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c-Jun NH2-terminal kinases (JNKs) and phosphatidylinositol 3-kinase (PI3-K) play critical roles in chronic diseases such as cancer, type II diabetes, and obesity. We describe here the binding of quercetagetin (3,3′,4′,5,6,7-hydroxyflavone), related flavonoids, and SP600125 to JNK1 and PI3-K by ATP-competitive and immobilized metal ion affinity-based fluorescence polarization assays and measure the effect of quercetagetin on JNK1 and PI3-K activities. Quercetagetin attenuated the phosphorylation of c-Jun and AKT, suppressed AP-1 and NF-κB promoter activities, and also reduced cell transformation. It attenuated tumor incidence and reduced tumor volumes in a two-stage skin carcinogenesis mouse model. Our crystallographic structure determination data show that quercetagetin binds to the ATP-binding site of JNK1. Notably, the interaction between Lys55, Asp169, and Glu73 of JNK1 and the catechol moiety of quercetagetin reorients the N-terminal lobe of JNK1, thereby improving compatibility of the ligand with its binding site. The results of a theoretical docking study suggest a binding mode of PI3-K with the hydroxyl groups of the catechol moiety forming hydrogen bonds with the side chains of Asp964 and Asp841 in the p110γ catalytic subunit. These interactions could contribute to the high inhibitory activity of quercetagetin against PI3-K. Our study suggests the potential use of quercetagetin in the prevention or therapy of cancer and other chronic diseases.

Atopic dermatitis is an inflammatory and chronically relapsing skin disorder that commonly occurs in children; the number of atopic dermatitis patients is increasing. The cause and mechanism of atopic dermatitis have not been defined clearly, although many studies are ongoing. Epidemiological studies suggest that soybean and its isoflavones have immunoregulatory activities. Here, we report that 7,3′,4′-trihydroxyisoflavone (7,3′,4′-THIF), a major metabolite of daidzin, effectively inhibited lipopolysaccharide (LPS)-induced nitric oxide (NO), tumor necrosis factor (TNF)-α, and interleukin (IL)-6 production in RAW 264.7 cells, and also reducedβ-hexosaminidase secretion in RBL-2H3 cells. Moreover, 7,3′,4′-THIF significantly reduced scratching time, transepidermal water loss, and mast cell infiltration. It also decreased protease-activated receptor (PAR)-2 and IL-4 expression and increased filaggrin expression in skin lesions of NC/Nga mice. These results suggest that 7,3′,4′-THIF improvesDermatophagoides farinabody extract-induced atopic dermatitis in NC/Nga mice.

Quercetin is a major flavonoid compound found in red wine at a much higher concentration than the phytoalexin resveratrol. In this study, we examined potential anti-metastatic effects and found that compared to resveratrol, quercetin more potently inhibits H-Ras-induced invasion and migration in MCF10A human epithelial cells, an effect likely mediated by the mitigation of matrix metalloproteinase (MMP)-2 activation. We then measured Akt phosphorylation to investigate whether the decreased MMP-2 activation was attributable to the inhibition of phosphatidylinositol 3-kinase (PI3K)/Akt signalling. Quercetin, but not resveratrol at equivalent concentrations, suppressed the phosphorylation of Akt and was a more potent inhibitor of PI3K activity than resveratrol. An ex vivo binding assay further revealed that quercetin directly binds to PI3K. Collectively, these results suggest that PI3K is a molecular target of quercetin for the inhibition of H-Ras-induced invasion and migration of MCF10A cells.

Cell metastasis is a major cause of death from cancer and can arise from excessive levels of oxidative stress. The objective of this study was to investigate whether the natural flavonoid quercetin can inhibit matrix metalloproteinase (MMP)-2 and -9 activities through the attenuation of reactive oxygen species (ROS) formation, an event expected to lead to the inhibition of cell motility. To induce sustained ROS formation, cells were treated with phenazine methosulfate (PMS; 1 μM). Noncytotoxic concentrations of quercetin inhibited PMS-induced increases in cell motility in HT1080 human fibrosarcoma (HT1080) cells. While nearly 100% of cells were observed to migrate after 24 h of PMS treatment, quercetin significantly (P0.01) suppressed this effect. We also found that quercetin, up to 10 μg/mL, attenuated PMS-induced MMP-2 activation. We then investigated whether the decreased levels of MMP-2 activation could be attributable to lower levels of ROS formation by quercetin. We found that quercetin treatments significantly attenuated PMS-induced ROS formation (P0.01) and resulted in decreased cell motility associated with a reduction in MMP-2 and -9 activitiy in HT1080 cells, even in the absence of PMS treatment. Collectively, these results suggest that quercetin inhibits cell motility via the inhibition of MMP activation in HT1080 cells in the presence and absence of PMS. This is likely to be a result of the suppression of intracellular ROS formation by quercetin.

Apigenin, a flavonoid abundant in various vegetables and fruits, including parsley and onions, has been reported to possess anticarcinogenic effects. However, the direct molecular target of apigenin and its chemopreventive effect on ultraviolet (UV)-induced skin inflammation are not understood fully. Herein, we examined the anti-inflammatory effect of apigenin and its associated mechanisms in JB6 P+ cell line and SKH-1 hairless mouse model. Apigenin inhibited UVB-induced cyclooxygenase-2 (COX-2) expression, which is a well-known key mediator of inflammation and cancer, and restored the upstream stimulatory factor level in JB6 P+ cells. Immunoblot and kinase assay data demonstrate that Src activity was attenuated by apigenin, and this led to subsequent inhibition of UVB-induced phosphorylation of epidermal growth factor receptor, mitogen-activated protein kinases and Akt signaling. Inhibitory effects of apigenin on UVB-induced signaling were also confirmed in HaCaT human keratinocytes. In addition, in vitro pull-down assays revealed that apigenin binds Src in an adenosine triphosphate-competitive manner. Results using in vivo skin model indicate apigenin significantly inhibits UVB-induced ear edema development, COX-2 expression and Src kinase activity in SKH-1 hairless mice. Collectively, these findings suggest that apigenin exerts potent chemopreventive activity against UVB-induced skin inflammation primarily by targeting Src.

cope Daidzein is one of the major soy isoflavones. Following ingestion, daidzein is readily metabolized in the liver and converted into hydroxylated metabolites. One such metabolite is 6,7,4′-trihydroxyisoflavone (6,7,4′-THIF), which has been the focus of recent studies due to its various health benefits, however, its anti-adipogenic activity has not been investigated. Our objective was to determine the effects of 6,7,4′-THIF on adipogenesis in 3T3-L1 preadipocytes and elucidate the mechanisms of action involved. Methods and results Adipogenesis was stimulated in 3T3-L1 preadipocytes. Both 6,7,4′-THIF and daidzein were treated in the presence and absence of mixture of isobutylmethylxanthine, dexamethasone, and insulin (MDI). We observed that 6,7,4′-THIF, but not daidzein, inhibited MDI-induced adipogenesis significantly at 40 and 80 μM, associated with decreased peroxisome proliferator-activated receptor-γ and C/EBP-α protein expression. 6,7,4′-THIF significantly suppressed MDI-induced lipid accumulation in the early stage of adipogenesis, attributable to a suppression of cell proliferation and the induction of cell cycle arrest. We also determined that 6,7,4′-THIF, but not daidzein, attenuated phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. 6,7,4′-THIF was found to inhibit PI3K activity via direct binding in an ATP-competitive manner. Conclusion Our results suggest that 6,7,4′-THIF suppresses adipogenesis in 3T3-L1 preadipocytes by directly targeting PI3K. Soy isoflavones like 6,7,4′-THIF may have potential for development into novel treatment strategies for chronic obesity.

