Effect of diallyl sulfide on in vitro and in vivo Nrf2-mediated pulmonic antioxidant enzyme expression via activation ERK/p38 signaling pathway

Increasing oxidative stress is intimately involved in the pathogenesis of lung failure. Nuclear factor-erythroid 2 related factor 2 (Nrf2) is a key element in redox homeostasis. Nrf2 regulates antioxidant-associated genes that are often the target of phytochemicals in chemoprevention. This study evaluated the effect of diallyl sulfide (DAS), which is present in garlic, on the expression of antioxidant enzymes in the rat lung and the Nrf2 modulation in MRC-5 lung cells. DAS increased the activities of glutathione S-transferase, glutathione reductase, and catalase as well as the GSH/GSSG ratio compared with the lung of untreated control rats (p < 0.05). The pulmonic superoxide dismutase, glutathione peroxidase, NAD(P)H:quinone oxidoreductase 1, and catalase mRNA levels were also significantly increased (p < 0.05) after DAS treatment. Following DAS treatment, DAS level was measured in the plasma after 7 days of oral administration, and the C(max) value was 15 ± 4.2 μM. The total amount of pulmonic Nrf2 and the nuclear translocation of Nrf2 were elevated in DAS-treated rats, clarifying the effect of DAS on the modulation of antioxidant enzymes. Furthermore, DAS could induce nuclear translocation of Nrf2 via ERK/p38 signaling pathway in lung MRC-5 cells. This study demonstrates that DAS administration can significantly induce the activity of antioxidant enzymes in rat lungs and suggests a possible use for DAS as a dietary preventive agent against oxidative stress-induced lung injury.     

Resveratrol upregulates nrf2 expression to attenuate methylglyoxal-induced insulin resistance in hep g2 cells

Oxidative stress can result in insulin resistance, a primary cause of type-2 diabetes. Methylglyoxal (MG), a highly reactive dicarbonyl metabolite generated during glucose metabolism, has also been confirmed to cause pancreatic injury and induce inflammation, thereby resulting in insulin resistance. Recently, resveratrol has been reported to exert antioxidant properties, protecting cells from the generation of reactive oxygen species (ROS). The aim of this study was to evaluate resveratrol activation of nuclear factor erythroid 2-related factor 2 (Nrf2) to attenuate MG-induced insulin resistance in Hep G2 cells. Therefore, the molecular signaling events affecting resveratrol-mediated heme oxygenase-1 (HO-1) and glyoxalase expression levels were further investigated in this study. Our findings indicated that resveratrol activated the extracellular signal-regulated kinase (ERK) pathway but not the p38 or c-Jun N-terminal kinase (JNK) pathways, subsequently leading to Nrf2 nuclear translocation and elevation of HO-1 and glyoxalase expression levels. Moreover, resveratrol significantly elevated glucose uptake and protected against MG-induced insulin resistance in Hep G2 cells. In contrast, depletion of Nrf2 by small interfering RNA (si-RNA) resulted in the abrogation of HO-1 and glyoxalase expression in the MG-treated resveratrol group in Hep G2 cells. Administration of an appropriate chemopreventive agent, such as resveratrol, may be an alternative strategy for protecting against MG-induced diabetes.     

Diallyl disulfide and diallyl trisulfide up-regulate the expression of the pi class of glutathione S-transferase via an AP-1-dependent pathway

Garlic organosulfur compounds are recognized as potential chemopreventive compounds. This protection is related to the induction of phase II detoxification enzymes. We previously reported that diallyl disulfide (DADS) and diallyl trisulfide (DATS) up-regulate the gene expression of the pi class of glutathione S-transferase (GSTP) and that an enhancer element named GPE I is required for this induction. In the present study, we further investigated the signal pathway involved in DADS and DATS up-regulation of this detoxification enzyme in Clone 9 cells. Cells were cultured with 25-200 micromol/L of DADS or DATS for 24 h. Western and Northern blots showed that both garlic allyl sulfides concentration dependently induced GSTP protein and mRNA expression, respectively. Changes in GST activity toward ethacrynic acid were consistent with the increase in GSTP expression (P < 0.05). Electromobility gel shift assay showed that the DNA binding activity of nuclear activator protein-1 (AP-1) is concentration-dependently increased in the presence of DADS and DATS as compared with that of the control cells. The phosphorylation of c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), but not of p38, was stimulated in the presence of both garlic allyl sulfides. Pretreatment with SP600125 and PD98059, which are JNK and ERK inhibitors, respectively, abolished the increase in AP-1-DNA binding activity and also the induction of GSTP protein by either allyl sulfide. Our results indicate that the effectiveness of DADS and DATS on GSTP expression is likely related to the JNK-AP-1 and ERK-AP-1 signaling pathways and, thus, that DADS and DATS enhance the binding of AP-1 to GPE I.     

