槲皮素对前列腺癌细胞增殖及转录因子Sp1功能的抑制作用

雄激素受体(androgenreceptor,AR)作为核转录因子,其高表达、基因突变以及AR辅激活因子的过表达等造成AR的异常激活与前列腺癌细胞的增殖、恶化转移、多药耐药等密切相关.天然黄酮槲皮素(quercetin),是一很有潜力的预防和治疗前列腺肿瘤的化合物.槲皮素不仅抑制前列腺癌细胞LNCaP的增殖,并呈剂量依赖性,而且下调前列腺癌中AR的表达、抑制AR的转录激活功能.GCbox是AR核心启动子的主要正调控元件,是转录因子Sp1的结合位点.细胞转染结果表明,槲皮素能抑制Sp1蛋白对AR启动子的激活作用,可能是槲皮素下调AR表达的机理之一.进一步研究显示,槲皮素还能明显抑制Sp1蛋白对AR转录激活功能的增强作用.Western印迹结果显示,槲皮素对Sp1蛋白表达无明显影响,但能够诱导c-Jun的高表达,而高表达的c-Jun蛋白能逆转Sp1蛋白对AR的转录激活作用,由此推测,槲皮素可能通过介导c-Jun与Sp1的蛋白质相互作用,抑制Sp1的功能,进而起到抑制AR表达和功能的作用.免疫沉淀结果又进一步证实了Sp1与c-Jun二者的相互作用.因此,槲皮素可能通过抑制前列腺癌细胞中AR的表达和功能抑制了细胞的增殖,其分子机理可能与槲皮素诱导的c-Jun与Sp1蛋白相互作用、降低Sp1对AR的转录激活作用有关.     

Early release of the density-dependent inhibition of phosphate uptake and ATP synthesis after src gene expression in chick embryo fibroblasts

Our results showed that the expression of the src gene in chick embryo fibroblasts (CEF) released the density-dependent inhibition (DDI) of phosphate metabolism (phosphate uptake and phosphorylation of small organic compounds). With increasing cell density, phosphate metabolism decreased by 58% in normal CEF and, in contrast, increased by 20% in Rous sarcoma virus (RSV)-transformed CEF. The same change in the DDI was observed in CEF infected by NY68 (a ts mutant for transformation of RSV) and maintained at the permissive temperature (37 degrees C) instead of the restrictive temperature (41.5 degrees C) for the expression of transformation. An interesting feature was that the release of the DDI of phosphate metabolism was an early event in the process of transformation, since it was almost concomitant with the stimulation of the pp60 src kinase activity following the shift from 41.5 to 37 degrees C of NY68 CEF. The phosphorylation of small organic compounds (Po) was more strongly increased by the change in temperature than was 32Pi accumulation. Furthermore, the percentage increases of Po and adenosine triphosphate (ATP) labelling with 32P were similar, suggesting that the expression of src gene enhanced ATP synthesis. In glucose-free medium, the stimulation of Po-labelling was still observed but was decreased. Therefore the activation of glycolytic activity is not an absolute requirement, but is necessary for the maximum effect of transformation on the release of DDI of phosphate metabolism. Oligomycin added in complete medium did not prevent the increase in Po-labelling. From these results, we assumed that ATP turnover was stimulated as a consequence of enhanced ATP degradation. We verified that the stimulation of Po phosphorylation was not a consequence of increased ATP utilization for RNA or protein synthesis. The stimulation of Po labelling was specifically abolished by quercetin. This drug inhibited the transformed cells more strongly than the non-transformed cells.     