Metastases are the primary cause of human cancer deaths. Luteolin, a naturally occurring phytochemical, has chemopreventive and/or anticancer properties in several cancer cell lines. However, anti-metastatic effects of luteolin in vivo and the underlying molecular mechanisms and target(s) remain unknown. Luteolin suppresses matrix metalloproteinase (MMP)-2 and -9 activities and invasion in murine colorectal cancer CT-26 cells. Western blot and kinase assay data revealed that luteolin inhibited Raf and phosphatidylinositol 3-kinase (PI3K) activities and subsequently attenuated phosphorylation of MEK and Akt. A pull-down assay indicated that luteolin non-competitively bound with ATP to suppress Raf activity and competitively bound with ATP to inhibit PI3K activity. GW5074, a Raf inhibitor, and LY294002, a PI3K inhibitor, inhibited MMP-2 and -9 activities and invasion in CT-26 cells. An in vivo mouse study showed that oral administration (10 or 50 mg/kg) of luteolin significantly inhibited tumor nodules and tumor volume of lung metastasis induced by intravenous injection of CT-26 cells. Luteolin also inhibited MMP-9 expression and activity in CT-26-induced mouse lung tissue. These results suggest that luteolin may have considerable potential for development as an anti-metastatic agent.

A cell-recycled air-lift bioreactor was studied for its performance in cultivation of Mentha piperita cells producing essential oils. The reactor system sustained a stable operation over 30 days with the aid of a cell settler. The maximum cell concentration reached 50% packed cell volume and it occurred at the dilution rate of 0.27 day-1. Volumetric productivity of essential oils in this system was substantially higher than that obtainable from batch culture.

Targeting tumor necrosis factor (TNF)-α-mediated signal pathways may be a promising strategy for developing chemopreventive agents, because TNF-α-mediated cyclooxygenase (COX)-2 expression plays a key role in inflammation and carcinogenesis. Luteolin [2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-chromenone] exerts anticarcinogenic effects, although little is known about the underlying molecular mechanisms and specific targets of this compound. In the present study, we found that luteolin inhibited TNF-α-induced COX-2 expression by down-regulating the transactivation of nuclear factor-κB and activator protein-1. Furthermore, luteolin inhibited TNF-α-induced phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase 1/ERK/p90(RSK), mitogen-activated protein kinase kinase 4/c-Jun N-terminal kinase/c-Jun, and Akt/p70(S6K). However, it had no effect on the phosphorylation of p38. These effects of luteolin on TNF-α-mediated signaling pathways and COX-2 expression are similar to those achieved by blocking tumor progression locus 2 serine/threonine kinase (TPL2) using pharmacologic inhibitors and small interfering RNAs. Luteolin inhibited TPL2 activity in vitro and in TPL2 immunoprecipitation kinase assays by binding directly in an ATP-competitive manner. Overall, these results indicate that luteolin exerts potent chemopreventive activities, which primarily target TPL2.

Neurodegenerative disorders are strongly associated with oxidative stress, which is induced by reactive oxygen species including hydrogen peroxide (H₂O₂). Epidemiological studies have suggested that coffee may be neuroprotective, but the molecular mechanisms underlying this effect have not been clarified. In this study, we investigated the protective effects of caffeinated coffee, decaffeinated coffee, and the phenolic phytochemical chlorogenic acid (5-O-caffeoylquinic acid), which is present in both caffeinated and decaffeinated coffee, against oxidative neuronal death. H₂O₂-induced apoptotic nuclear condensation in neuronal cells was strongly inhibited by pretreatment with caffeinated coffee, decaffeinated coffee, or chlorogenic acid. Pretreatment with caffeinated coffee, decaffeinated coffee, or chlorogenic acid inhibited the H₂O₂-induced down-regulation of anti-apoptotic proteins Bcl-2 and Bcl-X(L) while blocking H₂O₂-induced pro-apoptotic cleavage of caspase-3 and pro-poly(ADP-ribose) polymerase. We also found that caffeinated coffee, decaffeinated coffee, and chlorogenic acid induced the expression of NADPH:quinine oxidoreductase 1 (NQO1) in neuronal cells, suggesting that these substances protect neurons from H₂O₂-induced apoptosis by up-regulation of this antioxidant enzyme. The neuroprotective efficacy of caffeinated coffee was similar to that of decaffeinated coffee, indicating that active compounds present in both caffeinated and decaffeinated coffee, such as chlorogenic acid, may drive the effects.

3′-Methoxy-3,4′,5,7-tetrahydroxyflavone (isorhamnetin) is a plant flavonoid that occurs in fruits and medicinal herbs. Isorhamnetin exerts anticancer effects, but the underlying molecular mechanism for the chemopreventive potential of isorhamnetin remains unknown. Here, we report anti–skin cancer effects of isorhamnetin, which inhibited epidermal growth factor (EGF)-induced neoplastic cell transformation. It also suppressed anchorage-dependent and -independent growth of A431 human epithelial carcinoma cells. Isorhamnetin attenuated EGF-induced COX-2 expression in JB6 and A431 cells. In an in vivo mouse xenograft using A431 cells, isorhamnetin reduced tumor growth and COX-2 expression. The EGF-induced phosphorylation of extracellular signal-regulated kinases, p90 and p70 ribosomal S6 kinases, and Akt was suppressed by isorhamnetin. In vitro and ex vivo kinase assay data showed that isorhamnetin inhibited the kinase activity of MAP (mitogen-activated protein)/ERK (extracellular signal regulated kinase) kinase (MEK) 1 and PI3-K (phosphoinositide 3-kinase) and the inhibition was due to direct binding with isorhamnetin. Notably, isorhamnetin bound directly to MEK1 in an ATP-noncompetitive manner and to PI3-K in an ATP-competitive manner. This report is the first mechanistic study identifying a clear molecular target for the anticancer activity of isorhamnetin. Overall, these results indicate that isorhamnetin has potent anticancer activity and it primarily targets MEK and PI3-K, which might contribute to the chemopreventive potential of certain foods. Cancer Prev Res; 4(4); 582–91. ©2011 AACR.

Epidemiological studies suggest that coffee consumption reduces the risk of cancers, including colon cancer, but the molecular mechanisms and target(s) underlying the chemopreventive effects of coffee and its active ingredient(s) remain unknown. Based on serving size or daily units, coffee contains larger amounts of phenolic phytochemicals than tea or red wine. Coffee or chlorogenic acid inhibited CT-26 colon cancer cell-induced lung metastasis by blocking phosphorylation of ERKs. Coffee or caffeic acid (CaA) strongly suppressed mitogen-activated MEK1 and TOPK activities and bound directly to either MEK1 or TOPK in an ATP-noncompetitive manner. Coffee or CaA, but not caffeine, inhibited ERKs phosphorylation, AP-1 and NF-κB transactivation and subsequently inhibited TPA-, EGF- and H-Ras-induced neoplastic transformation of JB6 P+ cells. Coffee consumption was also associated with a significant attenuation of ERKs phosphorylation in colon cancer patients. These results suggest that coffee and CaA target MEK1 and TOPK to suppress colon cancer metastasis and neoplastic cell transformation.

