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Consumption of fruits and vegetables is correlated with a lower incidence of cancer. Here, we identified hyperoside as an active compound from Acanthopanax sessiliflorus , and investigated the effect of hyperoside on UVB-induced transactivation of activator protein 1 (AP-1) and on the mitogen-activated protein kinase signalling pathway in JB6 P + cells. Hyperoside inhibited UVB-induced AP-1 transactivation. It inhibited the UVB-induced phosphorylation of p90 RSK . Kinase assays revealed that hyperoside significantly inhibited ERK1/2 activity. Furthermore, hyperoside bound to ERK1/2 to suppress its activity. In addition, phosphorylation of cAMP response element binding protein (CREB) and signal transducers and activators of transcription (STAT) 3 were suppressed by hyperoside. Overall, these results indicate that hyperoside may be a promising chemopreventive agent that acts by suppressing the transactivation of AP-1 and the phosphorylation of p90 RSK , CREB, and STAT3 through the binding and inhibition of ERK1/2.

Although previous studies have reported that black soybean has chemopreventive potential, the underlying mechanisms remain unclear. Upregulation of cyclooxygenase-2 (COX-2) has been known to be a key mediator in the development of skin cancer. The present study investigated the effect of black soybean (Glycine max cv. Heugmi) seed coat extract (BSE) on 12-O-tetradecanoylphorbol-13-acetate (TPA) or ultraviolet-B-(UVB)-induced COX-2 expression, and its underlying mechanisms. The TPA- or UVB-induced COX-2 expression in mouse skin epithelial cells were dose-dependently inhibited by BSE treatment. BSE suppressed the COX-2 promoter activity. BSE also attenuated the transactivation of activator protein-1 (AP-1) and nuclear factor (NF)-κB, transcription factors of COX-2 expression in mouse skin epithelial cells transfected stably with AP-1 and NF-κB luciferase promoter, respectively. Furthermore, BSE inhibited the activation of MAPKKs/MAPKs pathways that otherwise induced by TPA or UVB. Collectively, BSE suppresses TPA or UVB-induced COX-2 expression by blocking the expressions of MAPKKs/MAPKs pathways, which may contribute to its chemopreventive potential.

Benzo[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE) is a well-known carcinogen that is associated with skin cancer. Abnormal expression of cyclooxygenase-2 (COX-2) is an important mediator in inflammation and tumor promotion. We investigated the inhibitory effect of cyanidin-3-glucoside (C3G), an anthocyanin present in fruits, on B[a]PDE-induced COX-2 expression in mouse epidermal JB6 P+ cells. Pretreatment with C3G resulted in the reduction of B[a]PDE-induced expression of COX-2 and COX-2 promoter activity. The activation of activator protein-1 (AP-1) and nuclear factor-κB (NF-κB) induced by B[a]PDE was also attenuated by C3G. C3G attenuated the B[a]PDE-induced phosphorylation of MEK, MKK4, Akt, and mitogen-activated protein kinases (MAPKs), but no effect on the phosphorylation of the upstream MAPK regulator Fyn. However, kinase assays demonstrated that C3G suppressed Fyn kinase activity and C3G directly binds Fyn kinase noncompetitively with ATP. By using PP2, a pharmacological inhibitor for SFKs, we showed that Fyn kinase regulates B[a]PDE-induced COX-2 expression by activating MAPKs, AP-1 and NF-κB. These results suggest that C3G suppresses B[a]PDE-induced COX-2 expression mainly by blocking the activation of the Fyn signaling pathway, which may contribute to its chemopreventive potential.

Myricetin is a widely distributed flavonol that is found in many plants, including tea, berries, fruits, vegetables, and medicinal herbs. Abundant sources provide interesting insights into the multiple mechanisms by which myricetin mediates chemopreventive effects on skin cancer. Myricetin strongly inhibited tumor promoter-induced neoplastic cell transformation by inhibiting MEK, JAK1, Akt, and MKK4 kinase activity directly. In a mouse skin model, myricetin attenuated the ultraviolet B (UVB)-induced COX-2 expression and skin tumor formation by regulating Fyn. Myricetin-mediated inactivation of Akt in the UVB response plays a role in regulating UVB-induced carcinogenesis. Recently, myricetin was found to inhibit UVB-induced angiogenesis by targeting PI3-K in an SKH-1 hairless mouse skin tumorigenesis model. Raf kinase is a critical target for myricetin in inhibiting the UVB-induced formation of wrinkles and suppression of type I procollagen and collagen levels in mouse skin. Accumulated data suggest that myricetin acts as a promising agent for the chemoprevention of skin cancer.

Multiple lines of evidences suggest that oxidative stress induced by reactive oxygen species are closely related to multi-stage carcinogenesis. Polyphenols, a group of chemicals with more than one phenol unit or building block per molecule, have been recognized for possessing many health benefits including cancer-preventive effects mainly due to their antioxidant activity. However, polyphenols can directly bind with signaling molecules involved in carcinogenesis and regulate its activity. Moreover, it is noteworthy that the binding between the polyphenol and the target protein is determined by their structural relationship, which implies that different polyphenols have different target proteins, leading to divergent chemopreventive effects. Extracellular stimuli transmit signals into a cell by activating their target signaling cascades involved in carcinogenesis. As an example, Src family kinase, a family of proto-oncogenic tyrosine kinases activated by a variety of oxidative stress and proinflammatory agents, is known to regulate cell proliferation, differentiation, survival and angiogenesis. Src family kinase subsequently activates downstream signal cascades including mitogen-activated protein kinase, phosphoinositol-3-kinase, and nuclear factor-kappaB, thereby inducing cell proliferation and causing cancer. Recent studies demonstrate that polyphenols can directly target signaling cascades involved in inflammation and the development of cancer. Inhibition of the kinases by polyphenols contributes to the attenuation of carcinogenesis. Therefore, the development of polyphenols as direct inhibitors against target proteins is regarded as a rational approach for chemoprevention. This review describes and discusses recent results about the direct interactions of polyphenols and protein kinases in cancer chemoprevention.

Heat-killed lactic acid bacteria (LAB) are known to be important immunomodulators that stimulate tumor necrosis factor-α (TNF-α) and nitric oxide (NO) production as well as increase phagocytic activity in macrophages. NK cells play a critical role in innate immune response and induce spontaneous killing of tumor cells and virus-infected cells. However, the effect of heat-killed LAB on NK cells is still unclear. In this study, we investigated the effect of heat-killed Lactobacillus acidophilus La205 (La205) on NK cytolytic activity. We found that heat-killed La205 directly stimulated NK cytolytic activity in dose- and time-dependent manners. To determine the mechanism underlying heat-killed La205-enhanced NK cytotoxicity, the expression of NK activating receptors was tested. Heat-killed La205 did not affect the expression of NK activating receptors. To investigate whether NK degranulation is related to heat-killed La205-enhanced NK cytotoxicity, NK degranulation inhibitor concanamycin A (CMA) was used. CMA effectively blocked heat-killed La205-induced NK cytotoxicity, and an assay for detection of a degranulation marker, CD107a, showed that heat-killed La205 increased granule exocytosis approximately 2-fold in comparison to non-treated NK cells. In addition, heat-killed La205 dramatically elevated mRNA expression of granulysin, a component of the cytolytic granule contents, in NK cells. However, other granule contents, including perforin and granzymes, were not changed by heat-killed La205. From these data, we concluded that heat-killed La205 stimulated NK cytolytic activity through enhancement of granule exocytosis, and granulysin may be a critical mediator in heat-killed La205-induced granule exocytosis.

