Depletion of mitochondrial methionine adenosyltransferase α1 triggers mitochondrial dysfunction in alcohol-associated liver disease.

MATα1 catalyzes the synthesis of S-adenosylmethionine, the principal biological methyl donor. Lower MATα1 activity and mitochondrial dysfunction occur in alcohol-associated liver disease. Besides cytosol and nucleus, MATα1 also targets the mitochondria of hepatocytes to regulate their function. Here, we show that mitochondrial MATα1 is selectively depleted in alcohol-associated liver disease through a mechanism that involves the isomerase PIN1 and the kinase CK2. Alcohol activates CK2, which phosphorylates MATα1 at Ser114 facilitating interaction with PIN1, thereby inhibiting its mitochondrial localization. Blocking PIN1-MATα1 interaction increased mitochondrial MATα1 levels and protected against alcohol-induced mitochondrial dysfunction and fat accumulation. Normally, MATα1 interacts with mitochondrial proteins involved in TCA cycle, oxidative phosphorylation, and fatty acid ß-oxidation. Preserving mitochondrial MATα1 content correlates with higher methylation and expression of mitochondrial proteins. Our study demonstrates a role of CK2 and PIN1 in reducing mitochondrial MATα1 content leading to mitochondrial dysfunction in alcohol-associated liver disease.

Theracurmin (Highly Bioavailable Curcumin) Prevents High Fat Diet-Induced Hepatic Steatosis Development in Mice.

Curcumin, a hydrophobic polyphenol isolated from the Curcuma longa L. plant, has many pharmacological properties, including antioxidant, anti-inflammatory, and chemo-preventive activities. Curcumin has been shown to have potential in preventing nonalcoholic fatty liver disease (NAFLD). However, the low bioavailability of curcumin has proven to be a major limiting factor in its clinical adoption. Theracurmin, a highly bioavailable curcumin that utilizes micronized technology showed improved biological absorbability in vivo. The aim of this study was to investigate the role of theracurmin in modulating hepatic lipid metabolism in vivo. A fatty liver mouse model was produced by feeding mice a high fat diet (HFD; 60% fat) for 12 weeks. We found that treatment for 12 weeks with theracurmin significantly lowered plasma triacylglycerol (TG) levels and reduced HFD-induced liver fat accumulation. Theracurmin treatment lowered hepatic TG and total cholesterol (T-CHO) levels in HFD-fed mice compared to controls. In addition, theracurmin administration significantly reduced lipid peroxidation and cellular damage caused by reactive oxygen species in HFD-fed mice. Overall, these results suggest that theracurmin has the ability to control lipid metabolism and can potentially serve as an effective therapeutic remedy for the prevention of fatty liver.

Protective effect of sestrin2 against iron overload and ferroptosis-induced liver injury.

Ferroptosis is the non-apoptotic form of cell death caused by small molecules or conditions that inhibit glutathione biosynthesis or resulting in iron-dependent accumulation of lipid peroxidation by lipid reactive oxygen species (ROS). Sestrin2 (Sesn2), a conserved antioxidant protein, is responsive to various stresses including genotoxic, metabolic, and oxidative stresses and acts to restore homeostatic balance. Sesn2 expression was reported to be regulated via stress-responsive transcription factors including p53, Nrf2, and HIF-1α. However, the role of Sesn2 in regulating ferroptosis is not known. In the current study, we investigated whether ferroptosis inducing compounds including erastin, sorafenib, and buthionine sulfoximine affect Sesn2 expression and the role of Sesn2 in cytoprotection against ferroptosis-mediated cell death. Our data demonstrate that ferroptosis inducers significantly increased Sesn2 in hepatocytes in a dose- and time-dependent manner. Treatment with erastin upregulated Sesn2 mRNA levels and luciferase reporter gene activity, and erastin-mediated Sesn2 induction was transcriptionally regulated by NF-E2-related factor 2 (Nrf2). Furthermore, deletion of the antioxidant response element (ARE) in the Sesn2 promoter or Nrf2 knockout or knockdown abolished erastin-induced Sesn2 expression. In cells expressing Sesn2, erastin-induced cell death, ROS formation, and glutathione depletion were almost completely inhibited compared to that in control cells. Treatment with phenylhydrazine in mice, well-reported iron overload liver injury model, increased ALT and AST levels and altered histological features, which were almost completely inhibited by adenoviral Sesn2 infection. Collectively, our results suggest that ferroptosis-mediated Sesn2 induction is dependent on Nrf2 and plays a protective role against iron overload and ferroptosis-induced liver injury.