Publisher Summary This chapter summarizes recent interesting and relevant findings regarding cocoa and its phytochemicals in human health. From over 3000 years ago to the present, cocoa has been used to treat anemia, mental fatigue, tuberculosis, fever, gout, kidney stones, and even poor sexual appetite. Several health effects, including improved heart function, relief of angina pectoris, stimulation of the nervous system, facilitated digestion, and improved kidney and bowel function have been considered for its medicinal use. Cocoa is unusually rich in phytochemicals. In dried and unfermented cocoa beans, pigment cells make up about 11 to 13% of the tissue. These cells are rich in polyphenolic compounds—in particular, catechins—although the characteristic purple color of cocoa beans is due to anthocyanins. The phytochemicals in cocoa fall into a number of types. The polyphenolics are quantitatively the largest group, comprising around 13.5% of the dried unfermented cocoa beans. Consuming chocolate has been reported to increase the total antioxidant capacity in human blood plasma. Cocoa polyphenols exhibit strong free radical scavenging activity. Data from numerous studies suggest that cocoa can effectively modify the inflammatory process. Cocoa monomer catechins and oligomeric procyanidins have a significant anti-inflammatory effect, inhibit neutrophil oxidative burst, and reduce the expression of adhesion molecules. Flavanol-rich cocoa increases cerebral blood flow, which suggests cocoa as a possible treatment for dementia. Cocoa has moisturizing properties in skin, improves skin appearance and texture, and confers substantial photoprotection from UV light.

Microglial hyperactivation is a hallmark of neurodegenerative diseases and the suppression of microglial hyperactivation is being investigated as a means to treat inflammation-mediated neurodegenerative disorders. Here we report that transcription factor Nrf2 in BV-2 microglia, which regulates the expression of phase II antioxidant enzyme genes, decreased the levels of LPS-induced inflammatory cytokines and mediators. These anti-inflammatory effects were not due to Nrf2-mediated up-regulation of phase II enzymes, since over-expression of these enzymes failed to suppress LPS-mediated microglial hyperactivation. However, Nrf2 inhibited LPS-derived increases in p38 MAPK phosphorylation and NF-κB activation. This suggests that Nrf2 inhibits microglial hyperactivation by suppressing p38 MAPK and NF-κB signaling pathway.

Epidemiologic and animal studies revealed that capsaicin can act as a carcinogen or cocarcinogen. However, the molecular mechanisms of the cancer-promoting effects of capsaicin are not clear. Here, we report that capsaicin has a cocarcinogenic effect on 12-O-tetradecanoylphorbol-13-acetate (TPA)–promoted skin carcinogenesis in vivo and is mediated through the epidermal growth factor receptor (EGFR), but not the transient receptor potential vanilloid subfamily member 1 (TRPV1). Topical application of capsaicin on the dorsal skin of 7,12-dimetylbenz(a)anthracene–initiated and TPA-promoted TRPV1 wild-type (WT) and TRPV1 knockout (KO) mice induced more and larger skin tumors in TRPV1/KO mice, suggesting a TRPV1-independent mechanism. Cyclooxygenase-2 (COX-2) was highly elevated by capsaicin treatment in tumors and murine embryonic fibroblasts from TRPV1/KO mice. Inhibitors of EGFR/MEK signaling suppressed TPA/capsaicin-induced COX-2 expression in TRPV1/KO cells, indicating that activation of EGFR and its downstream signaling is involved in COX-2 elevation. Capsaicin induced a further induction of TPA-increased COX-2 expression in EGFR/WT cells, but not in EGFR/KO cells. TPA/capsaicin cotreatment caused EGFR tyrosine phosphorylation and activated EGFR downstream signaling, including ERKs and Akt in EGFR/WT, but not EGFR/KO cells. Specific inhibition of EGFR and TRPV1 indicated that capsaicin-induced ERK activation in A431 cells was dependent on EGFR, but not TRPV1. Together, these findings suggest that capsaicin might act as a cocarcinogen in TPA-induced skin carcinogenesis through EGFR-dependent mechanisms. Cancer Res; 70(17); 6859–69. ©2010 AACR.

Ultraviolet (UV) radiation is the primary environmental risk factor in the development of nonmelanoma skin cancer, and UVB in particular promotes tumor growth through various signaling pathways. Kaempferol, a flavonoid with anti-inflammatory and anti-oxidative properties, has been studied as a chemopreventive agent; however, little is known regarding its effects on UVB-induced photo-carcinogenesis. Here, we examined the effect of kaempferol on UVB-induced skin inflammation. We found that kaempferol suppressed UVB-induced cyclooxygenase-2 (COX-2) protein expression in mouse skin epidermal JB6 P+ cells and attenuated the UVB-induced transcriptional activities of cox-2 and activator protein-1 (AP-1). Kaempferol attenuated the UVB-induced phosphorylation of several mitogen-activated protein kinases (MAPKs), including ERKs, p38, and JNKs, but had no effect on the phosphorylation of the upstream MAPK regulator Src. However, in vitro and ex vivo kinase assays demonstrated that kaempferol suppressed Src kinase activity. Furthermore, in vivo data from mouse skin support the idea that kaempferol suppresses UVB-induced COX-2 expression by blocking Src kinase activity. A pull-down assay revealed that kaempferol competes with ATP for direct binding to Src. Docking data suggest that kaempferol docks easily into the ATP-binding site of Src, which is located between the N and the C lobes of the kinase domain. Taken together, these results suggest that kaempferol is a potent chemopreventive agent against skin cancer through its inhibitory interaction with Src.

Cocoa polyphenols have antioxidant and anti-inflammatory effects. TNF-α is a pro-inflammatory cytokine that has a vital role in the pathogenesis of inflammatory diseases such as cancer and psoriasis. Vascular endothelial growth factor (VEGF) expression is associated with tumorigenesis, CVD, rheumatoid arthritis and psoriasis. We tested whether cocoa polyphenol extract (CPE) inhibited TNF-α-induced VEGF expression in promotion-sensitive JB6 mouse epidermal cells. CPE significantly inhibited TNF-α-induced up-regulation of VEGF via reducing TNF-α-induced activation of the nuclear transcription factors activator protein-1 (AP-1) and NF-κB, which are key regulators of VEGF expression. CPE also inhibited TNF-α-induced phosphorylation of protein kinase B (Akt) and extracellular signal-regulated kinase. CPE blocked activation of their downstream kinases, p70 kDa ribosomal protein S6 kinase and p90 kDa ribosomal protein S6 kinase. CPE suppressed phosphoinositide 3-kinase (PI3K) activity via binding PI3K directly. CPE did not affect TNF-α-induced phosphorylation of mitogen-activated protein kinase kinase-1 (MEK1) but suppressed TNF-α-induced MEK1 activity. Collectively, these results indicate that CPE reduced TNF-α-induced up-regulation of VEGF by directly inhibiting PI3K and MEK1 activities, which may contribute to its chemopreventive potential.

Abnormal expression of cyclooxygenase-2 (COX-2) and prostaglandin (PG)E(2) is an important mediator in inflammation and tumor promotion. Arsenite is a well-known metalloid carcinogen that is strongly associated with increased risk of liver cancer, but the underlying mechanism remains to be clarified. The present study demonstrates that COX-2 expression and PGE(2) secretion are up-regulated by arsenite in rat liver epithelial (RLE) cells. The possible inhibitory effect of quercetin, a naturally occurring dietary flavonol, on arsenite-induced COX-2 expression and PGE(2) production was investigated. Pretreatment with quercetin resulted in the reduction of arsenite-induced expression of COX-2 and production of PGE(2). The arsenite-induced phosphorylation of Akt, p70S6K, and extracellular signal-regulated protein kinases (ERKs), but not p38, was inhibited by quercetin treatment. An ex vivo kinase assay revealed that quercetin suppressed arsenite-induced phosphoinositide 3-kinase (PI3K) activity upstream of Akt in RLE cell lysates. Ex vivo pull-down assays demonstrated that quercetin directly bound with PI3K to inhibit PI3K activity. Moreover, LY294002 (a PI3K inhibitor) significantly attenuated COX-2 expression and PGE(2) production in arsenite-treated RLE cells. These results suggest that quercetin suppresses arsenite-induced COX-2 expression mainly by blocking the activation of the PI3K signaling pathway, which may contribute to its chemopreventive potential.

