Prostaglandins biosynthesis and nitric oxide production have been implicated in the process of carcinogenesis and inflammation. In this study, we investigated the effect of various flavonoids and (-)-epigallocatechin-3-gallate on the activities of inducible cyclooxygenase (COX-2) and inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. Apigenin, genistein and kaempferol were markedly active inhibitors of transcriptional activation of COX-2, with IC(50) < 15 microM. In addition, apigenin and kaempferol were also markedly active inhibitors of transcriptional activation of iNOS, with IC(50) < 15 microM. Of those compounds tested, apigenin was the most potent inhibitor of transcriptional activation of both COX-2 and iNOS. Western and northern blot analyses demonstrated that apigenin significantly blocked protein and mRNA expression of COX-2 and iNOS in LPS-activated macrophages. Transient transfection experiments showed that LPS caused an approximately 4-fold increase in both COX-2 and iNOS promoter activities, these increments were suppressed by apigenin. Moreover, electrophoretic mobility shift assay (EMSA) experiments indicated that apigenin blocked the LPS-induced activation of nuclear factor-kB (NF-kB). The inhibition of NF-kB activation occurs through the prevention of inhibitor kB (IkB) degradation. Transient transfection experiments also showed that apigenin inhibited NF-kB-dependent transcriptional activity. Finally, we showed that apigenin could inhibit the IkB kinase activity induced by LPS or interferon-gamma. The results of further studies suggest that suppression of transcriptional activation of COX-2 and iNOS by apigenin might mainly be mediated through inhibition of IkB kinase activity. This study suggests that modulation of COX-2 and iNOS by apigenin and related flavonoids may be important in the prevention of carcinogenesis and inflammation.

1 Resveratrol, naringenin and naringin are naturally occurring¯avonoids in grapes and grapefruits. The anti-in¯ammatory e ects of these¯avonoids have been well documented, but the mechanism is poorly characterized. High concentration of NO are produced by inducible NO synthase (iNOS) in in¯ammation, and the prevention of the expression of iNOS may be an important anti-in¯ammatory mechanism. In this study, the e ects of these¯avonoids on the induction of NO synthase (NOS) in RAW 264.7 cells activated with bacterial lipopolysaccharide (LPS, 50 ng ml 71 ) were investigated. 2 Resveratrol was found strongly to inhibit NO generation in activated macrophages, as measured by the amount of nitrite released into the culture medium, and resveratrol strongly reduced the amount of cytosolic iNOS protein and steady state mRNA levels. However, the inhibitory abilities of naringenin were lower, and the inhibitory abilities of naringin were almost negligible. 3 In electrophoretic mobility shift assays, the activation of NFkB induced by LPS for 1 h was inhibited by resveratrol (30 mM). Furthermore, in immunoblotting analysis, cells treated with LPS plus resveratrol showed an inhibition of phosphorylation as well as degradation of IkBa, and a reduced nuclear content of NFkB subunits. 4 The¯avonoids may be of value for inhibiting the enhanced expression of iNOS in in¯ammation through down-regulation of NFkB binding activity.

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Recent studies indicate that arsenic may generate reactive oxygen species to exert its toxicity. However, the mechanism is still unclear. In this study, we demonstrate that arsenite is able to induce apoptosis in a concentration- and time-dependent manner; however, arsenate is unable to do so. An increase of intracellular peroxide levels was accompanied with arsenite-induced apoptosis, as demonstrated by flow cytometry using DCFH-DA. N-Acetyl-L-cysteine (a thiol-containing antioxidant), diphenylene iodonium (an inhibitor of NADPH oxidase), 4,5-dihydro-1,3-benzene disulfonic acid (a selective scavenger of O2-), and catalase significantly inhibit arsenite-induced apoptosis and intracellular fluorescence intensity. In contrast, allopurinol (an inhibitor of xanthine oxidase), indomethacin (an inhibitor of cyclooxygenase), superoxide dismutase, or PDTC had no effect on arsenite-induced cell death. Activation of CPP32 activity, PARP (a DNA repair enzyme) degradation, and release of cytochrome c from mitochondria to the cytosol are involved in arsenite-induced apoptosis, and Bcl-2 antagonize arsenite-induced apoptosis by a mechanism that interferes in the activity of CPP32. These results lead to a working hypothesis that arsenite-induced apoptosis is triggered by the generation of hydrogen peroxide through activation of flavoprotein-dependent superoxide-producing enzymes (such as NADPH oxidase), and hydrogen peroxide might play a role as a mediator to induce apoptosis through release of cytochrome c to cytosol, activation of CPP32 protease, and PARP degradation.