CpG-B oligodeoxynucleotide promotes cell survival via up-regulation of Hsp70 to increase Bcl-xL and to decrease apoptosis-inducing factor translocation

Unmethylated CpG oligodeoxynucleotides (ODNs) activate immune responses in a TLR9-dependent manner. In this study, stimulation of mouse macrophages with CpG-B ODN increased cellular Hsp70 expression and prevented apoptosis induced by serum starvation or staurosporine treatment. CpG-B ODN-induced Hsp70 expression depended on TLR9, MyD88, and phosphatidylinositol 3-kinase. Inhibition of Hsp70 synthesis by an inhibitor (quercetin) or antisense hsp70 attenuated not only Hsp70 expression but also the anti-apoptotic capacity of CpG-B ODN. Ectopic expression of Hsp70 rescued the inhibitory effect of quercetin on CpG-B ODN-induced anti-apoptosis. Additional experiments demonstrated that quercetin and anti-sense hsp70 modulated CpG-B ODN-induced anti-apoptosis via a caspase-3-independent pathway by down-regulating the survival gene bcl-x(L) and by increasing translocation of apoptosis-inducing factor. These findings suggest that CpG-B ODN may up-regulate Hsp70 via a TLR9/MyD88/phosphatidylinositol 3-kinase pathway to increase Bcl-x(L) and to decrease apoptosis-inducing factor nuclear translocation, resulting in anti-apoptosis.     

Quercetin Attenuates Inflammatory Responses in BV-2 Microglial Cells: Role of MAPKs on the Nrf2 Pathway and Induction of Heme Oxygenase-1

A large group of flavonoids found in fruits and vegetables have been suggested to elicit health benefits due mainly to their anti-oxidative and anti-inflammatory properties. Recent studies with immune cells have demonstrated inhibition of these inflammatory responses through down-regulation of the pro-inflammatory pathway involving NF-κB and up-regulation of the anti-oxidative pathway involving Nrf2. In the present study, the murine BV-2 microglial cells were used to compare anti-inflammatory activity of quercetin and cyanidin, two flavonoids differing by their alpha, beta keto carbonyl group. Quercetin was 10 folds more potent than cyanidin in inhibition of lipopolysaccharide (LPS)-induced NO production as well as stimulation of Nrf2-induced heme-oxygenase-1 (HO-1) protein expression. In addition, quercetin demonstrated enhanced ability to stimulate HO-1 protein expression when cells were treated with LPS. In an attempt to unveil mechanism(s) for quercetin to enhance Nrf2/HO-1 activity under endotoxic stress, results pointed to an increase in phospho-p38MAPK expression upon addition of quercetin to LPS. In addition, pharmacological inhibitors for phospho-p38MAPK and MEK1/2 for ERK1/2 further showed that these MAPKs target different sites of the Nrf2 pathway that regulates HO-1 expression. However, inhibition of LPS-induced NO by quercetin was not fully reversed by TinPPIX, a specific inhibitor for HO-1 activity. Taken together, results suggest an important role of quercetin to regulate inflammatory responses in microglial cells and its ability to upregulate HO-1 against endotoxic stress through involvement of MAPKs.     

Quercetin inhibits matrix metalloproteinase-1 expression in human vascular endothelial cells through extracellular signal-regulated kinase.

Studies have shown that intake of quercetin was inversely associated with mortality from coronary heart disease. Since recent studies documented that disruption of atherosclerotic plaques is the key event triggering acute myocardial infarction, and vascular endothelium-derived matrix metalloproteinase-1 (MMP-1) contributes to plaque destabilization, we examined the effect of quercetin on MMP-1 expression in human vascular endothelial cells. Our results showed that quercetin significantly inhibited basal and oxidized LDL (oxLDL)-stimulated MMP-1 expression. Our data also indicated that extracellular signal-regulated kinase (ERK) mediated the basal and oxLDL-stimulated expression of MMP-1, and quercetin is a potent inhibitor of ERK, suggesting that quercetin may inhibit MMP-1 expression by blocking the ERK pathway. Finally, we showed that quercetin stimulated tissue inhibitor of metalloproteinase-1 expression in oxLDL- and PMA-treated cells. In conclusion, the present study demonstrated for the first time that quercetin inhibited MMP-1 expression in vascular endothelial cells, suggesting that quercetin might contribute to plaque stabilization.     