Nontoxic small molecules with multitargeting effects are believed to have potential in cancer prevention. Dietary phytochemicals were shown to exhibit cancer-preventive effects attributed to their antioxidant capacities. In this report, we show that the natural compound 5-deoxykaempferol (5-DK) exerts a chemopreventive effect on UVB-induced skin carcinogenesis by targeting multiple signaling molecules. 5-DK suppressed the UVB-induced expression of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor in mouse skin epidermal JB6 P+ cells. Moreover, 5-DK inhibited phosphorylation of MKK3/6, MKK4, and Akt, but had no effect on phosphorylation of Src, extracellular signal–regulated kinases, or ribosomal S6 kinase (RSK). However, 5-DK affected multiple targets by reducing Src, phosphoinositide 3-kinase (PI3K), and RSK2 activities. In particular, pull-down assays revealed that 5-DK specifically bound to and competed with ATP for binding with Src, PI3K, and RSK2. Exposure to 5-DK significantly suppressed UVB-induced tumorigenesis in mouse skin in a dose-dependent manner, and it inhibited the UVB-induced expression of COX-2, proliferating cell nuclear antigen, vascular endothelial growth factor, and matrix metalloproteinase-9. Our data suggest that 5-DK docks at the ATP-binding site of Src, PI3K, and RSK2. For RSK2, the ATP-binding site is located between the N- and C-lobes of the kinase domain. Taken together, our results indicate that 5-DK holds promise for the treatment of UVB-induced skin cancer by targeting Src, PI3K, and RSK2 signaling. Cancer Prev Res; 3(4); 454–65. ©2010 AACR.

Luteolin, a flavonoid present in various vegetables including onion and broccoli, has been reported to possess anticarcinogenic effects. However, its chemopreventive effect on UV-induced skin cancer and its mechanism are not fully understood. Herein, we examined the chemopreventive effect and associated mechanisms of luteolin in the JB6 P+ cell line and the SKH-1 hairless mouse model. Luteolin suppressed UVB-induced cyclooxygenase-2 expression and activator protein-1 and nuclear factor-κB activity in JB6 P+ cells. Immunoblot and kinase assay data showed that luteolin attenuated protein kinase Cϵ (PKCϵ) and Src kinase activities and subsequently inhibited UVB-induced phosphorylation of mitogen-activated protein kinases and the Akt signaling pathway. In addition, pull-down assays revealed that luteolin binds directly to PKCϵ and Src in an ATP-competitive manner. Importantly, luteolin suppressed tumor incidence, multiplicity, and overall size in SKH-1 hairless mice. Analysis of the skin by immunohistochemistry and immunoblotting showed that luteolin-treated groups had a substantial reduction in the levels of cyclooxygenase-2, tumor necrosis factor-α, and proliferating cell nuclear antigen compared with groups treated with only UVB. Further analysis using skin lysates showed that luteolin inhibited PKCϵ and Src kinase activity. Together, these data suggest that luteolin exerts potent chemopreventive activity against UVB-induced skin cancer mainly by targeting PKCϵ and Src. Cancer Res; 70(6); 2415–23

J. Neurochem. (2010) 112, 1415–1430. Abstract Alzheimer’s disease (AD) is an age-related neurodegenerative disease increasingly recognized as one of the most important medical problems affecting the elderly. Although a number of drugs, including several cholinesterase inhibitors and an NMDA receptor antagonist, have been approved for use, they have been shown to produce diverse side effects and yield relatively modest benefits. To overcome these limitations of current therapeutics for AD, extensive research and development are underway to identify drugs that are effective and free of undesirable side effects. Certain naturally occurring dietary polyphenolic phytochemicals have received considerable recent attention as alternative candidates for AD therapy. In particular, curcumin, resveratrol, and green tea catechins have been suggested to have the potential to prevent AD because of their anti-amyloidogenic, anti-oxidative, and anti-inflammatory properties. These polyphenolic phytochemicals also activate adaptive cellular stress responses, called ‘neurohormesis’, and suppress disease processes. In this commentary, we describe the amyloid-β-induced pathogenesis of AD, and summarize the intracellular and molecular targets of selected dietary phytochemicals that might slow the progression of AD.

Myricetin is one of the principal phytochemicals in onions, berries and red wine. Previous studies showed that myricetin exhibits potent anticancer and chemopreventive effects. The present study examined the effect of myricetin on ultraviolet (UV) B-induced angiogenesis in an SKH-1 hairless mouse skin tumorigenesis model. Topical treatment with myricetin inhibited repetitive UVB-induced neovascularization in SKH-1 hairless mouse skin. The induction of vascular endothelial growth factor, matrix metalloproteinase (MMP)-9 and MMP-13 expression by chronic UVB irradiation was significantly suppressed by myricetin treatment. Immunohistochemical and western blot analyses revealed that myricetin inhibited UVB-induced hypoxia inducible factor-1alpha expression in mouse skin. Western blot analysis and kinase assay data revealed that myricetin suppressed UVB-induced phosphatidylinositol-3 (PI-3) kinase activity and subsequently attenuated the UVB-induced phosphorylation of Akt/p70(S6K) in mouse skin lysates. A pull-down assay revealed the direct binding of PI-3 kinase and myricetin in mouse skin lysates. Our results indicate that myricetin suppresses UVB-induced angiogenesis by regulating PI-3 kinase activity in vivo in mouse skin.

Skin cancer is the most frequently diagnosed cancer in the United States. Ultraviolet B (UVB) rays (wavelength: 280–320 nm) play a pivotal role in the development of skin cancer by inducing the expression of inflammatory proteins such as cyclooxygenase-2 (COX-2). Cyanidin, the most plentiful of the plant pigments known as anthocyanidins, is a potent chemopreventive agent. In the present study, we examined the molecular mechanisms underlying the chemopreventive activity of cyanidin and identified its molecular targets. Cyanidin inhibited UVB-induced COX-2 expression and prostaglandin E2 secretion in the epidermal skin cell line JB6 P+ by suppressing the transactivation of nuclear factor-κB and activator protein-1 which are well-known transcription factors regulated by mitogen-activated protein kinase. Cyanidin markedly inhibited the phosphorylation of JNK1/2, ERK1/2, and MEK1/2 than the of MKK4 and Raf-1, two upstream kinases of JNK1/2, ERK1/2, and MEK1/2. Cyanidin significantly suppressed the activities of MKK4, MEK1, and Raf-1 through direct binding. Transient transfection of a small interfering RNA specific for MKK4 inhibited the UVB-induced expression of COX-2 in JB6 P+ cells, as did the expression of a dominant-negative ERK2 mutant. We conclude that MKK4, MEK1, and Raf-1 are targets of cyanidin for the suppression of UVB-induced COX-2 expression.

