A hydroxyl group of flavonoids affects oral anti-inflammatory activity and inhibition of systemic tumor necrosis factor-alpha production.
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We previously reported that oral administration of luteolin can inhibit serum tumor necrosis factor (TNF)-alpha production and several inflammatory and allergic models. We investigated here the effect of various flavonoids which resemble luteolin in structure. Lipopolysaccharide (LPS)-induced TNF-alpha production from macrophages was inhibited by treatment with flavone (luteolin, apigenin, and chrysin), flavonol (quercetin and myricetin), flavanonol (taxifolin), and anthocyanidin (cyanidin chloride) in vitro. Most of these, however, did not affect mice when administered orally. Serum TNF-alpha production was inhibited only by luteolin or apigenin, and only luteolin or quercetin inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced ear edema. These results suggest that the structure of luteolin: 3',4',5,7-tetrahydroxyflavone, is most suitable for the oral anti-inflammatory activity and that existence or disappearance of a hydroxy group may cause a loss of efficiency. (+info)
Phosphorylation on Thr-55 by TAF1 mediates degradation of p53: a role for TAF1 in cell G1 progression.
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The largest subunit of TFIID, TAF1, possesses an intrinsic protein kinase activity and is important for cell G1 progression and apoptosis. Since p53 functions by inducing cell G1 arrest and apoptosis, we investigated the link between TAF1 and p53. We found that TAF1 induces G1 progression in a p53-dependent manner. TAF1 interacts with and phosphorylates p53 at Thr-55 in vivo. Substitution of Thr-55 with an alanine residue (T55A) stabilizes p53 and impairs the ability of TAF1 to induce G1 progression. Furthermore, both RNAi-mediated TAF1 ablation and apigenin-mediated inhibition of the kinase activity of TAF1 markedly reduced Thr-55 phosphorylation. Thus, phosphorylation and the resultant degradation of p53 provide a mechanism for regulation of the cell cycle by TAF1. Significantly, the Thr-55 phosphorylation was reduced following DNA damage, suggesting that this phosphorylation contributes to the stabilization of p53 in response to DNA damage. (+info)
Interactions between sulforaphane and apigenin in the induction of UGT1A1 and GSTA1 in CaCo-2 cells.
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The isothiocyanate, sulforaphane and the flavonoid, apigenin modulate gene expression including phase II detoxifying enzymes, such as glutathione S-transferases (GST) and UDP-glucuronosyltransferases (UGT). Using undifferentiated CaCo-2 cells, we have examined the interactions between sulforaphane and apigenin in the regulation of UGT and GST expression. We show that apigenin induces UGT1A1 transcription (4-fold) but not GSTA1, and that sulforaphane induces both UGT1A1 (3.7-fold) and GSTA1 (2.8-fold) transcription in both dose- and time-dependent manners. The combination of sulforaphane and apigenin resulted in a synergistic induction of UGT1A1 mRNA up to 12-fold, although this interaction was not seen for GSTA1. Nuclear factor kappa B (NF-kappaB) mRNA was induced by apigenin and sulforaphane (2.5- and 2-fold, respectively). NF-kappaB translocation inhibitor SN50 and phosphatidylinositol 3-kinase (PI3) inhibitor LY294002 decreased the induction of GSTA1 by sulforaphane almost to baseline level. However, the MEK inhibitor PD98059 enhanced significantly the induction of GSTA1 by sulforaphane. This suggests that NF-kappaB and PI3-kinase signaling pathways play a role in GSTA1 gene expression. Conversely, the induction of UGT1A1 transcription by sulforaphane was totally abolished by PD98059, although PD98059 slightly enhanced (20%) the induction of UGT1A1 by apigenin implying that the induction of UGT1A1 by sulforaphane is mediated by MAPK/extracellular signal-regulated kinase kinase, whereas UGT1A1 induction by apigenin may be associated with NF-kappaB translocation since the NF-kappaB translocation inhibitor, SN50 enhanced the induction of UGT by apigenin. The results show that UGT1A1 and GSTA1 are regulated by sulforaphane through different signal transduction pathways and the differences in the mechanisms of modulation of UGT1A1 transcription by sulforaphane and apigenin resulted in a synergistic effect between these two compounds in the induction of UGT1A1. (+info)
Flavones mitigate tumor necrosis factor-alpha-induced adhesion molecule upregulation in cultured human endothelial cells: role of nuclear factor-kappa B.