The mitochondrial chaperone Prohibitin 1 negatively regulates interleukin-8 in human liver cancers.

Prohibitin 1 (PHB1) is a mitochondrial chaperone whose expression is dysregulated in cancer. In liver cancer, PHB1 acts as a tumor suppressor, but the mechanisms of tumor suppression are incompletely understood. Here we aimed to determine PHB1 target genes to better understand how PHB1 influences liver tumorigenesis. Using RNA-Seq analysis, we found interleukin-8 (IL-8) to be one of the most highly up-regulated genes following PHB1 silencing in HepG2 cells. Induction of IL-8 expression also occurred in multiple liver and nonliver cancer cell lines. We examined samples from 178 patients with hepatocellular carcinoma (HCC) and found that IL-8 mRNA levels were increased, whereas PHB1 mRNA levels were decreased, in the tumors compared with adjacent nontumorous tissues. Notably, HCC patients with high IL-8 expression have significantly reduced survival. An inverse correlation between PHB1 and IL-8 mRNA levels is found in HCCs with reduced PHB1 expression. To understand the molecular basis for these observations, we altered PHB1 levels in liver cancer cells. Overexpression of PHB1 resulted in lowered IL-8 expression and secretion. Silencing PHB1 increased c-Jun N-terminal kinase (JNK) and NF-κB activity, induced nuclear accumulation of c-JUN and p65, and enhanced their binding to the IL-8 promoter containing AP-1 and NF-κB elements. Conditioned medium from PHB1-silenced HepG2 cells increased migration and invasion of parental HepG2 and SK-hep-1 cells, and this was blocked by co-treatment with neutralizing IL-8 antibody. In summary, our findings show that reduced PHB1 expression induces IL-8 transcription by activating NF-κB and AP-1, resulting in enhanced IL-8 expression and release to promote tumorigenesis.

Role of the CYP3A4-mediated 11,12-epoxyeicosatrienoic acid pathway in the development of tamoxifen-resistant breast cancer.

Epoxyeicosatrienoic acid (EET) production via cytochrome P450 (CYP) epoxygenases closely correlates with the progression of breast cancer. However, its role in the development of chemoresistant breast cancers has yet to be elucidated. Here, we found that CYP3A4 expression and its epoxy-product, 11,12-epoxyeicosatrienoic acid (11,12-EET) was enhanced in tamoxifen (TAM)-resistant MCF-7 (TAMR-MCF-7) breast cancer cells compared to control MCF-7 cells. Treatment of TAMR-MCF-7 cells with ketoconazole and azamulin (selective CYP3A4 inhibitors) or 14,15-epoxyeicosa-5(Z)-enoic acid (14,15-EEZE, an EET antagonist) inhibited cellular proliferation and recovered the sensitivity to 4-hydroxytamoxifen. Chick chorioallantoic membrane and trans-well migration analyses revealed that the enhanced angiogenic, tumorigenic, and migration intensities of TAMR-MCF-7 cells were also significantly suppressed by ketoconazole and 14,15-EEZE. We previously reported that Pin1, a peptidyl prolyl isomerase, is a crucial regulator for higher angiogenesis and epithelial-mesenchymal transition characteristics of TAMR-MCF-7 cells. EET inhibition suppressed E2F1-dependent Pin1 gene transcription, and Pin1 silencing also blocked cell proliferation, angiogenesis, and migration of TAMR-MCF-7 cells. Our findings suggest that the CYP3A4-mediated EET pathway represents a potential therapeutic target for the treatment of tamoxifen-resistant breast cancer.

Induction of methionine adenosyltransferase 2A in tamoxifen-resistant breast cancer cells.