Arsenite is a well-known human carcinogen that especially targets skin. The tumor progression locus 2 (Tpl2) gene encodes a serine/threonine protein kinase that is overexpressed in various cancer cells. However, the relevance of Tpl2 in arsenite-induced carcinogenesis and the underlying mechanisms remain to be explored. We show that arsenite increased Tpl2 kinase activity and its phosphorylation in mouse epidermal JB6 P+ cells in a dose- and time-dependent manner. Exposure to arsenite resulted in a marked induction of cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2), important mediators of inflammation and tumor promotion. Treatment with a Tpl2 kinase inhibitor or Tpl2 short hairpin RNA suppressed COX-2 expression and PGE2 production induced by arsenite treatment, suggesting that Tpl2 is critical in arsenite-induced carcinogenesis. We also found that arsenite-induced phosphorylation of extracellular signal-regulated kinases (ERK) or c-Jun NH2-terminal kinases (JNK) was markedly suppressed by Tpl2 kinase inhibitor or Tpl2 short hairpin RNA. Inhibition of arsenite-induced ERK or JNK signaling using a pharmacologic inhibitor of ERK or JNK substantially blocked COX-2 expression. Furthermore, inhibition of Tpl2 reduced the arsenite-induced promoter activity of NF-κB and activator protein-1 (AP-1), indicating that NF-κB and AP-1 are downstream transducers of arsenite-triggered Tpl2. Our results show that Tpl2 plays a key role in arsenite-induced COX-2 expression and PGE2 production and further elucidate the role of Tpl2 in arsenite signals that activate ERK/JNK and NF-κB/AP-1 in JB6 P+ cells. [Cancer Res 2009;69(20):8043–9]

Cyclooxygenase-2 (COX-2), a key mediator of inflammation, and its product, prostaglandin E(2) (PGE(2)), enhance carcinogenesis, particularly in skin. Ultraviolet (UV) B is the most carcinogenic component of solar irradiation, and a crucial role of COX-2 in UVB-mediated skin carcinogenesis has been reported. Here, we investigated the effects of delphinidin, an abundant dietary anthocyanin, on UVB-induced COX-2 upregulation and the underlying molecular mechanism. We found that delphinidin suppressed UVB-induced COX-2 expression in JB6 P+ mouse epidermal cells. COX-2 promoter activity and PGE(2) production were also suppressed by delphinidin treatment within non-cytotoxic concentrations. Activator protein-1 and nuclear factor-kappaB, crucial transcription factors involved in COX-2 expression, were activated by UVB and delphinidin abolished this activation. UVB-induced phosphorylation of c-Jun N-terminal kinase, p38 kinase and Akt was inhibited by delphinidin. The activities of mitogen-activated protein kinase kinase (MAPKK) 4 and phosphatidylinositol-3 kinase (PI-3K) were inhibited markedly by delphinidin. A pull-down assay using delphinidin-Sepharose beads revealed that delphinidin binds directly with MAPKK4 or PI-3K in a manner that was competitive with adenosine triphosphate. Moreover, in vivo investigations using mouse skin revealed that the upregulation of COX-2 expression, MAPKK4 activity and PI-3K activity induced by UVB was abolished with delphinidin treatment. Collectively, our results demonstrated that delphinidin targets MAPKK4 and PI-3K directly to suppress COX-2 overexpression, suggesting a potential protective role for delphinidin against UVB-mediated skin carcinogenesis.

Mutations in Ras play a critical role in the development of human cancers, including breast cancer. We investigated the possible antiproliferative effects of the naturally occurring dihydrochalcone phloretin [2',4',6'-trihydroxy-3-(4-hydroxyphenyl)-propiophenone] on H-Ras-transformed MCF10A human breast epithelial (H-Ras MCF10A) cells. Phloretin suppressed H-Ras MCF10A cell proliferation in a dose-dependent manner and induced nuclear condensation in the cells, indicating that phloretin-induced cell death occurs mainly via the induction of apoptosis. Prominent upregulation of p53 and Bax and cleavage of poly (ADP)-ribose polymerase were also detected in the phloretin-treated cells. Finally, phloretin markedly increased caspase-3 activity as well as JNK and p38 mitogen-activated protein kinase signaling. Our findings suggest that the phloretin-induced apoptosis of breast tumor cells contributes to the chemopreventive potential of phloretin against breast cancer.

There is accumulating evidence that a moderate consumption of red wine has health benefits, such as the inhibition of neurodegenerative diseases. Although this is generally attributed to resveratrol, the protective mechanisms and the active substance(s) remain unclear. We examined whether and how red wine extract (RWE) and red wine flavonols quercetin and myricetin inhibited 4-hydroxynonenal (HNE)-induced apoptosis of rat pheochromocytoma PC 12 cells. RWE attenuated HNE-induced PC12 cell death in a dose-dependent manner. HNE induced cleavage of poly(ADP-ribose) polymerase, which is involved in DNA repair in the nucleus, and this was inhibited by RWE treatment. Treatment with RWE also inhibited HNE-induced nuclear condensation in PC 12 cells. Data of +2',7'-dichlorofluorescin diacetate showed that RWE protected against apoptosis of PC12 cells by attenuating intracellular reactive oxygen species. The cytoprotective effects on HNE-induced cell death were stronger for quercetin and myricetin than for resveratrol. HNE-induced nuclear condensation was attenuated by quercetin and myricetin. These results suggest that the neuroprotective potential of red wine is attributable to flavonols rather than to resveratrol.

Alzheimer's disease (AD) is an age-related neurodegenerative disorder in which apoptosis plays a potentially important role. 4-Hydroxynonenal (HNE) is a major lipid peroxidation product produced by oxidative stress, and its level is elevated in the AD brain. In the present study, piceatannol (but not resveratrol) at the concentration of 20 micromol/L inhibited HNE-induced PC12 cell death. Treatment with HNE induced nuclear condensation in PC12 cells, and this was attenuated by piceatannol treatment. HNE induced poly(ADP-ribose) polymerase cleavage and decreased Bcl-2 expression, with both of these effects being attenuated by piceatannol. Piceatannol also inhibited the phosphorylation of c-Jun N-terminal kinase, which is a key regulator of HNE-induced PC12 cell death. These results indicate that piceatannol has therapeutic potential in the prevention of AD.

We investigated the antiproliferative effects of the cytoplasmic fraction of Lactococcus lactis ssp. lactis (L.lac CF) on the SNU-1 human stomach cancer cell line. The proliferation of SNU-1 cells was inhibited by treatment with L.lac CF in a time- and dose-dependent manner. L.lac CF caused G0/G1 cell cycle arrest, which was associated with an increase in p53 and p21 expression, the reduction of cyclin D1 expression, and retinoblastoma protein phosphorylation. L.lac CF induced apoptosis in SNU-1 cells, as demonstrated by increased nucleus condensation and a sub-G1 peak. Caspase-3 activation, the induction of p53, and the downregulation of Bcl-2 were also observed in L.lac CF-treated cells. Thus, the inhibitory effect of L.lac CF on SNU-1 cell growth is mainly attributable to the induction of G0/G1 cell cycle arrest and apoptosis.