α-Mangostin induces apoptosis in human chondrosarcoma cells through downregulation of ERK/JNK and Akt signaling pathway

Chondrosarcoma is a malignant primary bone tumor that is resistant to chemotherapy and radiation therapy. α-Mangostin, a component of Garcinia mangostana Linn, is a xanthone derivative shown to have antioxidant and antitumor properties. This study is the first to investigate anticancer effects of α-mangostin in the human chondrosarcoma cell line SW1353. We showed that α-mangostin inhibited cell proliferation of SW1353 cells in a time- and dose-dependent manner by using the trypan blue exclusion method. Hoechst 33342 nuclear staining and nucleosomal DNA-gel electrophoresis revealed that α-mangostin could induce nuclear condensation and fragmentation, typically seen in apoptosis. Flow cytometry using Annexin V/PI double staining assessed apoptosis, necrosis and viability. α-Mangostin activated caspase-3, -8, -9 expression, decreased Bcl-2 and increased Bax. This promotes mitochondrial dysfunction, leading to the release of cytochrome c from the mitochondria to the cytoplasm. In addition, total and phosphorylated ERK and JNK were downregulated in α-mangostin-treated SW1353 cells but no changes in p38. α-Mangostin also decreased phosphorylated Akt without altering total Akt. These results suggest that α-mangostin inhinbited cell proliferation and induced apoptosis through downregulation of ERK, JNK and Akt signaling pathway in human chondrosarcoma SW1353 cells.     

Fucoxanthin enhances HO-1 and NQO1 expression in murine hepatic BNL CL.2 cells through activation of the Nrf2/ARE system partially by its pro-oxidant activity

To determine whether fucoxanthin, a major carotenoid in brown sea algae, may activate cellular antioxidant enzymes via up-regulation of the Nrf2/antioxidant-response element (ARE) pathway, we incubated mouse hepatic BNL CL.2 cells with fucoxanthin (0.5-20 μM) for 0-24 h. We found that fucoxanthin (≥5 μM) significantly increased cellular reactive oxygen species (ROS) at 6 h of incubation, whereas preincubation with α-d-tocopherol (30 μM) significantly attenuated the increase of ROS, indicating the pro-oxidant nature of fucoxanthin. Fucoxanthin significantly increased the phosphorylation of ERK and p38 and markedly increased nuclear Nrf2 protein accumulation after incubation for 12 h. Moreover, fucoxanthin significantly enhanced binding activities of nuclear Nrf2 with ARE and increased mRNA and protein expression of HO-1 and NQO1 after incubation for 12 h. siRNA inhibition of Nrf2 led to markedly decreased HO-1 and NQO1 protein expression. Thus, fucoxanthin may exert its antioxidant activity, at least partly, through its pro-oxidant actions.     

Miyabenol A inhibits LPS-induced NO production via IKK/IkappaB inactivation in RAW 264.7 macrophages: possible involvement of the p38 and PI3K pathways

The anti-inflammatory effect of miyabenol A, a stilbene isolated from Vitis thunbergii, on lipopolysaccaride (LPS)-induced nitric oxide (NO) production in RAW264.7 macrophages was studied. Miyabenol A inhibited NO production (EC 50: 2.7 muM) and iNOS protein and mRNA expression in a parallel concentration-dependent manner. LPS-evoked NF-kappaB nuclear translocation and associated IkappaB degradation were abrogated by miyabenol A treatment. Phosphorylations of IKKalpha/beta, ERK1/2, JNK p38 MAPK, and Akt were observed in LPS-stimulated cells; nevertheless, miyabenol A selectively blocked IKKalpha/beta, p38, and Akt phosphorylation. Furthermore, LPS-stimulated IKKalpha/beta and Akt phosphorylation was abolished by p38 inhibitor SB203580. Wortmannin (a PI3K inhibitor) also attenuated LPS-induced IKKalpha/beta phosphorylation, although to a less extent than SB203580, but failed to affect p38 phosphorylation. These observations suggested that PI3K/Akt might lie downstream of p38 MAPK to coregulate LPS-induced IKKalpha/beta phosphorylation. Taken together, miyabenol A acted via interfering with p38 MAPK-related signal pathways to down-regulate IKK/IkappaB activation and NO production.     

