Mitochondria accumulate large amounts of quercetin: prevention of mitochondrial damage and release upon oxidation of the extramitochondrial fraction of the flavonoid

Quercetin uptake in Jurkat cells is extremely rapid and associated with a remarkable accumulation of the flavonoid, dependent on its binding to intracellular components. Cell-associated quercetin is biologically active, quantitatively consumed to promote survival in the presence of reactive species, such as peroxynitrite (ONOO^?), or reduction of extracellular oxidants via activation of plasma membrane oxidoreductases. In alternative, quercetin is very slowly released upon post-incubation in drug-free medium, an event significantly accelerated by extracellular albumin. Quercetin uptake is also observed in isolated mitochondria, resulting in an enormous accumulation of the flavonoid, consumed under conditions associated with prevention of lipid peroxidation induced by ONOO^?. Interestingly, remarkable quercetin accumulation is also detected in the mitochondria isolated from quercetin-pre-loaded cells, and exposure to either ONOO^? or extracellular oxidants caused the parallel loss of both the mitochondrial and cytosolic fractions of the flavonoid. In conclusion, Jurkat cells accumulate large amounts of quercetin and even larger amounts of the flavonoid further accumulate in their mitochondria. Intramitochondrial quercetin appears to be functional for prevention of mitochondrial damage as well as for redistribution to the cytosol, when the fraction of the flavonoid therein retained is progressively consumed either by cell-permeant oxidants or by activation of plasma membrane oxidoreductases.     

Quercetin suppresses hypoxia-induced accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) through inhibiting protein synthesis

Quercetin, a ubiquitous bioactive plant flavonoid, has been shown to inhibit the proliferation of cancer cells and induce the accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha) in normoxia. In this study, under hypoxic conditions (1% O(2)), we examined the effect of quercetin on the intracellular level of HIF-1alpha and extracellular level of vascular endothelial growth factor (VEGF) in a variety of human cancer cell lines. Surprisingly, we observed that quercetin suppressed the HIF-1alpha accumulation during hypoxia in human prostate cancer LNCaP, colon cancer CX-1, and breast cancer SkBr3 cells. Quercetin treatment also significantly reduced hypoxia-induced secretion of VEGF. Suppression of HIF-1alpha accumulation during treatment with quercetin in hypoxia was not prevented by treatment with 26S proteasome inhibitor MG132 or PI3K inhibitor LY294002. Interestingly, hypoxia (1% O(2)) in the presence of 100 microM quercetin inhibited protein synthesis by 94% during incubation for 8 h. Significant quercetin concentration-dependent inhibition of protein synthesis and suppression of HIF-1alpha accumulation were observed under hypoxic conditions. Treatment with 100 microM cycloheximide, a protein synthesis inhibitor, replicated the effect of quercetin by inhibiting HIF-1alpha accumulation during hypoxia. These results suggest that suppression of HIF-1alpha accumulation during treatment with quercetin under hypoxic conditions is due to inhibition of protein synthesis.     

The effect of quercetin on apoptosis and necrosis induction in human colon adenocarcinoma cell line LS180

Quercetin is a very common flavonoid widely distributed in many plants. The flavonoid intake has been linked to the prevention of some human diseases including cancer. Quercetin inhibits heat shock protein expression and in this way triggers apoptosis of tumor cells. The present study was designed to investigate whether quercetin exerts cytotoxic activity against human colon adenocarcinoma cells. The studies have shown that quercetin alone and in combination with the heat shock can induce apoptosis and necrosis in vitro in human colon adenocarcinoma cells (LS 180). Relationships between heat shock proteins and quercetin in this phenomenon are discussed.     