IL-32 was recently identified as a proinflammatory cytokine that is induced by IL-18 in natural killer (NK) cells and is highly correlated with inflammatory disorders. However, the relationship between IL-32 and tumor progression is still unknown. In this study, we investigated whether overexpression of IL-32 affects susceptibility of chronic myeloid leukemia (CML) cells to NK cells. Interestingly, IL-32α-overexpressing CML cell lines, K562, Kcl22, and BV173, showed higher NK cell-mediated killing. Flow cytometry analysis revealed that overexpression of IL-32α induced increased expression of Fas and UL16-binding protein 2 (ULBP2) in CML cells. The direct relationship between overexpression of surface molecules by IL-32α and increased NK cell-mediated killing was confirmed by Fas or ULBP2 siRNA transfection. IL-32α-induced Fas and ULBP2 expression are regulated p38 MAPK. In addition, the transcription factor Ets1 plays a key role in ULBP2 specific expression by IL-32α overexpression in ULBP family members. Taken together, these data show that IL-32α stimulates Fas and ULBP2 expression via activation of p38 MAPK, which increases NK susceptibility of CML cells. Enhanced NK cell susceptibility of CML cells by IL-32α overexpression may improve the efficiency of NK cell-based immunotherapy.

Nonmelanoma skin cancer is one of the most frequently occurring cancers in the United States. Chronic exposure to UVB irradiation is a major cause of this cancer. Daidzein, along with genistein, is a major isoflavone found in soybeans; however, little is known about the chemopreventive effects of daidzein and its metabolites in UVB-induced skin cancer. Here, we found that 7,3′,4′-trihydroxyisoflavone (THIF), a major metabolite of daidzein, effectively inhibits UVB-induced cyclooxygenase 2 (COX-2) expression through the inhibition of NF-κB transcription activity in mouse skin epidermal JB6 P+ cells. In contrast, daidzein had no effect on COX-2 expression levels. Data from Western blot and kinase assays showed that 7,3′,4′-THIF inhibited Cot and MKK4 activity, thereby suppressing UVB-induced phosphorylation of mitogen-activated protein kinases. Pull-down assays indicated that 7,3′,4′-THIF competed with ATP to inhibit Cot or MKK4 activity. Topical application of 7,3′,4′-THIF clearly suppressed the incidence and multiplicity of UVB-induced tumors in hairless mouse skin. Hairless mouse skin results also showed that 7,3′,4′-THIF inhibits Cot or MKK4 kinase activity directly, resulting in suppressed UVB-induced COX-2 expression. A docking study revealed that 7,3′,4′-THIF, but not daidzein, easily docked to the ATP binding site of Cot and MKK4, which is located between the N- and C-lobes of the kinase domain. Collectively, these results provide insight into the biological actions of 7,3′,4′-THIF, a potential skin cancer chemopreventive agent.

Kaempferol, a natural flavonoid isolated from various plant sources, has been identified as a potential neuroprotectant. In this study, we investigated the protective effect of kaempferol against 4-hydroxynonenal (HNE)-induced apoptosis in PC12 rat pheochromocytoma cells. Kaempferol inhibited 4-HNE-mediated apoptosis, characterized by nuclear condensation, down-regulation of antiapoptotic protein Bcl-2, and activation of proapoptotic caspase-3. Kaempferol inhibited 4-HNE-induced phosphorylation of c-Jun N-terminal protein kinase (JNK). More importantly, kaempferol directly bound p47 phox , a cytosolic subunit of NADPH oxidase (NOX), and significantly inhibited 4-HNE-induced activation of NOX. The antiapoptotic effects of kaempferol were replicated by the NOX inhibitor apocynin, suggesting that NOX is an important enzyme in its effects. Our results suggest that kaempferol attenuates 4-HNE-induced activation of JNK and apoptosis by binding p47 phox of NOX and potently inhibiting activation of the NOX-JNK signaling pathway in neuron-like cells. Altogether, these results suggest that kaempferol may be a potent prophylactic against NOX-mediated neurodegeneration.

Aspergillus sojae isolated from a traditional Korean fermented soybean product exhibited strong naringinase activity. The naringinase enzyme was purified and had a molecular weight of 70 kDa. The α- l -rhamnosidase activity of this enzyme was optimal at pH 6.0 and stable in the pH range of 5.5–8.0. The purified enzyme also had β- d -glucosidase activity, but the activity was relatively weak compared to the activity of the naringinase from Penicillium decumbens . The enzymatic bioconversion by A. sojae naringinase of naringin to prunin was efficiently performed with a 91% yield and a negligible amount of naringenin. The bioconversion was achieved by repetitive batch reactions with enzyme recycling. Prunin exhibited a markedly enhanced solubility compared to naringenin and naringin while maintaining the in vitro inhibition of HMG-CoA reductase. The results reported in this paper show that the naringinase produced by A. sojae will be useful in enhancing the potential bioavailability of naringin by efficiently converting it to prunin as a food component in citrus.

Numerous in vitro and in vivo studies have shown that isoflavones exhibit anti-proliferative activity against epidermal growth factor (EGF) receptor-positive malignancies of the breast, colon, skin, and prostate. 7,3',4'-Trihydroxyisoflavone (7,3',4'-THIF) is one of the metabolites of daidzein, a well known soy isoflavone, but its chemopreventive activity and the underlying molecular mechanisms are poorly understood. In this study, 7,3',4'-THIF prevented EGF-induced neoplastic transformation and proliferation of JB6 P+ mouse epidermal cells. It significantly blocked cell cycle progression of EGF-stimulated cells at the G(1) phase. As shown by Western blot, 7,3',4'-THIF suppressed the phosphorylation of retinoblastoma protein at Ser-795 and Ser-807/Ser-811, which are the specific sites of phosphorylation by cyclin-dependent kinase (CDK) 4. It also inhibited the expression of G(1) phase-regulatory proteins, including cyclin D1, CDK4, cyclin E, and CDK2. In addition to regulating the expression of cell cycle-regulatory proteins, 7,3',4'-THIF bound to CDK4 and CDK2 and strongly inhibited their kinase activities. It also bound to phosphatidylinositol 3-kinase (PI3K), strongly inhibiting its kinase activity and thereby suppressing the Akt/GSK-3beta/AP-1 pathway and subsequently attenuating the expression of cyclin D1. Collectively, these results suggest that CDKs and PI3K are the primary molecular targets of 7,3',4'-THIF in the suppression of EGF-induced cell proliferation. These insights into the biological actions of 7,3',4'-THIF provide a molecular basis for the possible development of new chemoprotective agents.

Kaempferol (KF), which is a natural dietary flavonoid, has potential beneficial effects as a chemopreventive agent for critical health conditions, such as cancer. However, the molecular mechanisms underlying the activity of KF remain unknown. We report on the inhibition of neoplastic cell transformation by KF through the suppression of phosphatidylinositol 3-kinase (PI3K) activity. Epidermal growth factor (EGF)-induced neoplastic transformation of mouse epidermal JB6 P+ cells was inhibited by 40 microM KF. The activation of activator protein-1 and nuclear factor-kappaB induced by EGF was also attenuated by KF. The EGF-induced phosphorylation of Akt (protein kinase B) was completely suppressed by KF, although extracellular signal-regulated kinase, p38, c-Jun N-terminal kinase and p90 ribosomal S6 kinase were unaffected by KF. Kinase assay data revealed that KF bound directly to PI3K, which is upstream of Akt, and suppressed its activity. Furthermore, KF inhibited ultraviolet B (UVB)-induced PI3K activity and attenuated UVB-induced phosphorylation of Akt. Our results suggest that KF docks at the adenosine triphosphate-binding site of PI3K, which is located between the N-lobe and C-lobe of the kinase domain. Inhibition by KF of PI3K, which is an important factor in carcinogenesis, and its downstream effects may explain the chemopreventive action of KF.

Chronic exposure to solar ultraviolet (UV) light causes skin photoaging. Many studies have shown that naturally occurring phytochemicals have anti-photoaging effects, but their direct target molecule(s) and mechanism(s) remain unclear. We found that myricetin, a major flavonoid in berries and red wine, inhibited wrinkle formation in mouse skin induced by chronic UVB irradiation (0.18J/cm(2), 3 days/week for 15 weeks). Myricetin treatment reduced UVB-induced epidermal thickening of mouse skin and also suppressed UVB-induced matrix metalloproteinase-9 (MMP-9) protein expression and enzyme activity. Myricetin appeared to exert its anti-aging effects by suppressing UVB-induced Raf kinase activity and subsequent attenuation of UVB-induced phosphorylation of MEK and ERK in mouse skin. In vitro and in vivo pull-down assays revealed that myricetin bound with Raf in an ATP-noncompetitive manner. Overall, these results indicate that myricetin exerts potent anti-photoaging activity by regulating MMP-9 expression through the suppression of Raf kinase activity.

