MK571 inhibits phase-2 conjugation of flavonols by Caco-2/TC7 cells, but does not specifically inhibit their apical efflux

AbstractMK571 is a multidrug resistance protein-2 (ABCC2, Mrp2) inhibitor and has been widely used to demonstrate the role of Mrp2 in the cellular efflux of drugs, xenobiotics and their conjugates. Numerous reports have described modulation of Caco-2 cellular efflux and transport of flavonoids in the presence of MK571. Since flavonoids are efficiently conjugated by Caco-2/TC7 cells, we investigated the effects of MK571 on the efflux of flavonoid conjugates. The flavonol aglycones kaempferol, quercetin and galangin were efficiently taken up, conjugated and effluxed by Caco-2/TC7 cells. Apically-applied MK571 caused significant reductions in both the apical and basolateral efflux of flavonol conjugates from Caco-2/TC7 monolayers. MK571 did not significantly alter the apical:basolateral efflux ratio for flavonol conjugates, however, which is not consistent with MK571 specifically inhibiting only apical Mrp2. Since MK571 decreased the total amounts of conjugates formed, and increased cellular flavonol aglycone concentrations, we explored the possibility that MK571 also inhibits phase-2 conjugation of flavonols. MK571 dose-dependently inhibited the intracellular biosynthesis of all flavonol glucuronides and sulphates by Caco-2 cells. MK571 significantly inhibited phase-2 conjugation of kaempferol by cell-free extracts of Caco-2, and production of kaempferol-4′-O-glucuronide was competitively inhibited. These data show that MK571, in addition to inhibiting MRP2, is a potential inhibitor of enterocyte phase-2 conjugation

The Effects of Anthocyanins and Their Microbial Metabolites on the Expression and Enzyme Activities of Paraoxonase 1, an Important Marker of HDL Function

High circulating HDL concentrations and measures of various HDL functions are inversely associated with cardiovascular disease (CVD) risk. Paraoxonase 1 (PON1) contributes to many of the athero-protective functions of HDL, such as promoting the reverse cholesterol transport process and reducing the levels of oxidized LDL. PON1 activities are influenced by several factors, the most important being diet and genetic polymorphisms. Reported data from randomized controlled trials have shown that anthocyanin consumption increased PON1 activity. However, the underlying molecular mechanisms by which anthocyanins increase PON1 activity are not understood. Therefore, the aim of this research was to investigate the ability of anthocyanins and their metabolites to increase PON1 gene expression and/or enzyme activities as potential mechanisms. The effect of the two predominant dietary anthocyanins and 18 of their recently identified microbial metabolites including their phase-II conjugates on PON1 gene expression was studied using a PON1-Huh7 stably-transfected cell line and reporter gene assay. The effects of these compounds on PON1 arylesterase and lactonase activities were investigated using two isoforms of the PON1 enzyme that are the phenotypes of the 192Q/R polymorphism. None of the compounds caused even modest changes in PON1 promoter activity (p ≥ 0.05). Further, none of the compounds at physiological concentrations caused any significant changes in the arylesterase or lactonase activity of either of the iso-enzymes. Cyanidin reduced the lactonase activity of the PON1-R192R enzyme at high concentrations (-22%, p < 0.001), but not at physiologically achievable concentrations. In conclusion, none of the data reported here support the notion that anthocyanins or their metabolites affect PON1 transactivation or enzyme activities

Differential effects of quercetin and its two derivatives (isorhamnetin and isorhamnetin-3- glucuronide) in inhibiting proliferation of human breast cancer MCF-7 cells

Quercetin (Que) has consistently been reported to be useful cytotoxic compound in vivo and in vitro, but little is known on its metabolites. Here we examined and compared cytotoxic effect of Que and its water-soluble metabolites, isorhamnetin (IS) and isorhamnetin-3-glucuronide (I3G) in human breast cancer MCF-7 cells, and explain their tumor-inhibitory mechanism and structure-function relationship. The results showed that Que, IS and I3G could dose-dependently inhibit the growth of MCF-7 cells, and the cytotoxic effect was ranked as Que > IS > I3G. Furthermore, Que, IS and I3G mediated the cell-cycle arrest principally in S phase, followed by the decrease in the number of G0/G1 and G2/M, and 70.8%, 68.9% and 49.8% MCF-7 tumor cells entered early phase apotosis when treated with 100 µM Que, IS and I3G for 48 h, respectively. Moreover, induction of apoptosis by Que, IS and I3G were accompanied with the marginal generation of intracellular ROS. Given these results, Que, IS and I3G possess strong cytotoxic effect through a ROS-dependent apoptosis pathway in MCF-7 cells

Fluorescence spectroscopic evaluation of the interactions of quercetin, isorhamnetin, and quercetin-3'-sulfate with different albumins