Hydroxytyrosol induces proliferation and cytoprotection against oxidative injury in vascular endothelial cells: role of Nrf2 activation and HO-1 induction

Hydroxytyrosol (HT), a phenolic compound in olive oil and leaves, has been reported to prevent various human pathologies including cardiovascular diseases. This study investigated the effects of HT on proliferation and protection against oxidative stress-induced damage in vascular endothelial cells (VECs) and the molecular mechanism(s) involved. Treatment of VECs with HT increased cell proliferation, promoted wound repair, and protected cells against H(2)O(2) cytotoxicity through the activation of Akt and ERK1/2, but not p38 MAPK. HT increased the expression and nuclear translocation of nuclear factor-E2-related factor-2 (Nrf2). Nrf2 expression was attenuated by LY294002 and U0126, inhibitors of phosphatidylinositol-3-kinase and MEK1/2, respectively. Nrf2 siRNA decreased both proliferative and cytoprotective effects of HT and abrogated HO-1 induction. Moreover, HO-1 inhibition with HO-1 siRNA or zinc protoporphyrin IX significantly prevented HT-induced cell proliferation, cytoprotection, and reduction in intracellular reactive oxygen species (ROS), suggesting that HO-1 is involved in these HT functions. The findings demonstrate that HT positively regulates the antioxidant defense system in VECs through the activation of Nrf2 followed by cell proliferation and resistance to vascular injury. The present study provides a molecular basis for the contribution of HT in the Mediterranean diet to the prevention of cardiovascular diseases.     

Cinnamaldehyde inhibits enzymatic browning of cut lettuce by repressing the induction of phenylalanine ammonia-lyase without promotion of microbial growth

Cinnamaldehyde treatment inhibited the browning of cut lettuce during cold storage. In this study, to clarify the mechanism of inhibitory action of cinnamaldehyde against the browning and to show its microbiological merit, its effect on the browning of cut lettuce was compared to that of mild heat treatment. Both cinnamaldehyde and mild heat treatments inhibited the induction of phenylalanine ammonia-lyase (PAL) activity because of cutting. As a result, the biosynthesis of polyphenols, which are substrates of polyphenol oxidase, was inhibited. This reduction of polyphenol synthesis caused the inhibition of the browning. Cinnamaldehyde treatment repressed the induction of PAL mRNA, while mild heat treatment did not repress its induction. The increase in microbes in cut lettuce treated with cinnamaldehyde was less than that treated with mild heat after 12 days.     

Alpha-chaconine-reduced metastasis involves a PI3K/Akt signaling pathway with downregulation of NF-kappaB in human lung adenocarcinoma A549 cells

Alpha-chaconine, isolated from Solanum tuberosum Linn., is a naturally occurring steroidal glycoalkaloid in potato sprouts. Some reports demonstrated that alpha-chaconine had various anticarcinogenic properties. The aim of this study is to investigate the inhibitory effect of alpha-chaconine on lung adenocarcinoma cell metastasis in vitro. We chose the highly metastatic A549 cells, which were treated with various concentrations of alpha-chaconine to clarify the potential of inhibiting A549 cells invasion and migration. Data showed that alpha-chaconine inhibited A549 cell invasion/migration according to wound healing assay and Boyden chamber assay. Our results also showed that alpha-chaconine could inhibit phosphorylation of c-Jun N-terminal kinase (JNK) and Akt, whereas it did not affected phosphorylation of extracellular signal regulating kinase (ERK) and p38. In addition, alpha-chaconine significantly decreased the nuclear level of nuclear factor kappa B (NF-kappaB) and the binding ability of NF-kappaB. These results suggested that alpha-chaconine inhibited A549 cell metastasis by a reduction of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) activities involving suppression of phosphoinositide 3-kinase/Akt/NF-kappaB (PI3K/Akt/NF-kappaB) signaling pathway. Inhibiting metastasis by alpha-chaconine might offer a pivotal mechanism for its effective chemotherapeutic action.     