We previously showed that S-adenosylmethionine-mediated hypermethylation of the PTEN promoter was important for the growth of tamoxifen-resistant MCF-7 (TAMR-MCF-7) cancer cells. Here, we found that the basal expression level of methionine adenosyltransferase 2A (MAT2A), a critical enzyme for the biosynthesis of S-adenosylmethionine, was up-regulated in TAMR-MCF-7 cells compared with control MCF-7 cells. Moreover, the basal expression level of MAT2A in T47D cells, a TAM-resistant estrogen receptor-positive cell line was higher compared to MCF-7 cells. Immunohistochemistry confirmed that MAT2A expression in TAM-resistant human breast cancer tissues was higher than that in TAM-responsive cases. The promoter region of human MAT2A contains binding sites for nuclear factor-κB, activator protein-1 (AP-1), and NF-E2-related factor 2 (Nrf2), and the activities of these three transcription factors were enhanced in TAMR-MCF-7 cells. Both the protein expression and transcriptional activity of MAT2A in TAMR-MCF-7 cells were potently suppressed by NF-κB inhibition but not by c-Jun/AP-1 or Nrf2 knock-down. Interestingly, the expression levels of microRNA (miR)-146a and -146b were diminished in TAMR-MCF-7 cells, and miR-146b transduction decreased NF-κB-mediated MAT2A expression. miR-146b restored PTEN expression via the suppression of PTEN promoter methylation in TAMR-MCF-7 cells. Additionally, miR-146b overexpression inhibited cell proliferation and reversed chemoresistance to 4-hydroxytamoxifen in TAMR-MCF-7 cells.

GPR119: a promising target for nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease is associated with metabolic syndrome and has the unique characteristic of excess lipid accumulation in liver. G-protein-coupled receptor 119 (GPR119) is a promising target for type 2 diabetes. However, the role of GPR119 activation in hepatic steatosis and its precise mechanism has not been investigated. In primary cultured hepatocytes from wild-type and GPR119 knockout (KO) mice, expression of lipogenic enzymes was elevated in GPR119 KO hepatocytes. Treatment of hepatocytes and HepG2 cells with GPR119 agonists in phase 2 clinical trials (MBX-2982 [MBX] and GSK1292263) inhibited protein expression of both nuclear and total sterol regulatory element binding protein (SREBP)-1, a key lipogenesis transcription factor. Oral administration of MBX in mice fed a high-fat diet potently inhibited hepatic lipid accumulation and expression levels of SREBP-1 and lipogenesis-related genes, whereas the hepatic antilipogenesis effects of MBX were abolished in GPR119 KO mice. MBX activated AMPK and increased Ser-372 phosphorylation of SREBP-1c, an inhibitory form of SREBP-1c. Moreover, inhibition of AMPK recovered MBX-induced down-regulation of SREBP-1. These findings demonstrate for the first time that the GPR119 ligand alleviates hepatic steatosis by inhibiting SREBP-1-mediated lipogenesis in hepatocytes.

Discovery of novel 2,5-dioxoimidazolidine-based P2X(7) receptor antagonists as constrained analogues of KN62.

Novel 2,5-dioxoimidazolidine-based conformationally constrained analogues of KN62 (1) were developed as P2X7 receptor (P2X7R) antagonists using a rigidification strategy of the tyrosine backbone of 1. SAR analysis of the 2,5-dioxoimidazolidine scaffold indicated that piperidine substitution at the N3 position and no substitution at N1 position were preferable. Further optimization of the substituents at the piperidine nitrogen and the spacer around the skeleton resulted in several superior antagonists to 1, including 1-adamantanecarbonyl analogue 21i (IC50 = 23 nM in ethidium uptake assay; IC50 = 14 nM in IL-1ß ELISA assay) and (3-CF3-4-Cl)benzoyl analogue (-)-21w (54 nM in ethidium uptake assay; 9 nM in IL-1ß ELISA assay), which was more potent than the corresponding (+) isomer. Compound 21w displayed potent inhibitory activity in an ex vivo model of LTP-induced pain signaling in the spinal cord and significant anti-inflammatory activity in in vivo models of carrageenan-induced paw edema and type II collagen-induced joint arthritis.

Nectandrin B, a lignan isolated from nutmeg, inhibits liver X receptor-α-induced hepatic lipogenesis through AMP-activated protein kinase activation.