There are multiple lines of evidence that lactic acid bacteria (LAB) exert cancer-preventive effects. However, the underlying mechanisms are poorly understood. In the present study we found that the cytoplasmic fraction ofLactococcus lactisssp.lactisAmerican Type Culture Collection (ATCC) 7962 exerted the strongest antiproliferative effects (half-maximal inhibitory concentration (IC50) = 17 μg/ml) in SNU-1 human stomach cancer cells and arginine deiminase (ADI;EC3.5.3.6) activity. We also cloned, expressed and purified ADI fromL. lactisssp.lactisATCC 7962 (LADI). Both purified ADI fromL. lactis(PADI; IC50 = 2 μg/ml) and recombinant ADI originating from LADI (IC50 = 0·6 μg/ml) inhibited the proliferation of SNU-1 cells. LADI induced G0/G1-phase arrest, sub-G1accumulation, DNA condensation and DNA fragmentation in SNU-1 cells. 4′,6-Diamidino-2-phenylindole (DAPI) staining and DNA fragmentation data provide evidence that LADI induces apoptosis in SNU-1 cells. LADI increased the expressions of p53 and p27Kip1, and decreased the expressions of cyclin D1, c-myc and Bcl-xL in SNU-1 cells. However, LADI had no effects on the expressions of p21Cip1and Bcl-2. Collectively, these data indicate that ADI induces apoptosis and G0/G1-phase arrest of SNU-1 cells, which might contribute to the chemopreventive potential of LAB.

Recombinant Lactobacillus strains have been constructed using gene splicing by overlap extension (SOE). Primers were designed of which one end of an amplified product contained complementary sequences for an end of other amplified fragment. For efficient matching, we used an asymmetric PCR step that was effective at generating an excess of strands that would anneal in the final PCR. CP12, a recombinant fragment consisting of the integrase gene and attachment site of the bacteriophage A2, was constructed and inserted into the genome of Lactobacillus casei ATCC 393, yielding Lb. casei ATCC 393::XCP12. Another recombinant Lb. casei strain was constructed, where the egfp gene was a part of the construction. The EGFP produced from Lb. casei ATCC 393::XCEGFP14 was detected by Western blot hybridization. This simple and widely applicable approach has significant advantages over standard recombinant DNA techniques for Lactobacillus species.

Caffeic acid (3,4-dihydroxycinnamic acid) is a well-known phenolic phytochemical present in many foods, including coffee. Recent studies suggested that caffeic acid exerts anticarcinogenic effects, but little is known about the underlying molecular mechanisms and specific target proteins. In this study, we found that Fyn, one of the members of the non-receptor protein tyrosine kinase family, was required for ultraviolet (UV) B-induced cyclooxygenase-2 (COX-2) expression, and caffeic acid suppressed UVB-induced skin carcinogenesis by directly inhibiting Fyn kinase activity. Caffeic acid more effectively suppressed UVB-induced COX-2 expression and subsequent prostaglandin E(2) production in JB6 P+ mouse skin epidermal (JB6 P+) cells compared with chlorogenic acid (5-O-caffeoylquinic acid), an ester of caffeic acid with quinic acid. Data also revealed that caffeic acid more effectively induced the downregulation of COX-2 expression at the transcriptional level mediated through the inhibition of activator protein-1 (AP-1) and nuclear factor-kappaB transcription activity compared with chlorogenic acid. Fyn kinase activity was suppressed more effectively by caffeic acid than by chlorogenic acid, and downstream mitogen-activated protein kinases (MAPKs) were subsequently blocked. Pharmacological Fyn kinase inhibitor (3-(4-chlorophenyl)1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine and leflunomide) data also revealed that Fyn is involved in UVB-induced COX-2 expression mediated through the phosphorylation of MAPKs in JB6 P+ cells. Pull-down assays revealed that caffeic acid directly bound with Fyn and non-competitively with adenosine triphosphate. In vivo data from mouse skin also supported the idea that caffeic acid suppressed UVB-induced COX-2 expression by blocking Fyn kinase activity. These results suggested that this compound could act as a potent chemopreventive agent against skin cancer.

Resveratrol, present in grapes and red wine, is reported to be a natural chemopreventive agent against cancer. However, the concentrations required to exert these effects may be difficult to achieve by drinking only one or two glasses of red wine a day. Therefore, developing more potent, nontoxic analogues of resveratrol may provide a feasible means of achieving an effective physiologic concentration. Here we report that the resveratrol analogue, 3,5,3′,4′,5′-pentahydroxy-trans-stilbene (RSVL2), inhibits 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced neoplastic transformation in JB6 P+ mouse epidermal cells. Further, we identified MEK/ERK signaling as the direct molecular target for the anticancer effects of RSVL2 and demonstrated that RSVL2 inhibited MEK1, but not Raf1 or ERK2 kinase activity. RSVL2 also dose-dependently suppressed MEK1 kinase activity induced by TPA and the inhibition of H-Ras-induced cell transformation was much stronger for RSVL2 than for PD098059 or resveratrol. Both in vitro and ex vivo pull-down assays indicated that RSVL2, but not resveratrol, directly bound with GST-MEK1, but did not compete with ATP for binding. Docking data indicated that the low inhibitory activity of resveratrol might be due to the lack of the hydroxyl group at the meta position of the B ring, thereby preventing resveratrol from forming a hydrogen bond with the backbone amide group of Ser212, which is the key interaction for stabilizing the inactive conformation of the activation loop.

Matrix metalloproteinases (MMPs) are involved in various skin disorders, including photoaging, dermatitis, and tumorigenesis. Tumor necrosis factor (TNF)-alpha is a key proinflammatory cytokine that acts to provoke inflammation, proliferation, and tumorigenesis. The present study investigated the possible inhibitory effects of red wine polyphenols on TNF-alpha-induced upregulation of MMP-9 and on the migratory phenotype of JB6 P+ mouse epidermal (JB6 P+) cells. Red wine extract (RWE) and quercetin, which is a major flavonoid present in red wine, inhibited significantly the TNF-alpha-induced upregulation of MMP-9 and cell migration, whereas resveratrol did not have significant inhibitory effects. The inhibitory effects of RWE and quercetin were mediated by suppression of the phosphorylation of Akt and the transactivation of activator protein-1 and nuclear factor-kappaB, as determined by Western blotting and luciferase assays, respectively. Aside from Akt, quercetin had no effect on the phosphorylation of other mitogen-activated protein kinases. Direct kinase assay data revealed that RWE and quercetin inhibited phosphatidylinositol 3-kinase (PI3K) activity. The results of direct and cell-based pull-down assays demonstrated that RWE and quercetin bound to PI3K, resulting in the inhibition of PI3K activity. Using chemical inhibitors, it was confirmed that the PI3K-dependent Akt pathway was involved in TNF-alpha-induced MMP-9 upregulation and migration in JB6 P+ cells. Collectively, these results indicate that TNF-alpha-induced MMP-9 upregulation and the migratory phenotype are associated with the PI3K/Akt pathway, and that these effects are inhibited strongly by RWE and quercetin.

(-)-Epigallocatechin gallate (EGCG), a polyphenolic compound found in green tea, is a promising chemopreventive agent against cancer due to its strong antiproliferative effects on cancer cells; however, its possible toxicity and carcinogenicity must be investigated before EGCG can be used as a dietary supplement for chemoprevention. The inhibition of gap junctional intercellular communication (GJIC) is strongly associated with carcinogenesis, particularly the tumor promotion process; thus, we investigated the effects of EGCG on GJIC in WB-F344 normal rat liver epithelial (RLE) cells. EGCG, but not (-)-epicatechin (EC), another polyphenol found in green tea, inhibited GJIC in a dose-dependent and reversible manner in RLE cells. EGCG also induced the phosphorylation of connexin 43 (Cx43), a major regulator of GJIC. The phosphorylation of extracellular signal-regulated protein kinase 1/2 (ERK1/2) was also observed in EGCG-treated RLE cells. The inhibition of GJIC and phosphorylation of Cx43 and ERK1/2 by EGCG were completely blocked by U0126, a pharmacological inhibitor of mitogen-activated protein kinase/ERK kinase. EGCG generated a larger amount of hydrogen peroxide than EC in a dose-dependent manner. Furthermore, catalase partially inhibited the EGCG-induced inhibition of GJIC and the phosphorylation of Cx43 and ERK1/2. These results indicated that EGCG inhibited GJIC mainly due to its prooxidant activity.