Cruciferous vegetable consumption is associated with decreased risk of several cancers, including prostate cancer. Gluconasturtiin, one of the predominant glucosinolates in cruciferous vegetables, is hydrolyzed to yield phenylethyl isothiocyanate (PEITC). PEITC absorption and metabolism in humans involves glutathione conjugation followed by conversion via the mercapturic acid pathway to an N-acetylcysteine (NAC) conjugate that is excreted in the urine. We observed an inhibitory effect of PEITC and its metabolite, NAC-PEITC, on cancer cell proliferation, cell-cycle progression, and apoptosis in LNCaP human prostate cancer cells. PEITC and NAC-PEITC suppressed LNCaP cell proliferation in a dose-dependent manner, and exposure to 5 microM PEITC or NAC-PEITC reduced cell proliferation by 25% and 30%, respectively. Cell-cycle analysis revealed that cells treated with 5 microM PEITC or NAC-PEITC arrested at the G(2)/M phase. In addition, the percentage of cells in the S phase decreased from 46% to 25% following 48 h of incubation with PEITC or NAC-PEITC. The G(2)/M-phase cell-cycle arrest of LNCaP cells grown in the presence of PEITC or NAC-PEITC is correlated with the downregulation of Cdk1 and cyclin B(1) protein expression. Apoptosis was observed at the later stages of 24-h and 48-h treatments with 5 microM PEITC and NAC-PEITC. In conclusion, PEITC and NAC-PEITC are potential chemopreventive/chemotherapeutic agents against LNCaP human prostate cancer cells.

The present study was performed to evaluate the effects of two different phenolic antioxidants, resveratrol (3,5,4′-trihydroxystilbene) and butylated hydroxyanisole (BHA), on the hydrogen peroxide (H2O2)-induced inhibition of gap-junction intercellular communication (GJIC) in WB-F344 rat liver epithelial cells (WB-F344). Resveratrol is a naturally occurring polyphenolic antioxidant; on the other hand, BHA is a synthetic phenolic compound. We found that only resveratrol protects WB-F344 cells from H2O2-induced inhibition of GJIC, and BHA has no effect. The extracellular-signal-regulated protein kinase 1/2 (ERK1/2)–connexin 43 (Cx43) signaling pathway is crucial for the regulation of GJIC in rat liver epithelial cells, and resveratrol, but not BHA, blocked the H2O2-induced phosphorylation of Cx43, a critical regulator of GJIC, and ERK1/2 in WB-F344 cells. Resveratrol appears to attenuate the H2O2-mediated ERK1/2–Cx43 signaling pathway and consequently reverses H2O2-mediated inhibition of GJIC. DPPH and ABTS radical-scavenging assays revealed that the protective effect of resveratrol on the H2O2-mediated inhibition of GJIC was not mediated through its free radical-scavenging activity.

Aims Abnormal migration and proliferation of human aortic smooth muscle cells (HASMCs) to the intima causes intimal thickening of the aorta, which is strongly related to the development of atherosclerosis. Previous studies have suggested that red wine polyphenols, particularly resveratrol, have great protective effects against cardiovascular diseases. Here, we compared the anti-atherosclerotic effect of piceatannol, a metabolite of resveratrol, and its underlying mechanisms. Methods and results We demonstrated that piceatannol inhibited platelet-derived growth factor (PDGF)-BB-induced cell migration using a modified Boyden chamber assay and wound healing assay. Western blot analysis showed that PDGF-BB-induced phosphorylation of Akt, p70S6K, and p38 was inhibited by piceatannol, but not resveratrol. In vitro and ex vivo phosphoinositide 3-kinase (PI3K) assays demonstrated that piceatannol suppressed PI3K activity more effectively than resveratrol. PDGF-BB-induced migration and proliferation of HASMCs were inhibited by treatment with a commercial PI3K inhibitor, LY294002. Both in vitro and ex vivo pull-down assays revealed that piceatannol directly binds with sepharose 4B-PI3K beads in an ATP-competitive manner. Conclusion The results of the present study demonstrate that piceatannol directly binds with PI3K in an ATP-competitive manner and suppresses PI3K activity with anti-atherosclerotic effects.

Oxidative stress induced by reactive oxygen species (ROS) is strongly associated with the pathogenesis of various neurodegenerative disorders, including Alzheimer's disease. We investigated the possible combined effects of gallic acid and resveratrol, which are major antioxidants present in fruit, including grapes, on PC12 rat pheochromocytoma (PC12) cell death. Gallic acid did not protect against H(2)O(2)-induced PC12 cell death; it reduced the viability of PC12 cells in a dose-dependent manner. Gallic acid also induced cleavage of poly (ADP-ribose) polymerase, which is strongly related to apoptosis in neurons. Gallic acid induced the phosphorylation of c-Jun N-terminal protein kinase (JNK) and the downregulation of Bcl-2 in PC12 cells. Treatment of PC12 cells with resveratrol increased their viability in a dose-dependent manner by blocking the activation of JNK and the downregulation of Bcl-2. Furthermore, gallic acid led to a progressive reduction in the viability of vector-transfected PC12 cells, which was delayed in PC12 cells that overexpressed Bcl-2. The JNK inhibitor SP600125 protected against gallic acid-induced PC12 cell death. Collectively, these findings suggest that the combined effects of dietary phenolic phytochemicals on oxidative neuronal cell death and antioxidants differ in ROS-mediated neuronal cell death.

Tumor necrosis factor (TNF)-alpha-mediated cyclooxygenase (COX)-2 expression plays key roles in inflammation and tumorigenesis, particularly skin carcinogenesis, and hence targeting the TNF-alpha-mediated signaling pathway might be a promising strategy for developing chemopreventive agents against skin cancer and other skin disorders. Here we report that Fyn kinase - one of the members of the nonreceptor protein tyrosine kinase family - is involved in TNF-alpha-induced COX-2 expression, and that delphinidin - a major anthocyanidin present in red wine and berries - inhibits these effects by directly inhibiting Fyn kinase activity. Delphinidin strongly inhibited TNF-alpha-induced COX-2 expression in JB6 P+ mouse epidermal (JB6 P+) cells, whereas two other major phenolic compounds (resveratrol and gallic acid) did not exert significant inhibitory effects. Delphinidin inhibited the TNF-alpha-induced phosphorylations of JNK, p38 MAP kinase, Akt, p90RSK, MSK1, and ERK, and subsequently blocked the activation of the eukaryotic transcription factors AP-1 and NF-kappaB. Kinase and pull-down assay data revealed that delphinidin inhibited Fyn kinase activity and directly bound with Fyn kinase noncompetitively with ATP. By using PP2 (a commercial inhibitor of Fyn kinase) and siRNA-Fyn, we confirmed that Fyn kinase is involved in TNF-alpha-induced COX-2 expression mainly by down-regulating JNK in JB6 P+ cells. Together these findings suggest that the targeted inhibition of Fyn kinase activity and COX-2 expression by delphinidin contributes to the chemopreventive potential of red wine and berries.