Background-Diabetes mellitus causes multiple cardiovascular complications. High glucose can induce reactive oxygen species and apoptosis in endothelial cells. Little is known about the molecular mechanisms in high glucose-induced endothelial cell apoptosis. Methods and Results-We elucidated the signaling pathway of high glucose-induced apoptosis in human umbilical vein endothelial cells (HUVECs). HUVECs were treated with media containing 5.5, 19, or 33 mmol/L of glucose in the presence or absence of an antioxidant, ascorbic acid. The level of intracellular H 2 O 2 was measured by flow cytometry. For detection of apoptosis, the cell death detection ELISA assay and the morphological Hoechst staining were used. High glucose was capable of inducing the activity of c-Jun NH 2 -terminal kinase (JNK) but not extracellular signal-regulated kinase 1/2 or p38 mitogen-activated protein kinase during the treatment periods, as evidenced by immunocomplex kinase assay. Moreover, we found that the interleukin 1␤-converting enzyme (ICE)/CED-3 family protease (caspase-3) became activated in high glucose-induced apoptosis. Caspase-3/CPP32-specific inhibitor, Ac-DEVD-CHO, could inhibit high glucose-induced apoptosis. Furthermore, we found that JNK1 specific antisense oligonucleotide could suppress caspase-3 activity but not affect H 2 O 2 generation and could block apoptosis induced by high glucose. Also, H 2 O 2 generation, JNK activity, caspase-3 activity, and the subsequent apoptosis induced by high glucose could be suppressed by ascorbic acid. Conclusions-The present study indicates that reactive oxygen species induced by high glucose may be involved in JNK activation, which in turn triggers the caspase-3 that facilitates the apoptosis in HUVECs. (Circulation.

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Among the health-promoting effects of tea and tea polyphenols, the cancer-chemopreventive effects in various animal model systems have been intensively investigated; meanwhile, the hypolipidemic and antiobesity effects in animals and humans have also become a hot issue for molecular nutrition and food research. It has been demonstrated that the body weights of rats and their plasma triglyceride, cholesterol, and LDL-cholesterol have been significantly reduced by feedings of oolong, black, pu-erh, and green tea leaves to the animals. It has been suggested that the inhibition of growth and suppression of lipogenesis in MCF-7 breast cancer cells may be through down-regulation of fatty acid synthase gene expression in the nucleus and stimulation of cell energy expenditure in the mitochondria. The experimental data indicated that the molecular mechanisms of fatty acid synthase gene suppression by tea polyphenols (EGCG, theaflavins) may invite down-regulation of EGFR/PI3K/Akt/Sp-1 signal transduction pathways.

An isocratic HPLC procedure was developed for simultaneous determination of six catechins, gallic acid, and three methylxanthines in tea water extract. A baseline separation was achieved on a Cosmosil C18-MS packed column with a solvent mixture of methanol/doubly distilled water/formic acid (19.5:80.2:0.3, v/v/v) as mobile phase. A gradient HPLC procedure was also provided for the separation of these tea components. The contents of catechins, gallic acid, and methylxanthines have been measured in infusions of a range of green tea, oolong tea, and pu-erh tea products sold and consumed in the China, Japan, and Taiwan. When 15 Chinese green tea and 13 Japanese green tea products were analyzed by the HPLC method, the mean levels of the total catechins, (-)-epigallocatechin 3-gallate, (+)-catechin, and caffeine were found to be very similar in these two groups, but other minor catechins such as (-)-epigallocatechin, (-)-epicatechin, and (-)-gallocatechin 3-gallate were found to be higher in Japanese green tea products, whereas (-)-epicatechin 3-gallate, gallic acid, theophylline, and theobromine were found to be higher in Chinese green tea products. Oolong tea products possessed lower levels of catechins, whereas pu-erh tea products contained negligible amounts of these constituents. The new HPLC method is rapid, reliable, and reproducible and should be highly recommended to tea industries for routine analysis of commercial tea samples.