Nuruk, which is a natural inoculator and source of amylolytic enzymes, is used in Korean traditional rice wine. A methanol extract of nuruk (NE) attenuated lipopolysaccharide (LPS)- induced nitrite and interleukin (IL)-6 in RAW 264.7 cells. Both the n-hexane and water fractions from NE (MEH and MW, respectively) inhibited the production of nitrite and IL-6 in RAW 264.7 cells. MEH and MW also inhibited the LPS-induced inducible nitric oxide synthase expression. Further, and MEH protected against the LPS-induced activation of p38 mitogen-activated protein kinase. Together, these results indicate that nuruk may contribute to the anti-inflammatory and cancer-preventive effects of Korean traditional rice wine.

Tumor necrosis factor-alpha (TNF-alpha) is a mediator of multiple inflammatory diseases. Vascular endothelial growth factor (VEGF) plays a critical role in TNF-alpha-mediated diseases. We investigated the inhibitory effects of 3,3',4',5,5',7-hexahydroxyflavone (myricetin), an abundant natural flavonoid, on TNF-alpha-induced VEGF upregulation and the underlying molecular mechanism. Myricetin is a direct inhibitor of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase 1 (MEK1) and inhibits neoplastic cell transformation. We found that myricetin inhibited TNF-alpha-induced VEGF expression in JB6 P+ mouse epidermal cells by targeting MAPK kinase 4 (MKK4), as well as MEK1. The activation of activator protein-1 by TNF-alpha was inhibited by myricetin in a dose-dependent manner. The phosphorylation of c-Jun N-terminal kinase (JNK) and ERK was inhibited by myricetin, but not the phosphorylation of their upstream kinases MKK4 and MEK1. TNF-alpha-induced VEGF expression was inhibited by SP600125 and U0126, which are inhibitors of JNK and MEK, respectively. Myricetin inhibited TNF-alpha-induced MKK4 activity and bound glutathione S-transferase-MKK4 directly by competing with ATP. Computer modeling suggested that myricetin docks onto the ATP-binding site in MKK4, which is located between the N- and C-lobes of the kinase domain. Overall, our results indicate that myricetin has potent chemopreventive effects against TNF-alpha-related disease, mainly by targeting MKK4 and MEK1.

Skin cancer is currently the most common type of human cancer in Americans. Myricetin, a naturally occurring phytochemical, has potent anticancer-promoting activity and contributes to the chemopreventive potential of several foods, including red wine. Here, we show that myricetin suppresses UVB-induced cyclooxygenase-2 (COX-2) expression in mouse skin epidermal JB6 P+ cells. The activation of activator protein-1 and nuclear factor-κB induced by UVB was dose-dependently inhibited by myricetin treatment. Western blot and kinase assay data revealed that myricetin inhibited Fyn kinase activity and subsequently attenuated UVB-induced phosphorylation of mitogen-activated protein kinases. Pull-down assays revealed that myricetin competitively bound with ATP to suppress Fyn kinase activity. Importantly, myricetin exerted similar inhibitory effects compared with 4-amino-5-(4-chloro-phenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine, a well-known pharmacologic inhibitor of Fyn. In vivo mouse skin data also revealed that myricetin inhibited Fyn kinase activity directly and subsequently attenuated UVB-induced COX-2 expression. Mouse skin tumorigenesis data clearly showed that pretreatment with myricetin significantly suppressed UVB-induced skin tumor incidence in a dose-dependent manner. Docking data suggest that myricetin is easily docked to the ATP-binding site of Fyn, which is located between the N and C lobes of the kinase domain. Overall, these results indicated that myricetin exerts potent chemopreventive activity mainly by targeting Fyn in skin carcinogenesis. [Cancer Res 2008;68(14):6021–9]

Free radicals play a crucial role in the pathophysiology of human diseases such as cancer, atherosclerosis, and neurodegenerative diseases, and considerable attention has been focused on functional foods (or nutraceuticals) that are able to decrease the concentrations of free radicals and consequently protect against these diseases. The present study investigated an improved quantitative assay to measure antioxidant activity using the stable and fast-reacting chromogenic indicator [2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)] (ABTS). The ABTS-radical-scavenging activities of various antioxidants and apple extracts were measured in 96-well plates, and plots thereof were linearly interpolated, with the total radical-scavenging capacity quantified as the area under the curve. The first order of linear regression was obtained in a relationship between the absorbance reduction and various concentrations of the tested sample, and the total radical-scavenging capacity was expressed as the vitamin-C-equivalent antioxidant capacity. The advantages of this quantitative assay are that, first, it is fast, sensitive and confers little variation from experimental errors for single or mixed antioxidants; second, a large number of samples in a low quantity at a time can be run using 96-well plates.

Neurodegenerative disorders such as Alzheimer's disease (AD) are associated with oxidative stress, and it has been suggested that apoptosis is a crucial pathway in neuronal cell death in AD patients. 4-Hydroxynonenal (HNE), one of the aldehydic products of membrane lipid peroxidation, is reported to be elevated in the brains of AD patients and mediates the induction of neuronal apoptosis in the presence of oxidative stress. In this study, we investigated the HNE-induced apoptosis mechanism and the protective effects of the cocoa procyanidin fraction (CPF) and its major antioxidant procyanidin B2 against the apoptosis induced by HNE in rat pheochromocytoma (PC12) cells. HNE-induced nuclear condensation and increased sub-G1 fraction, both of which are markers of apoptotic cell death, were inhibited by CPF and procyanidin B2. Intracellular reactive oxygen species (ROS) accumulation was attenuated by pretreatment with CPF and procyanidin B2. CPF and procyanidin B2 also prevented HNE-induced poly(ADP-ribose) polymerase cleavage, antiapoptotic protein (Bcl-2 and Bcl-X(L)) down-regulation, and caspase-3 activation. Activation of c-Jun N-terminal protein kinase (JNK) and mitogen-activated protein kinase kinase 4 (MKK4) was attenuated by CPF and procyanidin B2. Moreover, CPF and procyanidin B2 bound directly to MKK4 and inhibited its activity. Data obtained with SP600125, a selective inhibitor of JNK, revealed that JNK is involved in HNE-induced apoptosis through the inhibition of PARP cleavage and caspase-3 activation in PC12 cells. Collectively, these results indicate that CPF and procyanidin B2 protect PC12 cells against HNE-induced apoptosis by blocking MKK4 activity as well as ROS accumulation.

Cocoa, a good source of dietary antioxidative polyphenols, exhibited anticarcinogenic activity in animal models, but the molecular mechanisms of the chemopreventive potential of cocoa remain unclear. Inhibition of gap-junction intercellular communication (GJIC) is strongly related to tumorigenesis. Cocoa polyphenol extracts (CPE) dose dependently attenuated hydrogen peroxide (H(2)O(2))-induced inhibition of GJIC in rat liver epithelial (RLE) cells. CPE inhibited the H(2)O(2)-induced phosphorylation and internalization of connexin 43, which is a regulating protein of GJIC in RLE cells. The H(2)O(2)-induced accumulation of reactive oxygen species and activation of extracellular signal-regulated kinase were inhibited by CPE treatment. However, CPE did not block H(2)O(2)-induced phosphorylation of p38 mitogen-activated protein kinase. An ex vivo kinase assay demonstrated that CPE inhibited the H(2)O(2)-induced mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) 1 activity in RLE cell lysates. Ex vivo pull-down assay data revealed that CPE directly bound with MEK1 to inhibit MEK1 activity. These results indicate that CPE protects against the H(2)O(2)-induced inhibition of GJIC through antioxidant activity and direct inhibition of MEK activity, which may contribute to its chemopreventive potential.