Inhibition of the peroxidation of linoleic acid by the flavonoid quercetin within their complex with human serum albumin

This work provides a quantitative kinetic analysis of oxidative pathways involving linoleic acid and the common dietary antioxidant quercetin (flavonoid), both bound to human serum albumin (HSA). In particular, it is shown that quercetin, although embedded in drug site I, is oxidized as quickly as free quercetin under a flux of hydrophilic peroxyl radicals. This observation suggests that efficient charge relays are established between the periphery of HSA and bound quercetin. Moreover, the peroxidation of HSA-bound linoleic acid is shown to take place at some specific fatty acid binding sites once one to two critical HSA residues are themselves oxidized. Quercetin efficiently delays the onset of lipid peroxidation. The inhibition persists long after the total consumption of quercetin, in agreement with some quercetin oxidation products exerting a residual antioxidant activity. Consistently, HSA markedly increases the maximal concentration of a two-electron oxidation product of quercetin that is accumulated and then consumed in the course of the peroxidation. The additional observation of the faster consumption of the single Trp residue in the presence of quercetin suggests that HSA enhances the antioxidant activity of quercetin by regenerating some of its oxidation products retaining a H-donating activity.     

The effect of quercetin on cell cycle progression and growth of human gastric cancer cells

Quercetin, a flavonoid, is found in many plants, including edible fruits and vegetables. We examined the effects on cell growth of human malignant cells derived from the gastrointestinal tract and on cell cycle progression. Quercetin markedly inhibited the growth of human gastric cancer cells and the IC50 value was 32-55 microM. DNA synthesis was suppressed to 14% of the control level by the treatment with 70 microM quercetin for 2 days. Furthermore, quercetin blocked cell progression from the G1 to the S phase.     

Modification of membranes by quercetin, a naturally occurring flavonoid, via its incorporation in the polar head group

Quercetin is a naturally occurring flavonoid that has a lot of beneficial properties to human health. In this report, using the spin label technique, the influence of quercetin on the fluidity of multilamellar DPPC liposomes was studied. The polarity of the environment preferred by quercetin was also examined by determining the dependence of the position of electronic absorption maxima on dielectric properties of different environments. Autofluorescence of quercetin was also used to examine its distribution in cells. An additional aim of the study was to find how quercetin presence affects human skin fibroblasts. The results showed that incorporation of quercetin at physiological pH into DPPC liposomes caused changes in the partition coefficient of the Tempo spin label between water and polar head group phases. By determining the electronic absorption maxima, we observed that the chromophore of quercetin is localized in the polar head region. Fluorescence microscopy of HSF cells showed quercetin presence in the membrane, cytoplasm and inside the nucleus. Ultrastructural observation revealed some changes, especially in membranous structures, after flavonol treatment. From the results we have concluded that quercetin present in the membrane and other structures can cause changes within cells crucial for its pharmacological activity.     

Modification of membranes by quercetin, a naturally occurring flavonoid, via its incorporation in the polar head group

Quercetin is a naturally occurring flavonoid that has a lot of beneficial properties to human health. In this report, using the spin label technique, the influence of quercetin on the fluidity of multilamellar DPPC liposomes was studied. The polarity of the environment preferred by quercetin was also examined by determining the dependence of the position of electronic absorption maxima on dielectric properties of different environments. Autofluorescence of quercetin was also used to examine its distribution in cells. An additional aim of the study was to find how quercetin presence affects human skin fibroblasts. The results showed that incorporation of quercetin at physiological pH into DPPC liposomes caused changes in the partition coefficient of the Tempo spin label between water and polar head group phases. By determining the electronic absorption maxima, we observed that the chromophore of quercetin is localized in the polar head region. Fluorescence microscopy of HSF cells showed quercetin presence in the membrane, cytoplasm and inside the nucleus. Ultrastructural observation revealed some changes, especially in membranous structures, after flavonol treatment. From the results we have concluded that quercetin present in the membrane and other structures can cause changes within cells crucial for its pharmacological activity.     

Properties of quercetin conjugates: modulation of LDL oxidation and binding to human serum albumin

