Regulation of Fas (CD95)-induced apoptosis by nuclear factor-kappaB and tumor necrosis factor-alpha in macrophages.
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The APO-1/Fas ligand (FasL) and tumor necrosis factor-alpha (TNF-alpha) are two functionally related molecules that induce apoptosis of susceptible cells. Although the two molecules have been reported to induce apoptosis via distinct signaling pathways, we have shown that FasL can also upregulate the expression of TNF-alpha, raising the possibility that TNF-alpha may be involved in FasL-induced apoptosis. Because TNF-alpha gene expression is under the control of nuclear factor-kappaB (NF-kappaB), we investigated whether FasL can induce NF-kappaB activation and whether such activation plays a role in FasL-mediated cell death in macrophages. Gene transfection studies using NF-kappaB-dependent reporter plasmid showed that FasL did activate NF-kappaB promoter activity. Gel shift studies also revealed that FasL mobilized the p50/p65 heterodimeric form of NF-kappaB. Inhibition of NF-kappaB by a specific NF-kappaB inhibitor, caffeic acid phenylethyl ester, or by dominant expression of the NF-kappaB inhibitory subunit IkappaB caused an increase in FasL-induced apoptosis and a reduction in TNF-alpha expression. However, neutralization of TNF-alpha by specific anti-TNF-alpha antibody had no effect on FasL-induced apoptosis. These results indicate that FasL-mediated cell death in macrophages is regulated through NF-kappaB and is independent of TNF-alpha activation, suggesting the antiapoptotic role of NF-kappaB and a separate death signaling pathway mediated by FasL. (+info)
Isolation and measurement of quercetin glucosides in flower buds of Japanese butterbur (Petasites japonicus subsp. gigantea Kitam.).
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Three quercetin glucosides were isolated from flower buds of Japanese butterbur (Petasites japonicus subsp. gigantea Kitam.) together with caffeic acid as the ingredients that had DPPH radical scavenging activity, using the DPPH-HPLC method for measuring the radical scavenging activity. These quercetin glucosides were identified as quercetin 3-O-beta-D-glucoside, quercetin 3-O-beta-D-6''-O-acetylglucoside, and rutin, and the amounts of the glucosides in flower buds were also examined by HPLC. The flower buds were harvested from four different sites, the total amount of quercetin glucosides in each site was 100-170 mg/100 g fr. wt., and there were no great differences of the amounts between growing fields. (+info)
Arabidopsis CYP98A3 mediating aromatic 3-hydroxylation. Developmental regulation of the gene, and expression in yeast.
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The general phenylpropanoid pathways generate a wide array of aromatic secondary metabolites that range from monolignols, which are ubiquitous in all plants, to sinapine, which is confined to crucifer seeds. The biosynthesis of these compounds involves hydroxylated and methoxylated cinnamyl acid, aldehyde, or alcohol intermediates. Of the three enzymes originally proposed to hydroxylate the 4-, 3-, and 5-positions of the aromatic ring, cinnamate 4-hydroxylase (C4H), which converts trans-cinnamic acid to p-coumaric acid, is the best characterized and is also the archetypal plant P450 monooxygenase. Ferulic acid 5-hydroxylase (F5H), a P450 that catalyzes 5-hydroxylation, has also been studied, but the presumptive 3-hydroxylase converting p-coumarate to caffeate has been elusive. We have found that Arabidopsis CYP98A3, also a P450, could hydroxylate p-coumaric acid to caffeic acid in vivo when expressed in yeast (Saccharomyces cerevisiae) cells, albeit very slowly. CYP98A3 transcript was found in Arabidopsis stem and silique, resembling both C4H and F5H in this respect. CYP98A3 showed further resemblance to C4H in being highly active in root, but differed from F5H in this regard. In transgenic Arabidopsis, the promoters of CYP98A3 and C4H showed wound inducibility and a comparable developmental regulation throughout the life cycle, except in seeds, where the CYP98A3 promoter construct was inactive while remaining active in silique walls. Within stem and root tissue, the gene product and the promoter activity of CYP98A3 were most abundant in lignifying cells. Collectively, these studies show involvement of CYP98A3 in the general phenylpropanoid metabolism, and suggest a downstream function for CYP98A3 relative to the broader and upstream role of C4H. (+info)
Differential production of meta hydroxylated phenylpropanoids in sweet basil peltate glandular trichomes and leaves is controlled by the activities of specific acyltransferases and hydroxylases.
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Sweet basil (Ocimum basilicum) peltate glandular trichomes produce a variety of small molecular weight phenylpropanoids, such as eugenol, caffeic acid, and rosmarinic acid, that result from meta hydroxylation reactions. Some basil lines do not synthesize eugenol but instead synthesize chavicol, a phenylpropanoid that does not contain a meta hydroxyl group. Two distinct acyltransferases, p-coumaroyl-coenzyme A:shikimic acid p-coumaroyl transferase and p-coumaroyl-coenzyme A:4-hydroxyphenyllactic acid p-coumaroyl transferase, responsible for the production of p-coumaroyl shikimate and of p-coumaroyl 4-hydroxyphenyllactate, respectively, were partially purified and shown to be specific for their substrates. p-Coumaroyl-coenzyme A:shikimic acid p-coumaroyl transferase is expressed in basil peltate glands that are actively producing eugenol and is not active in glands of noneugenol-producing basil plants, suggesting that the levels of this activity determine the levels of synthesis of some meta-hydroxylated phenylpropanoids in these glands such as eugenol. Two basil cDNAs encoding isozymes of cytochrome P450 CYP98A13, which meta hydroxylates p-coumaroyl shikimate, were isolated and found to be highly similar (90% identity) to the Arabidopsis homolog, CYP98A3. Like the Arabidopsis enzyme, the basil enzymes were found to be very specific for p-coumaroyl shikimate. Finally, additional hydroxylase activities were identified in basil peltate glands that convert p-coumaroyl 4-hydroxyphenyllactic acid to its caffeoyl derivative and p-coumaric acid to caffeic acid. (+info)
Daphnetin methylation by a novel O-methyltransferase is associated with cold acclimation and photosystem II excitation pressure in rye.