Nonalcoholic fatty liver disease is recognized as the most commonly occurring chronic liver disease. Liver X receptor α (LXRα) and sterol regulatory element-binding protein (SREBP)-1c play a central role in de novo fatty acid synthesis. This study investigated pharmacological effects of nectandrin B, a lignan isolated from nutmeg extract, on hepatic lipogenesis stimulated by LXRα-SREBP-1c-mediated pathway and the possible molecular basis. The reporter gene assay revealed that nectandrin B completely represses LXRα activity enhanced by a synthetic LXRα ligand (T0901317) in HepG2 cells. The inhibitory effect was further supported by the suppression of mRNA expression of LXRα target genes, SREBP-1c and LXRα itself. Nectandrin B also inhibited the increase in SREBP-1c expression promoted by insulin plus high glucose, major contributors to hepatic lipid accumulation. LXRα-SREBP-1c-mediated induction of acetyl-CoA carboxylase 1 and fatty acid synthase, major genes for de novo lipogenesis, was suppressed by nectandrin B. Moreover, Oil Red O staining showed that nectandrin B notably attenuates LXRα-induced lipid accumulation. AMP-activated protein kinase (AMPK) inhibits the activities of LXRα and SREBP-1c. Nectandrin B strongly activated AMPK signaling in HepG2 cells. Taken together, the suppressive effects of nectandrin B on lipogenic gene expression and lipid accumulation in hepatocytes may be due to its inhibitory effect on the LXRα-SREBP-1c pathway presumably via AMPK activation. These results suggest the potential of nectandrin B as a therapeutic candidate for fatty liver disease.

Pin1 induction in the fibrotic liver and its roles in TGF-ß1 expression and Smad2/3 phosphorylation.

BACKGROUND & AIMS: Therapeutic management of liver fibrosis remains an unsolved clinical problem. Hepatic accumulation of extracellular matrix, mainly collagen, is mediated by the production of transforming growth factor-ß1 (TGF-ß1) in stellate cells. Pin1, a peptidyl-prolyl isomerase, plays an important pathophysiological role in several diseases, including neurodegeneration and cancer. Herein, we determined whether Pin1 regulates liver fibrogenesis and examined its mechanism of action by focusing on TGF-ß1 signalling and hepatic stellate cell (HSC) activation. METHODS: Pin1 expression was assessed by immunohistochemistry, Western blot or real-time-polymerase chain reaction (RT-PCR) analyses of human and mouse fibrotic liver samples. The role of Pin1 during HSC activation was estimated using Pin1-null mouse embryonic fibroblast (MEF) cells and Pin1-overexpressing LX-2 human hepatic stellate cells. RESULTS: Pin1 expression was elevated in human and mouse fibrotic liver tissues, and Pin1 inhibition improved dimethylnitrosamine (DMN)-induced liver fibrosis in mice. Pin1 inhibition reduced the mRNA or protein expression of TGF-ß1 and α-smooth muscle actin (α-SMA) by DMN treatment. Pin1 knockdown suppressed TGFß1 gene expression in both LX-2 and MEF cells. Pin1-mediated TGFß1 gene transcription was controlled by extracellular signal-regulated kinase (ERK)- and phosphoinositide 3-kinase/Akt-mediated activator protein-1 (AP-1) activation. Moreover, TGFß1-stimulated Smad2/3 phosphorylation and plasminogen activator inhibitor-1 expression were inhibited by Pin1 knockdown. CONCLUSIONS: Pin1 induction during liver fibrosis is involved in hepatic stellate cell activation, TGFß1 expression, and TGFß1-mediated fibrogenesis signalling.

Nectandrin B suppresses the expression of adhesion molecules in endothelial cells: Role of AMP-activated protein kinase activation.