Quercetin is an important dietary flavonoid with in vitro antioxidant activity. However, it is found in human plasma as conjugates with glucuronic acid, sulfate or methyl groups, with no significant amounts of free quercetin present. The antioxidant properties of the conjugates found in vivo and their binding to serum albumin are unknown, but essential for understanding possible actions of quercetin in vivo. We, therefore, tested the most abundant human plasma quercetin conjugates, quercetin-3-glucuronide, quercetin-3'-sulfate and isorhamnetin-3-glucuronide, for their ability to inhibit Cu(II)-induced oxidation of human low density lipoprotein and to bind to human albumin, in comparison to free flavonoids and other quercetin conjugates. LDL oxidation lag time was increased by up to four times by low (<2 microM) concentrations of quercetin-3-glucuronide, but was unaffected by equivalent concentrations of quercetin-3'-sulfate and isorhamnetin-3-glucuronide. In general, the compounds under study prolonged the lag time of copper-induced LDL oxidation in the order: quercetin-7-glucuronide > quercetin > quercetin-3-glucuronide = quercetin-3-glucoside > catechin > quercetin-4'-glucuronide > isorhamnetin-3-glucuronide > quercetin-3'-sulfate. Thus the proposed products of small intestine metabolism (quercetin-7-glucuronide, quercetin-3-glucuronide) are more efficient antioxidants than subsequent liver metabolites (isorhamnetin-3-glucuronide, quercetin-3'-sulfate). Albumin-bound conjugates retained their property of protecting LDL from oxidation, although the order of efficacy was altered (quercetin-3'-sulfate > quercetin-7-glucuronide > quercetin-3-glucuronide > quercetin-4'-glucuronide = isorahmnetin-3-glucuronide). Kq values (concentration required to achieve 50% quenching) for albumin binding, as assessed by fluorescence quenching of Trp214, were as follows: quercetin-3'-sulfate (approximately 4 microM)= quercetin > or = quercetin-7-glucuronide > quercetin-3-glucuronide = quercetin-3-glucoside > isorhamnetin-3-glucuronide > quercetin-4'-glucuronide (approximately 20 microM). The data show that flavonoid intestinal and hepatic metabolism have profound effects on ability to inhibit LDL oxidation and a lesser but significant effect on binding to serum albumin.     

Inhibition of cytochrome P-450 activity in rat liver microsomes by the naturally occurring flavonoid, quercetin

The kinetic characteristics and mechanism of flavonoid inhibition of cytochrome P-450-mediated reactions were examined in rat liver microsomes, using the naturally occurring flavonoid, quercetin (3,3',4',5,7-pentahydroxyflavone). Quercetin inhibited the O-deethylation of ethoxyresorufin in beta-naphthoflavone-induced microsomes by 15-80% at concentrations of 10-250 nM. The pattern of inhibition was dependent on quercetin concentration. Quercetin also inhibited p-nitroanisole demethylation and benzo(a)-pyrene hydroxylation, but did not change the proportions of the individual benzo(a)pyrene metabolites in comparison to controls. Specific steps in the P-450 reaction pathway were tested for sensitivity to quercetin inhibition. The Km values of the P-450 substrates tested were increased in the presence of quercetin; competition for and/or alteration of the substrate binding site contributes to the mechanism of inhibition. In experiments under anaerobic, carbon monoxide-saturated conditions, quercetin did not inhibit cytochrome P-450 reduction by NADPH-cytochrome P-450 reductase. The cumene hydroperoxide-supported O-deethylation of ethoxyresorufin was inhibited by quercetin (15-60% inhibition at concentrations of 50-300 nM), suggesting that quercetin may interfere with the formation or breakdown of the oxygenated heme complex. Stoichiometry experiments established that quercetin is a potent uncoupler of P-450 reactions, elevating the rates of H2O2 formation almost twofold. Structure/activity studies indicated that certain other naturally occurring flavonoids were at least as potent inhibitors of ethoxyresorufin deethylation as quercetin. These findings are of interest in light of the significant dietary exposure of the human population to the flavonoids.     

Specific interactions of quercetin and other flavonoids with target proteins are revealed by elicited fluorescence

The fluorogenic properties of quercetin and similar flavonoids common in plants were exploited to analyse their interaction with target proteins. Quercetin produced a strong fluorescent signal upon binding to bovine serum albumin (BSA) and insulin. The fluorescent signal showed saturation kinetics with increasing flavonoid concentrations indicating the presence of defined peptide binding motifs. Other tested proteins showed no fluorescence with the flavonoids. In a comparative study including 22 flavonoids the compounds with fluorogenic properties were identified using our model proteins BSA and insulin and the structural requirements for the fluorogenic property were defined. Only flavones with a high degree of hydroxylation were able to elicit fluorescence. The emitted fluorescence was strongly enhanced at alkaline pH. Finally, an attempt was made to identify intracellular target molecules in live cells. Drosophila follicles showed a distinct staining pattern thus giving evidence that high concentrations of quercetin binding proteins are present in the nuclei and are associated with the ring canals. The presented biochemical and cytological data show that the interaction of the studied flavonoids with target proteins is specific and this finding opens up new experimental possibilities to systematically identify the cellular proteins with specific binding motifs for quercetin or other fluorogenic compounds of medical interest.     