Flavonoids and flavonoid sulphates as probes of auxin-transport regulation in Cucurbita pepo hypocotyl segments and vesicles

The accumulation of IAA by sealed microsomal vesicles prepared from hypocotyls of dark-grown Cucurbita pepo L. (zucchini) seedlings was stimulated by N-1-naphthylphthalamic acid (NPA: an inhibitor of carrier-mediated auxin efflux and hence of polar auxin transport) as well as by quercetin and certain other flavonoids with a specificity pattern similar to that previously shown for their NPA-like effects on auxin transport and inhibition of NPA binding to saturable sites. In contrast, putatively nonpenetrant negatively charged quercetinsulphate esters did not stimulate such auxin accumulation although they were able to oppose stimulation by NPA or quercetin itself. However, the binding of NPA to hypocotyl microsomes was 30- to 80-fold more strongly inhibited by the quercetin sulphates than by unsubstituted quercetin. As with vesicles, net IAA uptake by hypocotyl segments (2 mm) from dark-grown zucchini was stimulated less effectively by quercetin-sulphate esters than by quercetin itself. We discuss the implications of these observations for the accessibility of the NPA receptor from cell wall or cytoplasm and for the coupling of its occupancy to inhibition of the auxin efflux carrier.Abbreviations ION3 mixture of 4 M carbonylcyanide m-chlorophenylhydrazone, nigericin and valinomycin - NPA N-1-naphthylphthalamic acid - PMSF phenylmethylsulphonyl fluoride This work was supported by a Studentship (I.J.F.) from the Science and Engineering Research Council and by the Gatsby Charitable Foundation. We are particularly grateful to Dr. W. Michalke for a preprint and permission to use his method of microsome preparation in advance of publication.     

Inhibition by quercetin of thyroid hormone stimulation in vitro of human red blood cell Ca2+-ATPase activity

Human red blood cell membrane Ca2+-ATPase activity is stimulated in vitro by physiological concentrations of thyroid hormone. Quercetin, a flavonoid that inhibits several membrane-linked ATPases, suppressed thyroid hormone action on red cell Ca2+-ATPase activity and also interfered with binding of the hormone by red cell membranes. These effects of quercetin were dose-dependent over a range of concentrations (1-50 microM). In contrast, in the absence of thyroid hormone, quercetin at low concentrations stimulated Ca2+-ATPase activity and at 50 microM inhibited the enzyme. The effects of quercetin at low concentrations (1-10 microM), namely, stimulation of Ca2+-ATPase and inhibition of membrane-binding of thyroid hormone, mimic those of thyroid hormone and are consistent with the thyronine-like structure of quercetin. At high concentrations, quercetin is generally inhibitory of Ca2+-ATPase activity. Chalcone, fisetin, hesperetin and tangeretin are other flavonoids shown to reduce susceptibility of membrane Ca2+-ATPase to hormonal stimulation.     

Inhibitive effect of purple sweet potato leaf extract and its components on cell adhesion and inflammatory response in human aortic endothelial cells

This study investigated the effects of purple sweet potato leaf extract (PSPLE) and its components, cyanidin and quercetin, on human aortic endothelial cells (HAECs) during the inflammatory process. HAECs were pretreated with 100 μg/mL PSPLE or 10 μM quercetin, cyanidin or aspirin for 18 h followed by TNF-α (2 ng/mL) for 6 h, and U937 cell adhesion was determined. Adhesion molecule expression and CD40 were evaluated; NFκB p65 protein localization and DNA binding were assessed. PSPLE, aspirin, cyanidin and quercetin significantly inhibited TNF-α-induced monocyte-endothelial cell adhesion (p < 0.05). Cyanidin, quercetin and PSPLE also significantly attenuated VCAM-1, IL-8 and CD40 expression, and quercetin significantly attenuated ICAM-1 and E-selectin expression (p < 0.05). Significant reductions in NFκB expression and DNA binding by aspirin, cyanidin and quercetin were also observed in addition to decreased expression of ERK1, ERK2 and p38 MAPK (p < 0.05). Thus, PSPLE and its components, cyanidin and quercetin, have anti-inflammatory effects through modulation of NFκB and MAPK signaling. Further in vivo studies are necessary to explore the possible therapeutic effects of PSPLE on atherosclerosis.     