Since reactive oxygen species (ROS) play a key role in carcinogenesis, many studies have focused on the chemopreventive activities of naturally occurring antioxidants. However, the possibility that different antioxidants in food exert opposing effects on carcinogenesis has not been adequately investigated. Gap-junction intercellular communication (GJIC), which is strongly related to carcinogenesis (particularly the tumor promotion stage), may be a suitable model for investigating the tumor-promoting and antitumor-promoting effects of phytochemicals. The present study investigated the possible combined effects of resveratrol and gallic acid (GA), which are major antioxidants in red wine, on GJIC in WB-F344 rat liver epithelial (RLE) cells. GA at 100 microM, but not resveratrol, inhibited GJIC and generated hydrogen peroxide. The GA-induced inhibition of GJIC was recovered by resveratrol, but only partially recovered by catalase. Resveratrol did not attenuate GA-induced generation of hydrogen peroxide, but it did block GA-induced phosphorylation of connexin 43 (Cx43), a key modulator of GJIC. Furthermore, resveratrol down-regulated GA-induced phosphorylation of extracellular signal-regulated kinase (ERK)1/2, one of the critical regulators of Cx43. However, catalase partially blocked the GA-induced phosphorylation of Cx43 and ERK1/2. Collectively, these findings suggest that the combined effects of red wine phenolic phytochemicals on GJIC and antioxidants differ in ROS-mediated carcinogenesis depending on their dosages and structures.

Neurodegenerative disorders such as Alzheimer's disease (AD) are strongly associated with oxidative stress, which is induced by reactive oxygen species (ROS) including hydrogen peroxide (H(2)O(2)). Recent studies suggest that moderate coffee consumption may reduce the risk of neurodegenerative diseases such as AD, but the molecular mechanisms underlying this effect remain to be clarified. In this study, we investigated the protective effects of chlorogenic acid (5-O-caffeoylquinic acid; CGA), a major phenolic phytochemical found in instant decaffeinated coffee (IDC), and IDC against oxidative PC12 neuronal cell death. IDC (1 and 5 microg/ml) or CGA (1 and 5 microM) attenuated H(2)O(2)-induced PC12 cell death. H(2)O(2)-induced nuclear condensation and DNA fragmentation were strongly inhibited by pretreatment with IDC or CGA. Pretreatment with IDC or CGA also inhibited the H(2)O(2)-induced cleavage of poly(ADP-ribose) polymerase (PARP), and downregulation of Bcl-X(L) and caspase-3. The accumulation of intracellular ROS in H(2)O(2)-treated PC12 cells was dose-dependently diminished by IDC or CGA. The activation of c-Jun N-terminal protein kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) by H(2)O(2) in PC12 cells was also inhibited by IDC or CGA. Collectively, these results indicate that IDC and CGA protect PC12 cells from H(2)O(2)-induced apoptosis by blocking the accumulation of intracellular ROS and the activation of MAPKs.

Although the health benefits of dietary antioxidants have been extensively studied, their potential negative effects remain unclear. L-Ascorbic acid 6-palmitate (AAP), a synthetic derivative of ascorbic acid (AA), is widely used as an antioxidant and preservative in foods, vitamins, drugs, and cosmetics. Previously, we found that AA exerted an antitumor effect by protecting inhibition of gap-junctional intercellular communication (GJIC), which is closely associated with tumor progression. In this study, we examined whether AAP, an amphipathic derivative of AA, has chemopreventive effects using a GJIC model. AAP and AA exhibited dose-dependent free radical-scavenging activities and inhibited hydrogen peroxide (H(2)O(2))-induced intracellular reactive oxygen species (ROS) production in normal rat liver epithelial cells. Unexpectedly, however, AAP did not protect against the inhibition of GJIC induced by H(2)O(2); instead, it inhibited GJIC synergistically with H(2)O(2). AAP inhibited GJIC in a dose-dependent and reversible manner. This inhibitory effect was not due to the conjugated lipid structure of AAP, as treatment with palmitic acid alone failed to inhibit GJIC under the same conditions. The inhibition of GJIC by AAP was restored in the presence of mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor U0126, but not in the presence of other signal inhibitors and antioxidant (PKC inhibitors, EGFR inhibitor, NADPH oxidase inhibitor, catalase, vitamin E, or AA), indicating the critical involvement of MEK signaling in the GJIC inhibitory activity of AAP. Phosphorylation of ERK and connexin 43 (Cx43) was observed following AAP treatment, and this was reversed by U0126. These results suggest that the AAP-induced inhibition of GJIC is mediated by the phosphorylation of Cx43 via activation of the MEK-ERK pathway. Taken together, our results indicate that AAP has a potent carcinogenic effect, and that the influence of dietary antioxidants on carcinogenesis may be paradoxical.

Recent studies suggest that anthocyanidins play a pivotal role in the chemopreventive effects of fruits and vegetables. However, the underlying molecular mechanisms and cellular targets remain unknown. Neoplastic transformation of cells and inflammation are considered to be major events contributing to carcinogenesis. Here, we report that delphinidin, a major dietary anthocyanidin, inhibits tumor promoter–induced transformation and cyclooxygenase-2 (COX-2) expression in JB6 promotion-sensitive mouse skin epidermal (JB6 P+) cells by directly targeting Raf and mitogen-activated protein kinase kinase (MEK). Delphinidin inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)–induced neoplastic transformation and COX-2 expression at both the protein and transcriptional levels. The activation of activator protein-1 and nuclear factor-κB induced by TPA was dose dependently inhibited by delphinidin treatment. Delphinidin strongly suppressed Raf1 and MEK1 kinase activities and subsequently attenuated TPA-induced phosphorylation of MEK, extracellular signal-regulated kinase (ERK), p90RSK, and MSK. Although delphinidin suppressed ERK and c-Jun NH2-terminal kinase activities, it was more effective at inhibiting Raf1 or MEK1 activities. Pull-down and competition assays revealed that delphinidin binds with Raf1 or MEK1 noncompetitively with ATP. Delphinidin also dose dependently suppressed JB6 P+ cell transformation induced by epidermal growth factor and H-Ras, both of which are involved in the activation of Raf/MEK/ERK signaling. Together, these findings suggested that the targeted inhibition of Raf1 and MEK activities and COX-2 expression by delphinidin contribute to the chemopreventive potential of fruits and vegetables.

There is mounting evidence implicating the accumulation of intracellular reactive oxygen species (ROS) and reactive nitrogen species (RNS) in the pathogenesis of neurodegenerative disorders, including Alzheimer's disease. Recently, considerable attention has been focused on identifying naturally occurring antioxidants that are able to reduce excess ROS and RNS, thereby protecting against oxidative stress and neuron death. The present study investigated the possible protective effects of piceatannol (trans-3,4,3',5'-tetrahydroxystilbene), which is present in grapes and other foods, on hydrogen-peroxide- and peroxynitrite-induced oxidative cell death. PC12 rat pheochromocytoma (PC12) cells treated with hydrogen peroxide or SIN-1 (a peroxynitrite-generating compound) exhibited apoptotic death, as determined by nucleus condensation and cleavage of poly(ADP-ribose)polymerase (PARP). Piceatannol treatment attenuated hydrogen-peroxide- and peroxynitrite-induced cytotoxicity, apoptotic features, PARP cleavage and intracellular ROS and RNS accumulation. Treatment of PC12 cells with hydrogen peroxide or SIN-1 led to down-regulation of Bcl-X(L) and activation of caspase-3 and -8, which were also inhibited by piceatannol treatment. Hydrogen peroxide or SIN-1 treatment induced phosphorylation of the c-Jun-N-terminal kinase (JNK), which was inhibited by piceatannol treatment. Moreover, SP600125 (a JNK inhibitor) significantly inhibited hydrogen-peroxide- and peroxynitrite-induced PC12 cell death, revealing inactivation of the JNK pathway as a possible molecular mechanism for the protective effects of piceatannol against hydrogen-peroxide- and peroxynitrite-induced apoptosis of PC12 cells. Collectively, these findings suggest that the protective effect of piceatannol against hydrogen-peroxide- and peroxynitrite-induced apoptosis of PC12 cells is associated with blocking the activation of JNK and the down-regulation of Bcl-XL.