Underlying Mechanism of Quercetin-induced Cell Death in Human Glioma Cells

There has been considerable interest in recent years in the anti-tumor activities of flavonoids. Quercetin, a ubiquitous bioactive flavonoid, can inhibit proliferation and induce apoptosis in a variety of cancer cells. However, the precise molecular mechanism by which quercetin induces apoptosis in cancer cells is poorly understood. The present study was undertaken to examine the effect of quercetin on cell viability and to determine its underlying mechanism in human glioma cells. Quercetin resulted in loss of cell viability in a dose- and time-dependent manner and the decrease in cell viability was mainly attributed to cell death. Quercetin did not increase reactive oxygen species (ROS) generation and the quercetin-induced cell death was also not affected by antioxidants, suggesting that ROS generation is not involved in loss of cell viability. Western blot analysis showed that quercetin treatment caused rapid reduction in phosphorylation of extracellular signal-regulated kinase (ERK) and Akt. Transient transfection with constitutively active forms of MEK and Akt protected against the quercetin-induced loss of cell viability. Quercetin-induced depolarization of mitochondrial membrane potential. Caspase activity was stimulated by quercetin and caspase inhibitors prevented the quercetin-induced loss of cell viability. Quercetin resulted in a decrease in expression of survivin, antiapoptotic proteins. Taken together, these findings suggest that quercetin results in human glioma cell death through caspase-dependent mechanisms involving down-regulation of ERK, Akt, and survivin.     

Elastase release by stimulated neutrophils inhibited by flavonoids: importance of the catechol group

Pathogenesis of chronic inflammatory diseases is associated with excessive elastase release through neutrophil degranulation. In the present study, inhibition of human neutrophil degranulation by four flavonoids (myricetin, quercetin, kaempferol, galangin) was evaluated by using released elastase as a biomarker. Inhibitory potency was observed in the following order: quercetin > myricetin > kaempferol = galangin. Quercetin, the most potent inhibitor of elastase release also had a weak inhibitory effect on the enzyme catalytic activity. Furthermore, the observed effects were highly dependent on the presence of a catechol group at the flavonoid B-ring. The results of the present study suggest that quercetin may be a promising therapeutic agent in the treatment of neutrophil-dependent inflammatory diseases.     

Effects of quercetin and verapamil on membrane potential in the liverwort Conocephalum conicum

Quercetin is a very common flavonoid widely distributed in many plants. The flavonoid intake has been linked to the prevention of human diseases including cancer. Flavonoids possess also a broad spectrum of effects on plants. Quercetin is involved in Ca²⁺ transport and metabolism. The present study was designed to check the effects of quercetin alone and in combination with verapamil on the resting and action potentials in the liverwort Conocephalum conicum. The application of 59·10⁻⁶ mol·dm⁻³ quercetin caused an increase of action potential amplitudes. During the 3rd and 4th hour of treatment an increase by 20–22 % with respect to the control was observed. No changes were found in the resting potential in quercetin treated plants. Verapamil, a calcium channel inhibitor, caused gradual decrease of action potential amplitudes. Quercetin, when added together with verapamil, prevented its inhibitory effect. Interactions between quercetin and Ca²⁺ transport are discussed.     