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Flavones have been classified as anti-atherogenic agents that inhibit monocyte adhesion to stimulated endothelium, possibly by blocking induction of cell adhesion molecules (CAM). This anti-atherogenic feature of these flavonoids appears to be related to their chemical structures. Flavones may interfere with key signaling events involved in endothelial cell activation by inflammatory mediators. This study examined the effects of flavones on the induction of CAM and the translocation and DNA binding of nuclear factor-kappa B (NF-kappa B) in TNF-alpha-activated human umbilical vein endothelial cells (HUVEC). The effects of flavones, luteolin and apigenin, on adhesion of THP-1 monocytes to the TNF-alpha-activated HUVEC, protein expression and mRNA levels of vascular cell adhesion molecule-1 (VCAM-1), intracellular cell adhesion molecule-1 (ICAM-1) and E-selectin, and nuclear appearance and DNA binding activity of NF-kappa B were determined. Flavanols, flavonols, and flavanones were used for comparison. TNF-alpha significantly induced HUVEC protein expression of VCAM-1, ICAM-1, and E-selectin with increasing mRNA levels. Luteolin and apigenin inhibited the TNF-alpha-induced upregulation of THP-1 adhesion and VCAM-1 expression; these inhibitory effects were dose-dependent. The flavones at doses of > or =25 micromol/L almost completely abolished the increased CAM protein and mRNA regardless of their anti-oxidative activity. With the exception of the flavonol quercetin, flavonoids had no such effect; quercetin substantially attenuated the CAM induction. The flavones inhibited nuclear translocation and DNA binding activity of the NF-kappa B-containing binding site in the promoter region of the CAM genes in TNF-alpha-activated HUVEC. The inhibition of endothelial CAM induction by flavones is mediated by their interference with the NF-kappa B-dependent transcription pathway. Thus, the flavones may hamper initial atherosclerotic events involving endothelial CAM induction. (+info)
Suppression of constitutive and tumor necrosis factor alpha-induced nuclear factor (NF)-kappaB activation and induction of apoptosis by apigenin in human prostate carcinoma PC-3 cells: correlation with down-regulation of NF-kappaB-responsive genes.
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PURPOSE: Development of androgen independence and resistance to apoptosis in prostate cancer are often correlated with high levels of serum tumor necrosis factor (TNF)-alpha in these patients. The loss of sensitivity to TNF-alpha-induced apoptosis in androgen-insensitive prostate carcinoma cells is due in part to constitutive activation of Rel/nuclear factor (NF)-kappaB transcription factors that regulate several cell survival and antiapoptotic genes. Our previous studies have demonstrated growth inhibitory and apoptotic effects of apigenin, a common plant flavonoid, in a variety of human prostate carcinoma cells. Here we examined whether apigenin is effective in inhibiting NF-kappaB expression in androgen-insensitive human prostate carcinoma cells exhibiting high constitutive levels of NF-kappaB. EXPERIMENTAL DESIGN: Using androgen-insensitive human prostate carcinoma PC-3 cells, the effect of apigenin was assessed on NF-kappaB activation by electrophoretic mobility shift assay and reporter gene assay. Expression of NF-kappaB subunits p65 and p50, IkappaBalpha, p-IkappaBalpha, in-beads kinase assay and NF-kappaB-regulated genes were determined by Western blot analysis. Apoptosis was determined by annexin V/propidium iodide staining after fluorescence-activated cell-sorting analysis. RESULTS: Treatment of cells with 10-40- micro M doses of apigenin inhibited DNA binding and reduced nuclear levels of the p65 and p50 subunits of NF-kappaB. Apigenin inhibited IkappaBalpha degradation and IkappaBalpha phosphorylation and significantly decreased IKKalpha kinase activity. Apigenin also inhibited TNF-alpha-induced activation of NF-kappaB via the IkappaBalpha pathway, thereby sensitizing the cells to TNF-alpha-induced apoptosis. The inhibition of NF-kappaB activation correlated with a decreased expression of NF-kappaB-dependent reporter gene and suppressed expression of NF-kappaB-regulated genes [specifically, Bcl2, cyclin D1, cyclooxygenase-2, matrix metalloproteinase 9, nitric oxide synthase-2 (NOS-2), and vascular endothelial growth factor]. CONCLUSIONS: Our results indicate that inhibition of NF-kappaB by apigenin may lead to prostate cancer suppression by transcriptional repression of NF-kappaB-responsive genes as well as selective sensitization of prostate carcinoma cells to TNF-alpha-induced apoptosis. (+info)
Oncogenic Ras promotes butyrate-induced apoptosis through inhibition of gelsolin expression.