Cocoa was shown to inhibit chemically induced carcinogenesis in animals and exert antioxidant activity in humans. However, the molecular mechanisms of the chemopreventive potential of cocoa and its active ingredient(s) remain unknown. Here we report that cocoa procyanidins inhibit neoplastic cell transformation by suppressing the kinase activity of mitogen-activated protein kinase kinase (MEK). A cocoa procyanidin fraction (CPF) and procyanidin B2 at 5 μg/ml and 40 μm, respectively, inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced neoplastic transformation of JB6 P+ mouse epidermal (JB6 P+) cells by 47 and 93%, respectively. The TPA-induced promoter activity and expression of cyclooxygenase-2, which is involved in tumor promotion and inflammation, were dose-dependently inhibited by CPF or procyanidin B2. The activation of activator protein-1 and nuclear factor-κB induced by TPA was also attenuated by CPF or procyanidin B2. The TPA-induced phosphorylation of MEK, extracellular signal-regulated kinase, and p90 ribosomal s6 kinase was suppressed by CPF or procyanidin B2. In vitro and ex vivo kinase assay data demonstrated that CPF or procyanidin B2 inhibited the kinase activity of MEK1 and directly bound with MEK1. CPF or procyanidin B2 suppressed JB6 P+ cell transformation induced by epidermal growth factor or H-Ras, both of which are known to be involved in MEK/ERK signal activation. In contrast, theobromine (up to 80 μm) had no effect on TPA-induced transformation, cyclooxygenase-2 expression, the transactivation of activator protein-1 or nuclear factor-κB, or MEK. Notably, procyanidin B2 exerted stronger inhibitory effects compared with PD098059 (a well known pharmacological inhibitor of MEK) on MEK1 activity and neoplastic cell transformation.

Oxidative stress induced by reactive oxygen species has been strongly associated with the pathogenesis of neurodegenerative disorders, including Alzheimer's disease. In this study, we investigated the possible protective effects of a cocoa procyanidin fraction (CPF) and procyanidin B2 (epicatechin-(4beta-8)-epicatechin) - a major polyphenol in cocoa - against apoptosis of PC12 rat pheochromocytoma (PC12) cells induced by hydrogen peroxide (H(2)O(2)). CPF (1 and 5 microg/ml) and procyanidin B2 (1 and 5 microM) reduced PC12 cell death caused by H(2)O(2), as determined by MTT and trypan blue exclusion assays. CPF and procyanidin B2 attenuated the H(2)O(2)-induced fragmentation of nucleus and DNA in PC12 cells. Western blot data demonstrated that H(2)O(2) induced cleavage of poly(ADP-ribose)polymerase (PARP), downregulated Bcl-X(L) and Bcl-2 in PC12 cells. Pretreatment with CPF or procyanidin B2 before H(2)O(2) treatment diminished PARP cleavage and increased Bcl-X(L) and Bcl-2 expression compared with those only treated with H(2)O(2). Activation of caspase-3 by H(2)O(2) was inhibited by pretreatment with CPF or procyanidin B2. Furthermore, H(2)O(2)-induced rapid and significant phosphorylation of c-Jun N-terminal protein kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), and both of these effects were attenuated by CPF or procyanidin B2 treatment. These results suggest that the protective effects of CPF and procyanidin B2 against H(2)O(2)-induced apoptosis involve inhibiting the downregulation of Bcl-X(L) and Bcl-2 expression through blocking the activation of JNK and p38 MAPK.

Propyl gallate and its metabolite, gallic acid, are widely used as antioxidants in the food industry, but they have been shown to exhibit liver toxicity and enhance carcinogenesis. In the present study, we investigated the possible undesirable effects of propyl gallate and gallic acid on gap junctional intercellular communication (GJIC), inhibition of which is closely linked to carcinogenesis. Gallic acid and propyl gallate exhibited dose-dependent free-radical-scavenging activities as determined by 1,1-diphenyl-2-picrylhydrazyl- or 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)-radical-scavenging assays, and the free-radical-scavenging activity of gallic acid was stronger than that of propyl gallate. However, using WB-F344 rat liver epithelial cells, gallic acid inhibited GJIC in a dose-dependent manner, while propyl gallate had no significant effect compared with untreated controls. The gallic-acid-induced inhibition of GJIC was reversible, with a recovery of nearly 65% after 120 min. Gallic acid induced the phosphorylation of connexin 43 (Cx43) and phosphorylation of extracellular-signal-regulated kinase1/2 (ERK1/2). The gallic-acid-induced inhibition of GJIC was attenuated by treatment with mitogen-activated protein kinase kinase inhibitors (U0126 and PD098059). U0126 blocked the gallic-acid-induced phosphorylation of Cx43 and ERK1/2, indicating that the gallic-acid-induced inhibition of GJIC is mediated by phosphorylation of Cx43 via activation of ERK1/2. In addition, gallic-acid-induced inhibition of GJIC was protected by ascorbic acid and quercetin, which might represent a simple example of the different effects of natural antioxidants in carcinogenesis.

Background: Ginseng (Panax ginseng C.A. Meyer) has been used as a traditional herb in the treatment of many medical disorders. Ginsenosides, which are triterpene derivatives that contain sugar moieties, are the main pharmacological ingredients in ginseng. This study was designed to investigate the effect of ginsenoside Rg3-enriched ginseng extract (Rg3GE) on scopolamine-induced memory impairment in mice. Methods: Rg3GE (50 and 100 mg/kg) were administered to C57BL/6 mice by oral gavage for 14 days (days 1-14). Memory impairment was induced by scopolamine (1 mg/kg, intraperitoneal injection) for 6 days (days 914). The Morris water maze test was used to assess hippocampus-dependent spatial memory. The effects of scopolamine with or without Rg3GE on acetylcholinesterase and nuclear factor-kappa B (NF-kappa B) in the hippocampus were also examined. Results: Mice with scopolamine treatment alone showed impairments in the acquisition and retention of spatial memory. Mice that received Rg3GE and scopolamine showed no scopolamine-induced impairment in the acquisition of spatial memory. Oral administration of Rg3GE suppressed the scopolamine-mediated increase in acetylcholinesterase activity and stimulation of the NF-kappa B pathway (i.e., phosphorylation of p65) in the hippocampus. Conclusion: These findings suggest that Rg3GE may stabilize scopolamine-induced memory deficits through the inhibition of acetylcholinesterase activity and NF-kappa B signaling in the hippocampus.

Numerous studies have shown that the levels of matrix metalloproteinase (MMP)-2 and/or MMP-9 are associated with the invasive phenotypes of cancer cells. This study investigated the effects of caffeic acid phenethyl ester (CAPE), a chemopreventive phytochemical derived from honeybee propolis, on the invasive phenotype of SK-Hep1 human hepatocellular carcinoma cells (SK-Hep1 cells). CAPE effectively suppressed SK-Hep1 cell invasion in a dose-dependent manner. The constitutive expression of MMP-2 and MMP-9 in SK-Hep1 cells was almost completely abolished by treatment with 12.5 muM CAPE. CAPE also significantly inhibited nuclear factor kappa B (NF-kappaB) DNA-binding activity in SK-Hep1 cells. These results taken together suggest that CAPE exerts antimetastatic potential through inhibition of MMP-2 and MMP-9 expression, possibly by targeting NF-kappaB in hepatocellular carcinoma.

Daidzein and genistein are isoflavones found in soybean. Genistein is known to exhibit anticarcinogenic activities and inhibit tyrosine kinase activity. However, the underlying molecular mechanisms of the chemopreventive activities of daidzein and its metabolite, equol, are not understood. Here we report that equol inhibits 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced neoplastic transformation of JB6 P+ mouse epidermal cells by targeting the MEK/ERK/p90RSK/activator protein-1 signaling pathway. TPA-induced neoplastic cell transformation was inhibited by equol, but not daidzein, at noncytotoxic concentrations in a dose-dependent manner. Equol dose-dependently attenuated TPA-induced activation of activator protein-1 and c-fos, whereas daidzein did not exert any effect when tested at the same concentrations. The TPA-induced phosphorylation of ERK1/2, p90RSK, and Elk, but not MEK or c-Jun N-terminal kinase, was inhibited by equol but not by daidzein. In vitro kinase assays revealed that equol greatly inhibited MEK1, but not Raf1, kinase activity, and an ex vivo kinase assay also demonstrated that equol suppressed TPA-induced MEK1 kinase activity in JB6 P+ cell lysates. Equol dose-dependently inhibited neoplastic transformation of JB6 P+ cells induced by epidermal growth factor or H-Ras. Both in vitro and ex vivo pull-down assays revealed that equol directly bound with glutathione S-transferase-MEK1 to inhibit MEK1 activity without competing with ATP. These results suggested that the antitumor-promoting effect of equol is due to the inhibition of cell transformation mainly by targeting a MEK signaling pathway. These findings are the first to reveal a molecular basis for the anticancer action of equol and may partially account for the reported chemopreventive effects of soybean.