We have previously shown that nectandrin B, a potent natural activator of AMP-activated protein kinase (AMPK) results in endothelium-dependent relaxation via endothelial nitric oxide synthase phosphorylation. This study examined the effects of nectandrin B on monocyte adhesion and on the expression of adhesion molecules in endothelial cells, an initial event in atherogenesis. Nectandrin B inhibited tumor necrosis factor-α (TNFα)-induced monocytoid THP-1 cell adhesion to ECV 304 human endothelial cells. This lignan also suppressed TNFα-induced protein and mRNA expression of two cell adhesion molecules, vascular cell adhesion molecule-1 (VCAM-1) and intercellular cell adhesion molecule-1 (ICAM-1). In addition, expression of cyclooxygenase-2 and inducible nitric oxide synthase were diminished by nectandrin B treatment. Reporter gene and immunoblot analyses revealed that transcription factor activities of nuclear factor-κB (NF-κB), activator protein-1 (AP-1), and cyclic AMP response element binding protein (CREB) were inhibited by nectandrin B. Moreover, nectandrin B activated AMP-activated protein kinase (AMPK) in ECV 304 cells. Transfection of a dominant-negative mutant form of AMPK (DN-AMPK) partially reversed inhibitory effects of nectandrin B on the expression of VCAM-1 and ICAM-1, and on the transcriptional activity of CREB.

Enhancement of vascular endothelial growth factor-mediated angiogenesis in tamoxifen-resistant breast cancer cells: role of Pin1 overexpression.

Acquired resistance to tamoxifen (TAM) is a serious therapeutic problem in breast cancer patients. Here, we found that TAM-resistant MCF-7 cells (TAMR-MCF-7 cells) produced higher levels of vascular endothelial growth factor (VEGF) than control MCF-7 cells. Molecular analyses using reporter genes and Western blots supported the involvement of c-Jun/activator protein-1 and hypoxia-inducible factor 1alpha in enhanced VEGF transcription in TAMR-MCF-7 cells. Pin1, a peptidyl prolyl isomerase, was consistently overexpressed in TAMR-MCF-7 cells, and c-Jun/activator protein-1-dependent VEGF transcription in TAMR-MCF-7 cells was almost completely inhibited by Pin1 siRNA and by the Pin1 inhibitor juglone. Chick chorioallantoic membrane assays confirmed that the increased angiogenic intensity of TAMR-MCF-7 cells was significantly suppressed by Pin1 inhibition. These results show that Pin1 overexpression is closely associated with VEGF-mediated angiogenesis and suggest that Pin1 is a potential therapeutic target of excessive angiogenesis in TAM-resistant breast cancer cases.

Increased expression of Nrf2/ARE-dependent anti-oxidant proteins in tamoxifen-resistant breast cancer cells.

Acquired resistance to tamoxifen (TAM) is a serious therapeutic problem in breast cancer patients. In this study, we found that the expressions of anti-oxidant proteins (gamma-glutamylcysteine ligase heavy chain (gamma-GCL h), heme oxygenase-1, thioredoxin and peroxiredoxin1) in TAM-resistant MCF-7 (TAMR-MCF-7) cells were higher than control MCF-7 cells. Molecular analyses using antioxidant response element (ARE)-containing reporters and gel-shift supported the critical role of NF-E2-related factor2 (Nrf2)/ARE in the overexpression of antioxidant proteins in TAMR-MCF-7 cells. Intracellular peroxide production was significantly decreased in TAMR-MCF-7 cells and TAM resistance was partially reversed by Nrf2 siRNA. The basal phosphorylation of extracellular signal-regulated kinase (ERK) and p38 kinase were increased in the TAMR-MCF-7 cells and the inhibition of ERK significantly decreased the activity of minimal ARE reporter and gamma-GCL h protein expression in TAMR-MCF-7 cells. However, exposure of TAMR-MCF-7 cells to 17-beta-estradiol or ICI-182,780 did not significantly change gamma-GCL h expression. These results suggest that the persistent activation of Nrf2/ARE is critical for the enhanced expression of anti-oxidant proteins in TAM-resistant breast cancer cells and the pathway of ERK, but not of estrogen receptor signaling are involved in the up-regulation of Nrf2/ARE.

Differential regulation of ErbB2 expression by cAMP-dependent protein kinase in tamoxifen-resistant breast cancer cells.

