bond was suggested for the inhibitory effect. The binding of quercetin and the metabolites to a hydrophobic region at the entrance to the distal heme pocket of MPO was also proposed by a computer docking simulation. The current study provides the structure-activity relationships for flavonoids as the anti-inflammatory dietary constituents targeting the MPO-derived oxidative reactions in vivo.

Auraptene decreases the activity of matrix metalloproteinases in dextran sulfate sodium-induced ulcerative colitis in ICR mice.

Previously we reported that auraptene was a potent suppressant for matrix metalloproteinase (MMP)-7 expression in HT-29 human colon cancer cells. In the present study, we examined the effects of auraptene on MMP-2, -7, and -9 expression in colonic mucosa from dextran sulfate sodium (DSS)-induced ulcerative colitis mice. Auraptene remarkably suppressed the DSS-induced gelatinolytic activity of MMP-7 as well as the expression of MMP-2 and -9, suggesting that it might be useful in anti-metastatic therapies via the targeting of MMPs.

Citrus auraptene targets translation of MMP-7 (matrilysin) via ERK1/2-dependent and mTOR-independent mechanism.

Matrix metalloproteinase (MMP)-7 is considered to play essential roles in cancer progression. We examined the efficacy of auraptene, a citrus coumarin derivative, for suppressing MMP-7 expression in the human colorectal adenocarcinoma cell line HT-29. Auraptene remarkably inhibited the production of proMMP-7 protein, without affecting its mRNA expression level. Rapamycin, an inhibitor of mammalian target of rapamycin (mTOR), showed similar results, suggesting that auraptene suppresses mTOR-dependent proMMP-7 translation. Interestingly, however, auraptene showed no effects on the activation of Akt/mTOR signaling, whereas the phosphorylation levels of 4E binding protein (4EBP)1 and eukaryotic translation initiation factor (eIF)4B were substantially decreased. In addition, auraptene remarkably dephosphorylated constitutively activated extracellular signal-regulated kinase (ERK)1/2. Transfection of ERK1/2 siRNA led to a significant reduction of proMMP-7 protein production as well as of the phosphorylation of eIF4B. These results demonstrate that auraptene targets the translation step for proMMP-7 protein synthesis by disrupting ERK1/2-mediated phosphorylation of 4EBP1 and eIF4B.

(-)-Epigallocatechin-3-gallate promotes pro-matrix metalloproteinase-7 production via activation of the JNK1/2 pathway in HT-29 human colorectal cancer cells.

Matrix metalloproteinase (MMP)-7 (matrilysin-1) plays significant roles in the growth, invasion, and metastasis of colorectal tumors, while (-)-epigallocatechin-3-gallate (EGCG), a green tea polyphenol with chemopreventive properties, has been shown to be an inhibitor of MMP-2 and MMP-9. In the present study, HT-29 human colorectal cancer cells were treated with EGCG to examine its effects on pro-MMP-7 induction and production using RT-PCR and western blot analyses. Surprisingly, EGCG (10-100 microM) treatment increased both intracellular and extracellular pro-MMP-7 protein levels (2.6-8.4-fold and 1.9-6.4-fold, respectively) in dose- and time-dependent manner, with a significant upregulation of its mRNA expression. EGCG also activated extracellular signal-regulated protein kinase (ERK)1/2, c-JUN NH2-terminal kinase (JNK)1/2 and p38 mitogen-activated protein kinase (MAPK), as previously reported. In addition, the polyphenol triggered the phosphorylation of c-JUN (Ser63 and Ser73) and induced c-JUN/c-FOS, thereby increasing the DNA binding activity of activator protein-1 (AP-1), as shown by an AP-1 luciferase reporter assay. Pharmacological blockade of MAPK activities suggested that pro-MMP-7 expression was induced via JNK1/2 activation, but not in the case of ERK1/2 or p38 MAPK. N-Acetyl-L-cysteine, superoxide (O2-) dismutase and catalase attenuated the EGCG-induced pro-MMP-7 production, suggesting an involvement of oxidative stress in these events. Conversely, EGCG spontaneously generated O2- in a cell-free system that utilized a cytochrome C reduction method. Further, (-)-epicatechin-3-gallate (25 and 100 microM) and green tea polyphenols (33 and 132 microg/ml) induced pro-MMP-7 expression, whereas (-)-epicatechin and (-)-epigallocatechin (100 microM each) did not. Induction of pro-MMP-7 expression by EGCG was also shown in another human colorectal adenocarcinoma cell line, Caco-2. Our results suggest that some green tea catechins induce pro-MMP-7 production via O2- production and the activation of JNK1/2, c-JUN, c-FOS and AP-1 in HT-29 cells.