The interaction of quercetin with human serum albumin: a fluorescence spectroscopic study

Quercetin (3,3',4',5,7-pentahydroxyflavone), a ubiquitous, bioactive plant flavonoid, is known to possess anti-cancer, anti-tumor, and other important therapeutic activities of significant potency and low systemic toxicity. In this communication, we report for the first time a study on the interactions of quercetin with the plasma protein human serum albumin (HSA), exploiting the intrinsic fluorescence emission properties of quercetin as a probe. Quercetin is weakly fluorescent in aqueous buffer medium, with an emission maximum at approximately 538 nm. Binding of quercetin with HSA leads to dramatic enhancement in the fluorescence emission intensity and anisotropy (r), along with significant changes in the fluorescence excitation and emission profiles. The excitation spectrum suggests occurrence of efficient F?rster type resonance energy transfer (FRET) from the single tryptophan-214 residue of HSA to the protein bound quercetin. The emission, excitation, and anisotropy (r=0.18 at [HSA]=30 microM) data (using the native protein) along with emission studies of quercetin using partially denatured HSA (by 8M urea) indicate that the quercetin molecules bind at a motionally restricted site near tryptophan-214 in the interdomain cleft region of HSA. Furthermore, the binding constant (K=1.9 x 10(5)M(-1)) and Gibbs free energy change (deltaG(0)=-30.12 kJ/mol)) for quercetin-HSA interaction have been calculated from the relevant anisotropy data. Implications of these results are examined, particularly in relation to prospective applications in biomedical research.     

Flavonoid glucuronides are substrates for human liver beta-glucuronidase

Quercetin glucuronides are the main circulating metabolites of quercetin in humans. We hypothesise that the potential availability of the aglycone within tissues depends on the substrate specificity of the deconjugating enzyme beta-glucuronidase towards circulating flavonoid glucuronides. Human tissues (small intestine, liver and neutrophils) exhibited beta-glucuronidase against quercetin glucuronides. The various quercetin glucuronides were deconjugated at similar rates, but liver cell-free extracts were the most efficient and the activity was completely inhibited by saccharo-1,4-lactone (a beta-glucuronidase inhibitor). Furthermore, pure recombinant human beta-glucuronidase hydrolysed various flavonoid glucuronides, with a 20-fold variation in catalytic efficiency (k(cat)/K(m)=1.3x10(3) M(-1) s(-1) for equol-7-O-glucuronide and 26x10(3) M(-1) s(-1) for kaempferol-3-O-glucuronide). Similar catalytic efficiencies were obtained for quercetin O-glucuronides substituted at different positions. These results show that flavonoid glucuronides can be deconjugated by microsomal beta-glucuronidase from various human cells.     

DNA damage in human colonic mucosa cells evoked by nickel and protective action of quercetin - involvement of free radicals?

Nickel is a toxic and carcinogenic environmental and occupational pollutant and quercetin is a dietary flavonoid that is reported to modulate effects of many mutagens and carcinogens. We investigated the ability of nickel chloride to induce DNA damage in human colonic mucosa cells in the presence of quercetin, using the alkaline comet assay. Nickel chloride (5–250 μmol/L) evoked dose-dependent DNA damage and quercetin at 50 μmol/L decreased the extent of this damage. The cells exposed to nickel chloride progressively removed their DNA damage and the presence of 50 μmol/L quercetin in the repair-incubation medium did not affect the repair kinetics. Cells exposed to nickel and treated with endonuclease III, an enzyme recognizing oxidized bases, displayed a greater extent of DNA damage than those not treated with the enzyme. Quercetin did not exert a significant effect on the production of oxidized bases by nickel. Pretreatment of the cells with a nitrone spin trap, N-tert-butyl-α-phenylnitrone, decreased the extent of DNA damage evoked by nickel. Quercetin caused a further decrease in the extent of the damage in the presence of the trap. The results obtained suggest that reactive oxygen species, including free radicals, might be involved in the formation of DNA lesions induced by nickel chloride in colonic mucosa cells and that quercetin may exert protective effects in these cells. This revised version was published online in July 2006 with corrections to the Cover Date.     