Acquired resistance to tamoxifen (TAM) is a serious therapeutic problem in breast cancer patients, and Her-2/ErbB2 expression is associated with decreased sensitivity to TAM. We previously reported that cAMP-dependent protein kinase (PKA)-mediated activator protein-2 (AP-2) activation was responsible for the expression of Her-2/ErbB2 in p53-inactivated mammary epithelial cells (Yang et al., 2006). In the present study, we tested the hypothesis that PKA plays a role in the expression of ErbB2 in tamoxifen-resistant breast cancer cells. Treatment with H-89, a specific PKA inhibitor, suppressed 4-hydroxytamoxifen-induced ErbB2 expression in control MCF-7 cells. In contrast, PKA inhibition by H-89 or cAMP-dependent protein kinase inhibitor l gamma overexpression increased the expression levels of ErbB2 in TAM-resistant MCF-7 (TAMR-MCF-7) cells. Transcriptional regulation of the erbB2 gene depends on two transcription factors, AP-2 and polyomavirus enhancer activator3 (PEA3). H-89 decreased nuclear or total levels of PEA3 in TAMR-MCF-7 cells. Chromatin immunoprecipitation assay results revealed that H-89 treatment reduced PEA3 binding to the proximal Ets binding site of the erbB2 gene promoter. Reporter gene analyses using human erbB2 gene promoter supported the critical role of PEA3 in the overexpression of ErbB2 in TAMR-MCF-7 cells treated with H-89. This deregulated PKA signaling cascades required for the ErbB2 expression may be important for the differential response of TAM-resistant breast cancer cells to EGF/ErbB2 stimuli.

Induction of multidrug resistance associated protein 2 in tamoxifen-resistant breast cancer cells.

Acquired resistance to tamoxifen (TAM) is a serious therapeutic problem in breast cancer patients. The transition from chemotherapy-responsive breast cancer cells to chemotherapy-resistant cancer cells is mainly accompanied by the increased expression of multidrug resistance-associated proteins (MRPs). In this study, it was found that TAM-resistant MCF-7 (TAMR-MCF-7) cells expressed higher levels of MRP2 than control MCF-7 cells. Molecular analyses using MRP2 gene promoters supported the involvement of the pregnane X receptor (PXR) in MRP2 overexpression in TAMR-MCF-7 cells. Although CCAAT/enhancer-binding protein beta was overexpressed continuously in TAMR-MCF-7 cells, this might not be responsible for the transcriptional activation of the MRP2 gene. In addition, the basal activities of phosphatidylinositol 3-kinase (PI3-kinase) were higher in the TAMR-MCF-7 cells than in the control cells. The inhibition of PI3-kinase significantly reduced both the PXR activity and MRP2 expression in TAMR-MCF-7 cells. Overall, MRP2 induction plays a role in the additional acquisition of chemotherapy resistance in TAM-resistant breast cancer.

Induction of glutathione transferase in insulin-like growth factor type I receptor-overexpressed hepatoma cells.

Insulin-like growth factor type I receptor (IGF-IR) is frequently overexpressed in human hepatocellular carcinoma cells (HCC), and this overexpression has been correlated with increased tumor growth. The protective response of HCC to reactive oxygen species (ROS) produced by chemotherapeutic agents is mediated with the induction of phase II detoxifying genes including glutathione transferase (GST). To understand the roles of IGF-IR overexpression in HCC in terms of its detoxifying effect on ROS and conferred resistance to chemotherapy, we analyzed whether IGF-IR overexpressions affect IGF-1-inducible GST expression. GSTalpha was induced by exposure to IGF-1 in IGF-IR cells but not in cells expressing normal levels of IGF-IR. Furthermore, IGF-IR-overexpressed HCCs (IR-HCC) are more resistant to doxorubicin than control HCC cells, which was associated with the increased GST induction by IGF-1. Molecular analyses using GSTA2 promoter supported the involvement of xenobiotic response element (XRE) in GSTalpha induction. IGF-1 caused the nuclear translocation of CCAAT/enhancer-binding protein beta (C/EBPbeta), which might be responsible for XRE activation. In addition, IGF-1 increased the activities of phosphatidylinositol 3-kinase (PI3-kinase) and extracellular signal-regulated kinase in IR-HCCs. Moreover, the inhibition of PI3-kinase completely abolished the nuclear translocation of C/EBPbeta and the up-regulation of GSTalpha protein in IR-HCC treated with IGF-1. However, specific inhibitors against extracellular signal-regulated kinase, c-Jun N-terminal kinase, or p38 kinase did not alter IGF-1-inducible GSTalpha expression. These results provide evidence that one of the pathological consequences of IGF-IR overexpression in HCCs is the potentiation of GSTalpha inducibility by IGF-1. Moreover, this potentiation of GST may be associated with decreased susceptibility to chemotherapeutic agents such as doxorubicin.