Aim: To provide evidence that the production of bacteriocin by lactic acid bacteria can be enhanced by the presence of a bacteriocin-sensitive strain and identify the agent that is responsible for enhancing bacteriocin production. Methods and Results: One bacteriocin-producing lactic acid bacterium was isolated from kimchi. The strain GJ7 was designated as Leuconostoc citreum GJ7 based on Gram staining, biochemical properties, and 16S rRNA gene sequencing. The isolate produced a heat- and pH-stable bacteriocin (kimchicin GJ7), which has antagonistic activity against a broad spectrum of micro-organisms. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified kimchicin GJ7 showed a single band of molecular weight c. 3500 Da. Cultures of Leuc. citreum GJ7 in the presence of thermally inactivated kimchicin GJ7-sensitive strains, Lactobacillus plantarum KFRI 464, Lactobacillus delbrueckii KFRI 347, or Leuconostoc mesenteroides KCTC 1628, increased bacteriocin production. This inducing factor was characterized and purified from Lact. plantarum KFRI 464, which showed the greatest enhancement of kimchicin GJ7 activity. The inducing factor was purified using a DEAE (diethyl aminoethyl)-Sephacel column and high-performance liquid chromatography, and yielded a single band of c. 6500 Da. N-terminal sequencing of the inducing factor identified 16 amino acids. The N-terminal sequence of the inducing factor was synthesized and examined for the induction of kimchicin GJ7 activity, and was found to induce activity, but at a level about 10% lower than that of the entire molecule. Conclusions: The presence of a bacteriocin-sensitive strain, Lact. plantarum KFRI 464, acts as an environmental stimulus to activate the production of kimchicin GJ7 by Leuc. citreum GJ7. The inducing factor from Lact. plantarum KFRI 464 is highly homologous to the 30S ribosomal protein S16 from various micro-organisms. The N-terminal sequence of the inducing factor examined in this study is a very important sequence related to the inducing activity. Nevertheless, the inducing factor may not be part of the ribosomal protein S16 itself. Significance and Impact of the Study: We believe that the present study is the first to identify an agent that is produced by one micro-organism and influences bacteriocin production in another. The bacteriocin-enhancing system described in this study could be effectively used to control the growth of other micro-organisms (sensitive cells) in food systems. Moreover, this enhancement of bacteriocin production can be applied usefully in industrial production of natural food preservatives.

Evidence suggests that mitogen-activated protein kinase kinase (MEK) plays a role in cell transformation and tumor development and might be a significant target for chemoprevention. 3,5,4'-Trihydroxy-trans-stilbene (resveratrol), a non-flavonoid polyphenol found in various foods and beverages, including red wines, is reported to be a natural chemopreventive agent. However, the concentrations required to exert these effects might be difficult to achieve by drinking only one or two glasses of red wine a day. On the other hand, the flavonol content of red wine is approximately 30 times higher than that of resveratrol. Here we demonstrated that 3,3',4',5,5',7-hexahydroxyflavone (myricetin), one of the major flavonols in red wine, is a novel inhibitor of MEK1 activity and transformation of JB6 P+ mouse epidermal cells. Myricetin (10 microM) inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA) or epidermal growth factor (EGF)-induced cell transformation by 76 or 72%, respectively, compared with respective reductions of 26 or 19% by resveratrol (20 microM). A combination of myricetin and resveratrol exerted additive but not synergistic effects on either TPA- or EGF-induced transformation. Myricetin, but not resveratrol, attenuated tumor promoter-induced activation of c-fos or activator protein-1. Myricetin strongly inhibited MEK1 kinase activity and suppressed TPA- or EGF-induced phosphorylation of extracellular signal-regulated kinase (ERK) or p90 ribosomal S6 kinase, downstream targets of MEK. Moreover, myricetin inhibited H-Ras-induced cell transformation more effectively than either PD098059, a MEK inhibitor, or resveratrol. Myricetin directly bound with glutathione S-transferase-MEK1 but did not compete with ATP. Overall, these results indicated that myricetin has potent anticancer-promoting activity and mainly targets MEK signaling, which may contribute to the chemopreventive potential of several foods including red wines.

A 4661 bp cryptic plasmid, pFMBL1, was isolated from Leuconostoc mesenteroides SY2, an isolate from Kimchi, and characterized. Nucleotide sequence analysis revealed two open reading frames, orf1 and orf2. orf2 was 453 bp in size and its translation product had 58% identity with a putative protein possibly involved in the replication of pTXL1, a cryptic plasmid from L. mesenteroides ssp. mesenteroides Y110. RNA transcript from orf2 was detected but not from orf1 or intergenic region. Minimum 3.5 kb fragment encompassing orf1 and orf2 was required for the replication of pFMBL1 and employed for the construction of Escherichia coli-Leuconostoc shuttle vector, pSJ33E. L. mesenteroides SY1 (another Kimchi isolate), Leuconostoc ssp., and Lactobacillus brevis were successfully transformed with pSJ33E, and the transformation efficiencies were ranged between 1.1 × 101 and 4 × 105 transformants/μg DNA. No single-stranded DNA intermediate was detected from L. mesenteroides SY1 cells harboring pSJ33E, indicating that pFMBL1 probably replicated via θ-type mechanism. pSJ33E was stably maintained in L. mesenteroides SY1 in the absence of erythromycin (Em, 5 μg/ml) and after 1 month of daily subculturing in MRS broth without selective pressure, three percent of cells still retained pSJ33E.

Abnormal upregulation of cyclooxygenase-2 (COX-2) has been frequently observed in various types of transformed and cancerous cells. Numerous anti-inflammatory agents have been shown to exert chemopreventive effects by targeting COX-2, a rate-limiting enzyme involved in the inflammatory process. Anthocyanins are naturally occurring polyphenolic compounds that endow various fruits, vegetables, and plants with intense colors. Peonidin is another representative anthocyanidin, but its chemopreventive potential has not been fully described. This article investigated the effect of peonidin on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced COX-2 expression and transformation in JB6 P(+) mouse epidermal cells (JB6 P(+) cells). Treatment of JB6 P(+) cells with peonidin inhibited TPA-induced COX-2 expression, and also decreased TPA-induced neoplastic transformation and blocked TPA-induced phosphorylation of extracellular signal-regulated kinases (ERKs) in the cells. The inhibition of the signaling mechanism regulating the activation of ERKs strongly suggests that peonidin exhibits chemopreventive as well as anti-inflammatory activities.

Elevated levels of cyclooxygenase-2 (COX-2) and matrix metalloproteinases (MMPs) are frequently found in various types of cancerous and transformed cells, with recent studies implicating the upregulation of COX-2 and MMPs in the development of breast cancer. This article investigated the effects of jaceosidin (4',5,7-trihydroxy-3',6-dimethoxyflavone) isolated from Artemisia argyi on the upregulation of COX-2 and MMP-9 induced by the tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA) in MCF10A human breast epithelial cells (MCF10A cells). Treatment of MCF10A cells with TPA induced the upregulation of COX-2 and MMP-9, and this was attenuated by jaceosidin treatment. Jaceosidin also inhibited the invasive and migrative phenotypes of MCF10A cells induced by TPA. Furthermore, jaceosidin blocked the TPA-induced phosphorylation of extracellular signal-regulated protein kinase-1 and -2 (ERK-1 and -2), which is one of the signaling molecules regulating COX-2 and MMP. These results suggest that jaceosidin inhibits the TPA-induced upregulation of COX-2 and MMP-9 by blocking ERK-1 and -2 phosphorylation in human breast epithelial cells, which may be indicative of its chemopreventive potential.