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In plants, O-methylation of phenolic compounds plays an important role in such processes as lignin synthesis, flower pigmentation, chemical defense, and signaling. However, apart from phenylpropanoids and flavonoids, very few enzymes involved in coumarin biosynthesis have been identified. We report here the molecular and biochemical characterization of a gene encoding a novel O-methyltransferase that catalyzes the methylation of 7,8-dihydroxycoumarin, daphnetin. The recombinant protein displayed an exclusive methylation of position 8 of daphnetin. The identity of the methylated product was unambiguously identified as 7-hydroxy-8-methoxycoumarin by co-chromatography on cellulose TLC and coelution from high performance liquid chromatography, with authentic synthetic samples, as well as by UV, mass spectroscopy, (1)H NMR spectral analysis, and NOE correlation signals of the relevant protons. Northern blot analysis and enzyme activity assays revealed that the transcript and corresponding enzyme activity are up-regulated by both low temperature and photosystem II excitation pressure. Using various phenylpropanoid and flavonoid substrates, we demonstrate that cold acclimation of rye leaves increases O-methyltransferase activity not only for daphnetin but also for the lignin precursors, caffeic acid, and 5-hydroxyferulic acid. The significance of this novel enzyme and daphnetin O-methylation is discussed in relation to its putative role in modulating cold acclimation and photosystem II excitation pressure. (+info)
Curcumin activates the haem oxygenase-1 gene via regulation of Nrf2 and the antioxidant-responsive element.
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The transcription factor Nrf2, which normally exists in an inactive state as a consequence of binding to a cytoskeleton-associated protein Keap1, can be activated by redox-dependent stimuli. Alteration of the Nrf2-Keap1 interaction enables Nrf2 to translocate to the nucleus, bind to the antioxidant-responsive element (ARE) and initiate the transcription of genes coding for detoxifying enzymes and cytoprotective proteins. This response is also triggered by a class of electrophilic compounds including polyphenols and plant-derived constituents. Recently, the natural antioxidants curcumin and caffeic acid phenethyl ester (CAPE) have been identified as potent inducers of haem oxygenase-1 (HO-1), a redox-sensitive inducible protein that provides protection against various forms of stress. Here, we show that in renal epithelial cells both curcumin and CAPE stimulate the expression of Nrf2 in a concentration- and time-dependent manner. This effect was associated with a significant increase in HO-1 protein expression and haem oxygenase activity. From several lines of investigation we also report that curcumin (and, by inference, CAPE) stimulates ho-1 gene activity by promoting inactivation of the Nrf2-Keap1 complex, leading to increased Nrf2 binding to the resident ho-1 AREs. Moreover, using antibodies and specific inhibitors of the mitogen-activated protein kinase (MAPK) pathways, we provide data implicating p38 MAPK in curcumin-mediated ho-1 induction. Taken together, these results demonstrate that induction of HO-1 by curcumin and CAPE requires the activation of the Nrf2/ARE pathway. (+info)
15-Lipoxygenase-1 mediates cyclooxygenase-2 inhibitor-induced apoptosis in gastric cancer.
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It has been found that expression of 15-lipoxygenase-1 (15-LOX-1) and its main product, 13-S-hydroxyoctadecadienoic acid (13-S-HODE), are decreased in human colorectal and esophageal cancers and that non-steroidal anti-inflammatory drugs (NSAIDs) can therapeutically induce 15-LOX-1 expression to trigger apoptosis in those cancer cells. We found that a specific cyclooxygenase-2 (COX-2) inhibitor SC-236 similarly induced apoptosis in gastric cancer cells. In the present study, we tested whether SC-236 induced apoptosis through up-regulation of 15-LOX-1 in gastric cancer. We found that: (i) SC-236 inhibited growth of gastric cancer cells mainly by inducing apoptosis; (ii) SC-236 induced 15-LOX-1 expression and increased endogenous 13-S-HODE product, instead of 15-S-HETE during apoptosis; (iii) SC-236 did not affect expression of COX-1, COX-2, 5-LOX and 12-LOX; and (iv) 15-LOX-1 inhibition suppressed SC-236 induced apoptosis. These findings demonstrated that SC-236 induced apoptosis in gastric cancer cells via up-regulation of 15-LOX-1, and 13-S-HODE. These are potential and new targets for prevention and treatment of gastric cancer. (+info)
Pulse radiolysis, electron paramagnetic resonance spectroscopy and theoretical calculations of caffeic acid oligomer radicals.
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Seven representative compounds isolated from Salvia officinalis, among them caffeic acid, the dimer rosmarinic acid and oligomers of caffeic acid, were investigated with regard to their antioxidant potential both expressed by the radical scavenging activity and the stability and structure of the intermediate radicals. Pulse-radiolytic investigation revealed very high rate constants with both hydroxyl and azide radicals. Evidence from kinetic modelling calculations suggested unusual complex behavior due to the presence of both O(4)- and O(3)-semiquinones and - in two cases - formation and decay of a hydroxyl radical adduct at the vinyl side chain. EPR spectroscopy studies, which included dihydrocaffeic acid as a model for the saturated side chains of the oligomers, confirmed that the radical structures after oxidation in slightly alkaline solutions are representing dissociated O(4)-semiquinones. While according to calculations based on hybrid density-functional theory the other radical structures are valid intermediates, they cannot be observed except by pulse radiolysis due to their fast decay. (+info)