Accumulation of quercetin conjugates in blood plasma after the short-term ingestion of onion by women

Quercetin is a typical flavonoid present mostly as glycosides in plant foods; it has attracted much attention for its potential beneficial effects in disease prevention. In this study, we examined human volunteers after the short-term ingestion of onion, a vegetable rich in quercetin glucosides. The subjects were served diets containing onion slices (quercetin equivalent: 67.6-93.6 mg/day) with meals for 1 wk. Quercetin was only found in glucuronidase-sulfatase-treated plasma, and its concentration after 10 h of fasting increased from 0.04 +/- 0.04 microM before the trial to 0.63 +/- 0.72 microM after the 1-wk trial. The quercetin content in low-density lipoprotein (LDL) after glucuronidase-sulfatase treatment corresponded to <1% of the alpha-tocopherol content. Human LDL isolated from the plasma after the trial showed little improvement of its resistance to copper ion-induced oxidation. It is therefore concluded that conjugated metabolites of quercetin accumulate exclusively in human blood plasma in the concentration range of 10(-7) approximately 10(-6) M after the short-term ingestion of vegetables rich in quercetin glucosides, although these metabolites are hardly incorporated into plasma LDL.     

The effect of quercetin, a widely distributed flavonoid in food and drink, on cytosolic aldehyde dehydrogenase: a comparison with the effect of diethylstilboestrol

Quercetin is a flavonoid found in red wine and many other dietary sources. Observations concerning the state of ionisation and the stability of the compound over a range of pH are presented. Quercetin is a potent inhibitor of cytosolic aldehyde dehydrogenase at physiological pH when the concentration of either the substrate or the cofactor is relatively low, but it has an activatory effect when the concentrations of substrate and cofactor are both high (1 mM). Gel filtration experiments show that quercetin binds very tightly to the enzyme under conditions where the compound is neutral and when it is ionised. The binding is less in the presence of NAD(+). Quercetin cuts down the ability of the resorufin anion to bind to the enzyme. The observations are explained by a model in which quercetin binds competitively to both the coenzyme-binding site and the aldehyde-binding site; binding in the latter location, when the enzyme is in the form of the E-NADH complex, accounts for the activation. The effects of quercetin are significantly different in some respects from those of diethylstilboestrol; this is explained by the latter being able to bind to the aldehyde site but not the NAD(+) site. The possibility that quercetin may affect aldehyde dehydrogenase in vivo is discussed.     

Flavonol derivatives of Ichthyothere terminalis

The flavonoids of Ichthyothere terminalis are based upon quercetin, with minor amounts of kaempferol and dihydroquercetin. All glycosides are linked at position-3. Quercetin 3-glucoside, 3-galactoside, and 3-arabinoside comprise the monoglycoside fraction. The diglycoside fraction consists of quercetin 3-rutinoside, 3-rhamnosylgalactoside and 3-digalactoside. The single triglycoside present was shown to be quereetin 3-rhamnosylgalactosylgalactoside. A major constituent of the aglycone fraction was shown to be 3-0-methylquercetin. The flavonoid profile of Ichthyothere terminalis shows marked ditterences from those of the related genera Clibadium and Desmanthodium.     

Preventive effect of dietary quercetin on disuse muscle atrophy by targeting mitochondria in denervated mice

Quercetin is a major dietary flavonoid in fruits and vegetables. We aimed to clarify the preventive effect of dietary quercetin on disuse muscle atrophy and the underlying mechanisms. We established a mouse denervation model by cutting the sciatic nerve in the right leg (SNX surgery) to lack of mobilization in hind-limb. Preintake of a quercetin-mixed diet for 14 days before SNX surgery prevented loss of muscle mass and atrophy of muscle fibers in the gastrocnemius muscle (GM). Phosphorylation of Akt, a key phosphorylation pathway of suppression of protein degradation, was activated in the quercetin-mixed diet group with and without SNX surgery. Intake of a quercetin-mixed diet suppressed the generation of hydrogen peroxide originating from mitochondria and elevated mitochondrial peroxisome proliferator-activated receptor-γ coactivator 1α mRNA expression as well as NADH dehydrogenase 4 expression in the GM with SNX surgery. Quercetin and its conjugated metabolites reduced hydrogen peroxide production in the mitochondrial fraction obtained from atrophied muscle. In C2C12 myotubes, quercetin reached the mitochondrial fraction. These findings suggest that dietary quercetin can prevent disuse muscle atrophy by targeting mitochondria in skeletal muscle tissue through protecting mitochondria from decreased biogenesis and reducing mitochondrial hydrogen peroxide release, which can be related to decreased hydrogen peroxide production and/or improvements on antioxidant capacity of mitochondria.