An improved liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the determination of acrylamide in processed foods. The homogenized samples, spiked with 13 C 3 -acrylamide as an internal standard, were extracted with water and centrifuged. D 5 -3-chloropropanediol as a recovery standard was added to 1-ml aliquots, and the sample was purified with a C 18 -cartridge column. The extract was directly analyzed using LC-MS/MS without derivatization. The ion transitions of 72–55 m / z (acrylamide), 75–58 m / z ( 13 C 3 -acrylamide), and 116–98 m / z (d 5 -3-chloropropanediol) were found to be the most reliable for the identification and quantification of acrylamide in multiple reaction monitoring. The limit of quantification for acrylamide, defined as a signal-to-noise ratio of 10:1, was 2 μg/kg. The use of d 5 -3-chloropropanediol minimized the effects of variation in the sample matrixes and increased the quality of analysis. This method could be applied to the quantification of acrylamide in processed foods.

Cruciferous vegetables contain a series of relatively unique secondary metabolites of amino acids, called glucosinolates. Sinigrin, the predominant aliphatic glucosinolate in cruciferous vegetables, is hydrolyzed to yield allyl isothiocyanate (AITC), which, after absorption and metabolism in humans, is excreted in the urine as an N-acetyicysteine (NAC) conjugate. We have determined the inhibitory effects of AITC and its NAC conjugate on cell proliferation, the expression of matrix metalloproteinases (MMPs), adhesion, invasion, and migration in SK-Hep1 human hepatoma cells. Our results demonstrate that AITC and NAC-AITC suppress SK-Hep1 cell proliferation in a dose-dependent manner; by 25% and 30% for 10 μM AITC and 10 μM NAC-AITC, respectively. We examined the influence of AITC and NAC-AITC on the gene expression of MMPs and tissue inhibitors of metalloproteinase (TIMPs). Gelatin zymography also revealed a significant downregulation of MMP-2/-9 expression in SK-Hep1 cells treated with 0.1–5 μM AITC and NAC-AITC compared with controls. Reverse transcriptase polymerase chain reaction revealed dose-dependent decreases in MMP-2/-9 messenger RNA levels in both AITC-treated and NAC-AITC–treated cells. TIMP-1/-2 activities were unaffected by treatment with AITC or NAC-AITC in our experiments. NAC-AITC inhibited cancer cell adhesion and invasion much more potently than its parent compound. NAC-AITC at 5 μM caused excellent inhibition of cell migration for 48 hrs. These results demonstrate the potential of AITC and NAC-AITC as chemopreventive agents.

Lycopene, which is the predominant carotenoid in tomatoes and tomato-based foods, may protect humans against various cancers. Effects of lycopene on the adhesion, invasion, migration, and growth of the SK-Hep1 human hepatoma cell line were investigated. Lycopene inhibited cell growth in dose-dependent manners, with growth inhibition rates of 5% and 40% at 0.1 microM and 50 microM lycopene, respectively, after 24 hrs of incubation. Similarly, after 48 hrs of incubation, lycopene at 5 microM and 10 microM decreased the cell numbers by 30% and 40%, respectively. Lycopene decreased the gelatinolytic activities of both matrix metalloproteinase (MMP)-2 and MMP-9, which were secreted from the SK-Hep1 cells. Incubation of SK-Hep1 cells with 110 microM of lycopene for 60 mins significantly inhibited cell adhesion to the Matrigel-coated substrate in a concentration-dependent manner. To study invasion, SK-Hep1 cells were grown either on Matrigel-coated Transwell membranes or in 24-well plates. The cells were treated sequentially for 24 hrs with lycopene before the start of the invasion assays. Cell growth and death were assessed under the same conditions. The invasion of SK-Hep1 cells treated with lycopene was significantly reduced to 28.3% and 61.9% of the control levels at 5 microM and 10 microM lycopene, respectively (P < 0.05). In the migration assay, lycopene-treated cells showed lower levels of migration than untreated cells. These results demonstrate the antimetastatic properties of lycopene in inhibiting the adhesion, invasion, and migration of SK-Hep1 human hepatoma cells.

One of the most frequent events in carcinogenesis is uncontrolled activation of Ras signaling pathway. A previous study demonstrated that the introduction of H-Ras into the normal WB-F344 rat liver epithelial (WB) cell line and adult male F344 rats resulted in tumorigenicity. The present study investigated whether H-Ras induced the invasive and migrative phenotypes in WB cells, and subsequently aimed at characterizing the underlying mechanisms. H-Ras induced the invasive and migrative phenotypes of WB cells with a selective up-regulation of matrix metalloproteinase (MMP)-9, but not MMP-2. Cyclooxygenase (COX)-2 and the subsequent production of prostaglandin E2 (PGE2) were also induced by H-Ras. Treatment of H-Ras WB cells with GM6001 and NS398, the inhibitors of MMPs and COX-2, respectively, significantly inhibited the H-Ras-induced invasive and migrative phenotypes. DNA binding activity of nuclear factor (NF)-kappaB, but not that of activator protein (AP)-1, was increased by H-Ras. Caffeic acid phenethyl ester and Bay 11-7082, specific inhibitors of NF-kappaB and IKK, respectively, significantly inhibited the expression of MMP-9 and COX-2, invasion and migration of H-Ras WB cells, revealing NF-kappaB as a transcriptional factor responsible for H-Ras-induced malignant phenotypic conversion of WB cells. Activation of ERKs pathway was critical for H-Ras-induced invasive and migrative phenotypes, up-regulation of MMP-9 and COX-2 as well as enhanced DNA binding activity of NF-kappaB in WB cells. Taken together, these results demonstrate that H-Ras up-regulates MMP-9 and COX-2 through activation of ERKs and IKK-IkappaBalpha-NF-kappaB signal pathway which may contribute to the malignant progression of WB rat liver epithelial cells.

ylamide content in ylamide content in ylamide content in ylamide content in processed foods and apply them to commercial snacks. The formation of acrylamide in fried foods was found to processed foods and apply them to commercial snacks. The formation of acrylamide in fried foods was found to processed foods and apply them to commercial snacks. The formation of acrylamide in fried foods was found to processed foods and apply them to commercial snacks. The formation of acrylamide in fried foods was found to processed foods and apply them to commercial snacks. The formation of acrylamide in fried foods was found to depend on the composition of raw materials as well as the frying time and temperature. In potato chips, acrylamide depend on the composition of raw materials as well as the frying time and temperature. In potato chips, acrylamide depend on the composition of raw materials as well as the frying time and temperature. In potato chips, acrylamide depend on the composition of raw materials as well as the frying time and temperature. In potato chips, acrylamide depend on the composition of raw materials as well as the frying time and temperature. In potato chips, acrylamide was rapidly formed at over 160 °C, with the amount proportional to the heating duration and temperature. Free was rapidly formed at over 160 °C, with the amount proportional to the heating duration and temperature. Free was rapidly formed at over 160 °C, with the amount proportional to the heating duration and temperature. Free was rapidly formed at over 160 °C, with the amount proportional to the heating duration and temperature. Free was rapidly formed at over 160 °C, with the amount proportional to the heating duration and temperature. Free amino acids were used to reduce acrylamide, with lysine, glycine, and cysteine having the greatest effects in the amino acids were used to reduce acrylamide, with lysine, glycine, and cysteine having the greatest effects in the amino acids were used to reduce acrylamide, with lysine, glycine, and cysteine having the greatest effects in the amino acids were used to reduce acrylamide, with lysine, glycine, and cysteine having the greatest effects in the amino acids were used to reduce acrylamide, with lysine, glycine, and cysteine having the greatest effects in the aqueous system. L aqueous system. L aqueous system. L aqueous system. L aqueous system. 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Soaking potato In potato snacks, the addition of 0.5% glycine to pallets reduced acrylamide by more than 70%. Soaking potato slices in a 3% solution of either lysine or glycine reduced the formation of acrylamide by more than 80% in potato slices in a 3% solution of either lysine or glycine reduced the formation of acrylamide by more than 80% in potato slices in a 3% solution of either lysine or glycine reduced the formation of acrylamide by more than 80% in potato slices in a 3% solution of either lysine or glycine reduced the formation of acrylamide by more than 80% in potato slices in a 3% solution of either lysine or glycine reduced the formation of acrylamide by more than 80% in potato chips fried for 1.5 min at 185 °C. These results indicate that the addition of certain amino acids by soaking the chips fried for 1.5 min at 185 °C. These results indicate that the addition of certain amino acids by soaking the chips fried for 1.5 min at 185 °C. These results indicate that the addition of certain amino acids by soaking the chips fried for 1.5 min at 185 °C. These results indicate that the addition of certain amino acids by soaking the chips fried for 1.5 min at 185 °C. These results indicate that the addition of certain amino acids by soaking the uncooked products in appropriate solutions is an effective way of reducing acrylamide in processed foods. uncooked products in appropriate solutions is an effective way of reducing acrylamide in processed foods. uncooked products in appropriate solutions is an effective way of reducing acrylamide in processed foods. uncooked products in appropriate solutions is an effective way of reducing acrylamide in processed foods.