Downregulation of COX-2 and iNOS by amentoflavone and quercetin in A549 human lung adenocarcinoma cell line

Flavonoids are natural polyphenolic compounds ubiquitously present in the plant kingdom. They are reported to exhibit numerous beneficial health effects. In the present study, we demonstrate the potential effects of different flavonoids on cytokines mediated cyclooxygenase-2 and inducible nitric oxide synthase expression and activities in A549 cell line using quercetin, amentoflavone and flavanone. Our data revealed that quercetin, at 50 micro M concentration inhibited PGE(2) biosynthesis by A549 very strongly with little effect on COX-2 mRNA and protein expression. Unlike quercetin, amentoflavone inhibited both PGE(2) biosynthesis and COX-2 mRNA and protein expression strongly. In another set of experiment, quercetin inhibited iNOS protein expression completely without affecting iNOS mRNA expression. In contrast, amentoflavone although exerted no inhibitory effect on iNOS mRNA expression, did inhibit weakly iNOS protein expression. Flavanone had no inhibitory effect on either enzyme at the same concentration. Taken together, our data indicated that amentoflavone and quercetin differentially exerted supression of PGE(2) biosynthesis via downregulation of COX-2/iNOS expression.     

Differential effects of quercetin,a natural polyphenolic flavonoid,on L-type calcium current in pituitary tumor (GH3) cells and neuronal NG108-15 cells

The effects of quercetin, a natural polyphenolic compound, on voltage-dependent L-type Ca(2+) current (I(Ca,L)) in rat pituitary GH(3) cells were investigated with the aid of the whole-cell voltage-camp technique. Quercetin (0.5-200 microM) stimulated I(Ca,L) in a concentration-dependent manner. The current-voltage (I-V) relationship of I(Ca,L) was slightly shifted to more negative potentials in the presence of quercetin. The EC(50) value of the quercetin-induced stimulation of I(Ca,L) was about 7 microM. The presence of quercetin (5 microM) shifted the steady state inactivation curve of I(Ca,L) to a more negative potential by approximately -10 mV. Although quercetin might increase intracellular cyclic AMP, sp-cAMPS did not affect I(Ca,L). In addition, neither flavone nor wortmannin had any effect on the amplitude of I(Ca,L), while epicatechin and genistein slightly suppressed it. Quercetin (50 microM) decreased the amplitude of tetrodotoxin-sensitive Na(+) current in GH(3) cells. Under current-clamp configuration, quercetin could increase the firing frequency of actions potentials. Conversely, in NG108-15 neuronal cells, quercetin suppressed the amplitude of I(Ca,L). The quercetin-induced inhibition of I(Ca,L) was abolished in NG108-15 cells preincubated with t-butyl hydroperoxide (1 mM). Quercetin-mediated stimulation of I(Ca,L) in GH(3) cells was presumably not associated with the level of intracellular cyclic AMP, or with the activity of tyrosine or phosphoinositide 3-kinases. Therefore, the effects of quercetin on ion currents may, at least in part, contribute to the underlying mechanisms through which it affects neuronal or neuroendocrine function.     