Aims Expression and activation of matrix metalloproteinase (MMP)-2 play pivotal roles in the migration and invasion of human aortic vascular smooth muscle cells (VSMC) originating from normal human tissue, which is strongly linked to atherosclerosis. The present study investigated the possible inhibitory effects of cocoa procyanidin on thrombin-induced expression and activation of MMP-2 in VSMC. Methods and results Cocoa procyanidin fraction (CPF) and procyanidin B2, one of major procyanidins in cocoa (3 mg/mL and 5 mM, respectively), strongly inhibited thrombin-induced activation and expression of pro-MMP-2 in VSMC, as determined by zymography. The thrombin-induced invasion and migration of VSMC were inhibited by CPF or procyanidin B2 (P , 0.05), as assessed by a modified Boyden chamber and wound healing assays, respectively. An enzymatic assay data demonstrated that CPF and procyanidin B2 directly inhibited membrane type-1 (MT1)-MMP activity (P , 0.05), and this inhibition of CPF was greater than those of red wine polyphenols. Western blot data showed that CPF and procyanidin B2 inhibited thrombin-induced phosphorylation of extracellular signal-regulated protein kinase but not mitogen-activated protein kinase kinase (MEK) in VSMC. Kinase and pull-down data revealed that CPF and procyanidin B2 inhibited MEK1 activity and directly bound with glutathione-S-transferase-MEK1. In addition, the thrombin-induced invasion and migration and the activation and expression of proMMP-2 in VSMC were attenuated by U0126 (a well-known inhibitor of MEK1). Conclusion Cocoa procyanidins are potent inhibitors of MEK and MT1-MMP, and subsequently inhibit the expression and activation of pro-MMP-2, and also the invasion and migration of VSMC, which may in part explain the molecular action of antiatherosclerotic effects of cocoa.

Considerable attention has focused on the health-promoting effects of red wine and its nonflavonoid polyphenol compound resveratrol. However, the underlying molecular mechanisms and molecular target(s) of red wine or other potentially active ingredients in red wine remain unknown. Here, we report that red wine extract (RWE) or the red wine flavonoid quercetin inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced transformation of JB6 promotion-sensitive mouse skin epidermal (JB6 P+) cells. The activation of activator protein-1 and nuclear factor-κB induced by TPA was dose dependently inhibited by RWE or quercetin treatment. Western blot and kinase assay data revealed that RWE or quercetin inhibited mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) 1 and Raf1 kinase activities and subsequently attenuated TPA-induced phosphorylation of ERK/p90 ribosomal S6 kinase. Although either RWE or quercetin suppressed Raf1 kinase activity, they were more effective in inhibiting MEK1 activity. Importantly, quercetin exerted stronger inhibitory effects than PD098059, a well-known pharmacologic inhibitor of MEK. Resveratrol did not affect either MEK1 or Raf1 kinase activity. Pull-down assays revealed that RWE or quercetin (but not resveratrol) bound with either MEK1 or Raf1. RWE or quercetin also dose dependently suppressed JB6 P+ cell transformation induced by epidermal growth factor or H-Ras, both of which are involved in the activation of MEK/ERK signaling. Docking data suggested that quercetin, but not resveratrol, formed a hydrogen bond with the backbone amide group of Ser212, which is the key interaction for stabilizing the inactive conformation of the activation loop of MEK1. [Cancer Res 2008;68(3):946–55]

Arginine deiminase (ADI, E.C. 3.5.3.6), one of the arginine deprivation enzymes, exhibits anticarcinogenic activities. The present study investigated the anti-inflammatory activities of the purified recombinant ADI originating from Lactococcus lactis ssp. lactis ATCC7962 (LADI). LADI dose-dependently inhibited lipopolysaccharide (LPS)-induced upregulation of inducible nitric oxide synthase and the production of nitric oxide in RAW 264.7 murine macrophages. The induction of cyclooxygenase-2 expression and subsequent production of prostaglandin E2 by LPS was also attenuated by LADI treatment. Moreover, LADI inhibited the production of interleukin-6 in LPS-stimulated RAW 264.7 macrophages. These results indicate that LADI exerts anti-inflammatory effects, which may in part explain its chemopreventive potential.

A gene coding for phosphoketolase, a key enzyme of carbohydrate catabolism in heterofermentative lactic acid bacteria (LAB), was cloned from a Lactobacillus paraplantarum C7 and expressed in Escherichia coli. The gene is 2502 bp long and codes for a 788-amino-acids polypeptide with a molecular mass of 88.7 kDa. A Shine-Dalgamo sequence (aaggag) and an inverted-repeat terminator sequence are located upstream and downstream of the phosphoketolase gene, respectively. The gene exhibits an identity of52% with phosphoketolases of other LAB. The phosphoketolase of Lb. paraplantarum C7 (LBPK) contains several highly conserved phosphoketolase signature regions and typical thiamine pyrophosphate (TPP) binding sites, as reported for other TPP-dependent enzymes. The phosphoketolase gene was fused to a glutathione S-transferase (GST::LBPK) gene for purification. The GST::LBPK fusion protein was detected in the soluble fraction of a recombinant Escherichia coli BL21. The GST::LBPK fusion protein was purified with a yield of 4.32 mg/400 ml by GSTrap HP affinity column chromatography and analyzed by N-terminal sequencing. LBPK was obtained by factor Xa treatment of fusion protein and the final yield was 3.78 mg/400 ml. LBPK was examined for its N-terminal sequence and phosphoketolase activity. The K(M) and Vmax values for fructose-6-phosphate were 5.08 +/- 0.057 mM (mean +/- SD) and 499.21 +/- 4.33 micromol/min/mg, respectively, and the optimum temperature and pH for the production of acetyl phosphate were 45 degrees C and 7.0, respectively.

A Lactobacillus paraplantarum strain producing a bacteriocin was isolated from kimchi using the spot-on-the lawn method and named L. paraplantarum C7. The bacteriocin, paraplantaricin C7, was found to inhibit certain Lactobacillus strains, including L. plantarum, L. pentosus, and L. delbrueckii subsp. lactis. It also inhibited Enterococcus faecalis, yet did not inhibit most of the other LAB (lactic acid bacteria) tested. The maximum level of paraplantaricin C7 activity was observed under the culture conditions of 25 degrees C and a constant pH of 4.5. Paraplantaricin C7 retained 90% of its activity after 10 min of treatment at 100 degrees C and remained stable within a pH range of 2-8. Based on a culture supernatant, paraplantaricin C7 was purified by DEAE-Sephacel column chromatography and C18 reverse-phase HPLC. SDS-PAGE and activity staining were then conducted using the purified paraplantaricin C7, and its molecular mass determined to be about 3,800 Da. The 28 N-terminal amino acids from the purified paraplantaricin C7 were determined, and the structural gene encoding paraplantaricin C7, ppnC7, was cloned by PCR using degenerate primers based on the N-terminal amino acid sequence. The nucleotide sequences for ppnC7 and other neighboring orfs exhibited a limited homology to the previously reported plantaricin operon genes. Paraplantaricin C7 is a novel type II bacteriocin containing a double glycine leader sequence.