A bifunctional alcohol/acetaldehyde dehydrogenase (AdhE) gene (adhE) was cloned from Leuconostoc mesenteroides C7 (LMC7), which is the dominant lactic acid bacterium produced during heterofermentation of kimchi. The nucleotide sequence of the DNA fragment containing putative adhE, which is 2685 bp long and encodes an 886 amino acid polypeptide, exhibits 99% homology with Leu. mesenteroides sp. cremoris. The deduced AdhE comprises two conserved domains: alcohol dehydrogenase (Adh) and acetaldehyde dehydrogenase (Aldh). Moreover, two NAD-binding sites were observed, based on the presence of the GXGXXG motif. A pADHE containing the adhE gene expressed AdhE at the translational level in Escherichia coli BL21, which was at a higher level than in E. coli DH5alpha and E. coli JM109. The AdhE of LMC7 showed Adh and Aldh activities that, when expressed in E. coli. BL21, were 7.5 and 5.7 U mg(-1) , respectively.

There are multiple lines of evidence that a variety of natural fatty acids are effective in health promotion. Among these fatty acids, conjugated linoleic acid (CLA)--a collective term referring to a mixture of positional and geometric isomers of linoleic acid (LA, cis-9, cis-12-octadecadienoic acid)--is currently under intensive investigation due to its health-promotion potential. The antitumor activity of CLA is of special interest, since it shows inhibitory effects against multistage carcinogenesis at relatively low dietary levels. Many studies using in vivo and in vitro models have shown that CLA suppresses the development of multistage carcinogenesis at different sites. The research to date on CLA has provided a vast amount of information about the mechanism on how CLA functions in the prevention of cancer. This article discusses characteristics of CLA in the prevention of cancer in both in vivo and in vitro studies and the possible underlying chemoprevention mechanisms.

The utilization of cacao bean husk (CBH), a by-product of chocolate manufacture, would be both environmentally and economically beneficial. For this purpose, a process for effectively separating and fractionating CBH fractions having cancer preventive potential was developed in this study. For screening the fractions with potent cancer preventive activity, we examined the effect of extracts and fractions of CBH on the inhibition of gap-junction intercellular communication (GJIC) and the DNA synthesis of cancer cells, both of which are characteristics of the promotion and progression stages in carcinogenesis. The extracts of CBH (especially, the 60% ethanol fraction after extraction with 50% acetone) containing 43 wt.% polyphenol exerted an excellent protective effect on H 2 O 2 -induced inhibition of GJIC in WB-F344 rat liver epithelial cells as determined by the scrape-loading/dye transfer assay. The enhancement of GJIC by the extracts of CBH was approximately 10-fold higher than that of a well-known dietary chemopreventive component, vitamin C. The extracts of CBH (especially, the 60% ethanol fraction) also suppressed DNA synthesis in all liver, stomach, and colon cancer cells as demonstrated by the 3 H-thymidine incorporation assay, by approximately four-fold higher than that of vitamin C. These results imply that the polyphenol extracts and fractions of CBH are effective functional materials to be used in either preventing or inhibiting cancer.

One of the most frequent defects in human cancers is the uncontrolled activation of the ras signaling pathways. Increased expression of cyclooxygenase-2 (COX-2) and inhibition of gap junction intercellular communication (GJIC) have been frequently observed in several forms of human malignancies. The present study investigated the effects of caffeic acid phenethyl ester (CAPE), a chemopreventive phytochemical derived from honey propolis, on COX-2 expression and GJIC in Harvey-ras-transformed WB-F344 rat liver epithelial cells (H-ras WB cells). H-ras induced COX-2 expression in WB-F344 rat liver epithelial cells (WB cells). H-ras WB cells also exhibited complete inhibition of GJIC and predominant unphosphorylation of connexin 43 (Cx43), a major protein modulating GJIC. CAPE significantly inhibited the constitutive expression of COX-2 and restored the disrupted GJIC through the phosphorylation of Cx43 at a concentration of 12.5 microM in H-ras WB cells. Although the molecular basis for the cancer chemopreventive activity of CAPE is not completely understood, several studies suggest that CAPE is a potent and specific inhibitor of the transcription factor nuclear factor kappaB (NF-kappaB) activation. We also found that CAPE significantly inhibited H-ras-induced NF-kappaB DNA-binding activity without affecting the activation of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase, which are major intracellular molecules involved in the Ras signaling pathways. In conclusion, CAPE may exert cancer chemopreventive effects through the inhibition of COX-2 expression and the restoration of disrupted GJIC induced by H-ras, possibly by targeting NF-kappaB.

The re-utilization of cacao bean husk, a waste generated from the chocolate industry, would bring benefits both environmentaly and economicaly. This study relates to a process for effectively separating and fractionating a cacao bean husk fraction having high inhibitory activity against glucosyltransferase (GTF) for the prevention of tooth decay (anticaries activity). Since the GTF inhibitory activity is known to be rendered by polyphenols, the separation process was also able to aim at high recovery of polyphenols which benefits human health. In this study, cacao bean husk extract, obtained under optimal conditions, extraction with 50% (v/v) aq. (aqueous) acetone solution at 60 °C for 4 h followed by 50% (v/v) aq. ethanol using a styrene-based resin, showed significantly higher inhibitory activity (2 and 12 folds, respectively) against GTF and a similar polyphenol content, compared to two other commercial anti-GTF polyphenol products.

A cryptic plasmid pC7 was characterized, which was isolated from Lactobacillus paraplantarum C7 derived from Kimchi, a traditional korean vegetable food. The plasmid pC7 is a circular molecule of 2,134 base-pairs in length with a G + C content of 38.5%. The nucleotide sequence analysis revealed the presence of an open reading frame encoding a putative 317 amino acids protein homologous to replication proteins RepA. Furthermore, a putative double and a single-strand origin were identified. Together with features of nucleotide sequences, the detection of single-stranded intermediate DNA in Lb. paraplantarum demonstrated that pC7 replicates via a rolling circle mechanism. A cloning vector for lactic acid bacteria was developed on the basis of the pC7 replicon, into which were inserted an erythromycin resistance gene as a marker, multiple cloning sites, and Escherichia coli ColE1 replication origin. E. coli and several species of Lactobacillus and Leuconostoc can be transformed with the resultant vector. Therefore, pC7 derivatives may be useful LAB–E. coli shuttle vectors, which are essential in engineering important strains in food fermentation.