Isoenzyme-specific up-regulation of glutathione transferase and aldo-keto reductase mRNA expression by dietary quercetin in rat liver

The impact of quercetin on the mRNA expression of hepatic enzymes involved in drug metabolism was evaluated with a DNA microarray and real-time PCR. Male Sprague–Dawley rats were fed an experimental diet containing either 0, 2.5, 5, 10, or 20 g/kg of quercetin for 15 days. The DNA microarray analysis of the gene expression profile in pooled RNA samples from rats fed diets containing 0, 5, and 20 g/kg of quercetin revealed genes of some isoenzymes of glutathione transferase (Gst) and aldo-keto reductase (Akr) to be activated by this flavonoid. Real-time PCR conducted with RNA samples from individual rats fed varying amounts of quercetin together with the microarray analysis showed that quercetin caused marked dose-dependent increases in the mRNA expression of Gsta3, Gstp1, and Gstt3. Some moderate increases were also noted in the mRNA expression of isoenzymes belonging to the Gstm class. Quercetin also dose-dependently increased the mRNA expression of Akr1b8 and Akr7a3. However, it did not affect the parameters of the other Gst and Akr isoenzymes. It is apparent that quercetin increases the mRNA expression of Gst and Akr involved in drug metabolism in an isoenzyme-specific manner. Inasmuch as Gst and Akr isoenzymes up-regulated in their gene expression are involved in the prevention and attenuation of cancer development, this consequence may account for the chemopreventive propensity of quercetin.     

Quercetin treatment changes fluxes in the primary metabolism and increases culture longevity and recombinant α<Subscript>1</Subscript>-antitrypsin production in human AGE1.HN cells

Addition of the flavonoid quercetin to cultivations of the α₁-antitrypsin (A1AT) producing human AGE1.HN.AAT cell line resulted in alterations of the cellular physiology and a remarkable improvement of the overall performance of these cells. In a first screening in 96-well plate format, toxicity and the effect of quercetin on the lactate/glucose ratio was analyzed. It was found that quercetin treatment reduced the lactate/glucose ratio dose dependently. An increase in culture longevity, viable cell density (160% of control), and A1AT concentration (from 0.39 g/L in the control to 0.76 g/L with quercetin, i.e., 195% of the control) was observed in batch cultivation with 10 μM quercetin compared to the control. A detailed analysis of quercetin effects on primary metabolism revealed dose-dependent alterations in metabolic fluxes. Quercetin addition resulted in an improved channeling of pyruvate into the mitochondria accompanied by reduced waste product formation and stimulation of TCA cycle activity. The observed changes in cellular physiology can be explained by different properties of quercetin and its metabolites, e.g., inhibition of specific enzymes, stimulation of oxidation of cytoplasmic, and mitochondrial NADH resulting in reduced NADH/NAD⁺ ratio, and cytoprotective activity. The present study shows that the addition of specific effectors to the culture medium represents a promising strategy to improve the cellular metabolic phenotype and the production of biopharmaceuticals. The provided results contribute, additionally, to an improved understanding of quercetin action on the metabolism of human cells in a general physiological context.     

Quercetin induces apoptosis by activating caspase-3 and regulating Bcl-2 and cyclooxygenase-2 pathways in human HL-60 cells

Quercetin is one of the naturally occurring dietary flavonol compounds. It is present abundantly in plants and has chemopreventive and anticancer effects. To investigate its anticancer mechanism, we examined the activity of quercetin against acute leukemia cell line, HL-60. Our results showed that quercetin inhibited cell proliferation and induced apoptosis in a time- and dose-dependent manner. Furthermore, quercetin down-regulated the expression of anti-apoptosis protein Bcl-2 and up-regulated the expression of pro-apoptosis protein Bax. Caspase-3 was also activated by quercetin, which started a caspase-3-depended mitochodrial pathway to induce apoptosis. It was also found that quercetin inhibited the expression of the cycloocygenase-2 (Cox-2) mRNA and Cox-2 protein. Taken together, these findings suggested that quercetin induces apoptosis in a caspase-3-dependent pathway by inhibiting Cox-2 expression and regulates the expression of downstream apoptotic components, including Bcl-2 and Bax. Quercetin can be a potent and promising medicine which might be safely used in leukemia therapy.