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Activation of Ras promotes oncogenesis by altering a multiple of cellular processes, such as cell cycle progression, differentiation, and apoptosis. Oncogenic Ras can either promote or inhibit apoptosis, depending on the cell type and the nature of the apoptotic stimuli. The response of normal and transformed colonic epithelial cells to the short chain fatty acid butyrate, a physiological regulator of epithelial cell maturation, is also divergent: normal epithelial cells proliferate, and transformed cells undergo apoptosis in response to butyrate. To investigate the role of k-ras mutations in butyrate-induced apoptosis, we utilized HCT116 cells, which harbor an oncogenic k-ras mutation and two isogenic clones with targeted inactivation of the mutant k-ras allele, Hkh2, and Hke-3. We demonstrated that the targeted deletion of the mutant k-ras allele is sufficient to protect epithelial cells from butyrate-induced apoptosis. Consistent with this, we showed that apigenin, a dietary flavonoid that has been shown to inhibit Ras signaling and to reverse transformation of cancer cell lines, prevented butyrate-induced apoptosis in HCT116 cells. To investigate the mechanism whereby activated k-ras sensitizes colonic cells to butyrate, we performed a genome-wide analysis of Ras target genes in the isogenic cell lines HCT116, Hkh2, and Hke-3. The gene exhibiting the greatest down-regulation by the activating k-ras mutation was gelsolin, an actin-binding protein whose expression is frequently reduced or absent in colorectal cancer cell lines and primary tumors. We demonstrated that silencing of gelsolin expression by small interfering RNA sensitized cells to butyrate-induced apoptosis through amplification of the activation of caspase-9 and caspase-7. These data therefore demonstrate that gelsolin protects cells from butyrate-induced apoptosis and suggest that Ras promotes apoptosis, at least in part, through its ability to down-regulate the expression of gelsolin. (+info)
The flavone hispidulin, a benzodiazepine receptor ligand with positive allosteric properties, traverses the blood-brain barrier and exhibits anticonvulsive effects.
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The functional characterization of hispidulin (4',5,7-trihydroxy-6-methoxyflavone), a potent benzodiazepine (BZD) receptor ligand, was initiated to determine its potential as a modulator of central nervous system activity. After chemical synthesis, hispidulin was investigated at recombinant GABA(A)/BZD receptors expressed by Xenopus laevis oocytes. Concentrations of 50 nm and higher stimulated the GABA-induced chloride currents at tested receptor subtypes (alpha(1-3,5,6)beta(2)gamma(2)S) indicating positive allosteric properties. Maximal stimulation at alpha(1)beta(2)gamma(2)S was observed with 10 microm hispidulin. In contrast to diazepam, hispidulin modulated the alpha(6)beta(2)gamma(2)S-GABA(A) receptor subtype. When fed to seizure-prone Mongolian gerbils (Meriones unguiculatus) in a model of epilepsy, hispidulin (10 mg kg(-1) body weight (BW) per day) and diazepam (2 mg kg(-1) BW per day) markedly reduced the number of animals suffering from seizures after 7 days of treatment (30 and 25% of animals in the respective treatment groups, vs 80% in the vehicle group). Permeability across the blood-brain barrier for the chemically synthesized, (14)C-labelled hispidulin was confirmed by a rat in situ perfusion model. With an uptake rate (K(in)) of 1.14 ml min(-1) g(-1), measurements approached the values obtained with highly penetrating compounds such as diazepam. Experiments with Caco-2 cells predict that orally administered hispidulin enters circulation in its intact form. At a concentration of 30 microm, the flavone crossed the monolayer without degradation as verified by the absence of glucuronidated metabolites. (+info)
Involvement of cytosolic Cl- in osmoregulation of alpha-ENaC gene expression.
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Hypotonicity stimulates transepithelial Na(+) reabsorption in renal A6 cells, but the mechanism for this stimulation is not fully understood. In the present study, we found that hypotonicity stimulated Na(+) reabsorption through increases in mRNA expression of the alpha-subunit of the epithelial Na(+) channel (alpha-ENaC). Hypotonicity decreases cytosolic Cl(-) concentration; therefore, we hypothesized that hypotonicity-induced decreases in cytosolic Cl(-) concentration could act as a signal to regulate Na(+) reabsorption through changes in alpha-ENaC mRNA expression. Treatment with the flavone apigenin, which activates the Na(+)-K(+)-2Cl(-) cotransporter and increases cytosolic Cl(-) concentration, markedly suppressed the hypotonicity-induced increase in alpha-ENaC mRNA expression. On the other hand, blockade of the Na(+)-K(+)-2Cl(-) cotransporter decreases cytosolic Cl(-) concentration and increased alpha-ENaC mRNA expression and Na(+) reabsorption. Blocking Cl(-) channels with 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB) inhibited the hypotonicity-induced decrease in cytosolic Cl(-) concentration and suppressed the hypotonicity-induced increase in alpha-ENaC mRNA expression. Coapplication of NPPB and apigenin synergistically suppressed alpha-ENaC mRNA expression. Thus, in every case, changes in cytosolic Cl(-) concentration were associated with changes in alpha-ENaC mRNA expression and changes in Na(+) reabsorption: decreases in cytosolic Cl(-) concentration increased alpha-ENaC mRNA and increased Na(+) reabsorption, whereas increases in cytosolic Cl(-) concentration decreased alpha-ENaC mRNA and decreased Na(+) reabsorption. These findings support the hypothesis that changes in cytosolic Cl(-) concentration are an important and novel signal in hypotonicity-induced regulation of alpha-ENaC expression and Na(+) reabsorption. (+info)