ErbB2 overexpression in p53-inactivated mammary epithelial cells.

Functional loss of p53 and ErbB2 overexpression are the frequent genetic alterations in human breast carcinomas. Here, we found that ErbB2 expression was upregulated in primary cultured mammary epithelial cells (MECs) isolated from mice with a defect in exons 5 and 6 of the p53 gene (p53(Delta5,6)). The reporter gene activity in the p53(Delta5,6) MECs transfected with the -756bp flanking region of the hErbB2 gene was higher than the wild type MECs. p53 inactivation selectively increased the level of AP-2alpha, but not AP-2beta and AP-2gamma and a mutation of the two AP-2 binding sites completely inhibited the reporter activity.

Bifunctional effects of fucoidan on the expression of inducible nitric oxide synthase.

Algal fucoidan is a marine sulfated polysaccharide with a wide variety of biological activities including anti-thrombotic and anti-inflammatory effects. This study evaluated the effect of fucoidan on the expression of inducible nitric oxide synthase (iNOS) in a macrophage cell line, RAW264.7. Low concentration range of fucoidan (10 microg/ml) increased the basal expression level of iNOS in quiescent macrophages. However, we found for the first time that fucoidan inhibited the release of nitric oxide (NO) in RAW264.7 cells stimulated with lipopolysaccharide (LPS). Western blot analysis revealed that fucoidan suppressed the LPS-induced expression of the inducible nitric oxide synthase (iNOS) gene. Moreover, the activation of both nuclear factor-kappaB (NF-kappaB) and activator protein 1 (AP-1) are key steps in the transcriptional activation of the iNOS gene. Here, it was revealed that fucoidan selectively suppressed AP-1 activation, and that the activation of AP-1 appears to be essential for the induction of iNOS in activated macrophages. This inhibitory effect on AP-1 activation by fucoidan might be associated with its NO blocking and anti-inflammatory effects.

Inhibition of nuclear factor-kappaB activation by 2',8''-biapigenin.

Inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) play a key role in the inflammatory processes. Improper overproduction of NO and prostaglandins by both enzymes are also believed to be involved in the pathogenesis of certain human cancers. Crude extracts of Selaginella tamariscina are used as an oriental medicine, which has been reported to inhibit the production of proinflammatory cytokines and cause cell cycle arrest. We isolated 2',8''-biapigenin from S. tamariscina and investigated whether it modulates iNOS and COX-2 expressions in Raw264.7 macrophages stimulated with lipopolysaccharide (LPS). We found that 2',8''-biapigenin blocked the transactivations of iNOS and COX-2 genes via the inactivation of nuclear factor-kappaB by preventing the nuclear translocation of p65. Hence, it may be possible to develop S. tamariscina extracts or 2',8''-biapigenin as a useful agent for cancer chemoprevention or for the treatment of inflammatory diseases.

Potent inhibition of the inductions of inducible nitric oxide synthase and cyclooxygenase-2 by taiwaniaflavone.

The improper productions of nitric oxide and prostaglandins following the inductions of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) are involved in the pathogenesis of chronic inflammation. Selaginella tamariscina is used as an oriental medicine for its anti-inflammatory effects. Here, we isolated taiwaniaflavone from S. tamariscina and investigated whether taiwaniaflavone affects the induction of iNOS and COX-2 in RAW264.7 macrophages stimulated with lipopolysaccharide. We found that taiwaniaflavone blocks the transactivations of iNOS and COX-2 genes by blocking the nuclear translocation of p65 and subsequent nuclear factor-kappaB inactivation. It is known that NF-kappaB activation is controlled by the phosphorylation and subsequent degradation of I-kappaBalpha, and in the present study, we found that the phosphorylation and degradation of I-kappaBalpha were also inhibited by taiwaniaflavone. Our findings indicate that taiwaniaflavone may provide a developmental basis for an agent against inflammatory diseases.