Cancer chemopreventive effects of lycopene: suppression of MMP-7 expression and cell invasion in human colon cancer cells

Clinical studies indicate that high blood levels of leptin or matrix metalloproteinase-7 (MMP-7; matrilysin) proteins are associated with tumor progression of human colorectal cancer (CRC). Leptin could play an important role in cell migration and invasion of cancer cells. Our previous study indicated that lycopene could inhibit the proliferation of human colon cancer cells in vitro. However, the inhibitory effects of lycopene on the progression of human colon cancer cells have not been demonstrated yet. In this study, we investigated the inhibitory effects of lycopene on tumor progression including cell invasion and MMP-7 expression in leptin-stimulated human colon cancer cells in vitro. Our results demonstrated that lycopene significantly inhibited leptin-mediated cell invasion and MMP-7 expression in human colon cancer HT-29 cells. Lycopene could augment the expression and stability of E-cadherin proteins. Our results showed that MAPK/ERK and PI3K/Akt signaling pathways played important roles in leptin-mediated MMP-7 expression and cell invasion. Lycopene could effectively inhibit the phosphorylation of Akt, glycogen synthase kinase-3β (GSK-3β) and ERK 1/2 proteins. The molecular mechanisms of lycopene were in part through decreases in nuclear levels of AP-1 and β-catenin proteins. These novel findings suggested that lycopene could act as a chemopreventive agent to suppress MMP-7 expression and leptin-mediated cell invasion in human colon cancer HT-29 cells.     

Grapefruit and oroblanco enhance hepatic detoxification enzymes in rats: possible role in protection against chemical carcinogenesis

Citrus fruits are considered to be functional foods that promote good health. This study was carried out to assess the effect of oroblanco and grapefruit consumption on hepatic detoxification enzymes. Male Sprague-Dawley rats were provided with either regular drinking water (control) or experimental treatments of oroblanco juice, grapefruit juice, or a sugar mix for 6 weeks. After 1 week of treatment, half the animals in each group were injected with the procarcinogen 1,2-dimethylhydrazine. Grapefruit juice significantly increased activity and expression of the hepatic phase I enzyme, cytochrome P450 CYP1A1, with a marked trend toward enhanced NAD(P)H:quinone reductase (QR) activity. Oroblanco juice significantly increased glutathione S-transferase phase II enzyme activity along with CYP1A1 expression and a notable trend toward increased activity of both CYP1A1 and QR. These results suggest that these citrus fruits are bifunctional inducers, modulating both phase I and phase II drug-metabolizing enzymes to enhance hepatic detoxification.     

Selenocystine induces S-phase arrest and apoptosis in human breast adenocarcinoma MCF-7 cells by modulating ERK and Akt phosphorylation

Selenocystine (SeC) is a nutritionally available selenoamino acid with selective anticancer effects on a number of human cancer cell lines. The present study shows that SeC inhibited the proliferation of human breast adenocarcinoma MCF-7 cells in a time- and dose-dependent manner, through the induction of cell cycle arrest and apoptotic cell death. SeC-induced S-phase arrest was associated with a marked decrease in the protein expression of cyclins A, D1, and D3 and cyclin-dependent kinases (CDKs) 4 and 6, with concomitant induction of p21waf1/Cip1, p27Kip1, and p53. Exposure of MCF-7 cells to SeC resulted in apoptosis as evidenced by caspase activation, PARP cleavage, and DNA fragmentation. SeC treatment also triggered the activation of JNK, p38 MAPK, ERK, and Akt. Inhibitors of ERK (U0126) and Akt (LY294002), but not JNK (SP600125) and p38 MAPK (SB203580), suppressed SeC-induced S-phase arrest and apoptosis in MCF-7 cells. The findings establish a mechanistic link between the PI3K/Akt pathway, MAPK pathway, and SeC-induced cell cycle arrest and apoptosis in MCF-7 cells.