Quercetin is one of the most commonly occurring flavonoids in nature. Although, quercetin and its metabolites express negligible fluorescence, the albumin-bound form of quercetin has a strong fluorescence property. Considering the structural variance of different albumins, we hypothesized that the fluorescence of albumin complexes of quercetin and its metabolites may vary significantly. Therefore, in this study the fluorescence enhancement of quercetin and some of its major metabolites in the presence of bovine (BSA), human (HSA), porcine (PSA), and rat serum albumins (RSA) were investigated by steady-state fluorescence spectroscopy in PBS buffer (pH 7.4). Among the tested quercetin metabolites, significant fluorescence signal was shown by albumin complexes of quercetin, isorhamnetin, and quercetin-3’-sulfate, while other metabolites (tamarixetin, quercetin-3-glucuronide, and isorhamnetin-3-glucuronide) expressed negligible fluorescence. BSA was the most potent enhancer of quercetin-3’-sulfate but it showed poor effects regarding other flavonoids. The strongest enhancement of isorhamnetin was caused by HSA, while it was less effective enhancer of quercetin and quercetin-3’-sulfate. PSA showed a strong fluorescence enhancement of quercetin and quercetin-3’-sulfate but it was poorly effective regarding isorhamnetin. RSA was the most potent enhancer of quercetin but it caused only a weak enhancement of isorhamnetin and quercetin-3’-sulfate. Large changes of the pH (such as pH 5.0 and pH 10.0) almost completely abolished the fluorescence signals of the complexes. Nevertheless, slight decrease (pH 7.0) reduced and slight increase (pH 7.8) generally enhanced the fluorescence of flavonoid-albumin complexes (only exceptions were quercetin-PSA and quercetin-RSA). Complex formations were also investigated by fluorescence quenching studies. Based on our results, the formations of quercetin-BSA, quercetin-HSA, isorhamnetin-BSA, isorhamnetin-HSA, isorhamnetin-PSA, and quercetin-3’-sulfate – HSA complexes followed 1:1 stoichiometry, while the presence of a secondary binding site of flavonoids was assumed regarding other tested albumin complexes. Our study highlights that albumins can induce significantly different fluorescence enhancement of flavonoids, and even the stoichiometry of flavonoid-albumin complexes may differ

Anti-Inflammatory Effects of Quercetin on High-Glucose and Pro-Inflammatory Cytokine Challenged Vascular Endothelial Cell Metabolism

SCOPE: Pro-inflammatory stimuli such as hyperglycemia and cytokines have been shown to negatively affect endothelial cell functions. The aim of this study is to assess the potential of quercetin and its human metabolites to overcome the deleterious effects of hyperglycemic or inflammatory conditions on the vascular endothelium by modulating endothelial cell metabolism. METHODS AND RESULTS: A metabolomics approach enabled identification and quantification of 27 human umbilical vein endothelial cell (HUVEC) metabolites. Treatment of HUVECs with high-glucose concentrations causes significant increases in lactate and glutamate concentrations. Quercetin inhibits glucose-induced increases in lactate and adenosine 5'-triphosphate (ATP) and also increased inosine concentrations. Tumor necrosis factor α-treatment (TNFα) of HUVECs causes increases in asparagine and decreases in aspartate concentrations. Co-treatment with quercetin reduces pyruvate concentrations compared to TNFα-only treated controls. Subsequently, it was shown that quercetin and its HUVEC phase-2 conjugates inhibit adenosine deaminase, xanthine oxidase and 5'nucleotidase (CD73) but not ectonucleoside triphosphate diphosphohydrolase-1 (CD39) or purine nucleoside phosphorylase activities. CONCLUSION: Quercetin was shown to alter the balance of HUVEC metabolites towards a less inflamed phenotype, both alone and in the presence of pro-inflammatory stimuli. These changes are consistent with the inhibition of particular enzymes involved in purine metabolism by quercetin and its HUVEC metabolites

Quantitative Dietary Fingerprinting (QDF)-A Novel Tool for Comprehensive Dietary Assessment Based on Urinary Nutrimetabolomics

Accurate dietary assessment is a challenge in nutritional research, needing powerful and robust tools for reliable measurement of food intake biomarkers. In this work, we have developed a novel quantitative dietary fingerprinting (QDF) approach, which enables for the first time the simultaneous quantitation of about 350 urinary food-derived metabolites, including (poly)phenolic aglycones, phase II metabolites, and microbial-transformed compounds, as well as other compounds (e.g., glucosinolates, amino acid derivatives, methylxanthines, alkaloids, and markers of alcohol and tobacco consumption). This method was fully validated for 220 metabolites, yielding good linearity, high sensitivity and precision, accurate recovery rates, and negligible matrix effects. Furthermore, 127 additional phase II metabolites were also included in this method after identification in urines collected from acute dietary interventions with various foods. Thus, this metabolomic approach represents one-step further toward precision nutrition and the objective of improving the accurateness and comprehensiveness in the assessment of dietary patterns and lifestyles

Accumulation of Dietary S-Methyl Cysteine Sulfoxide in Human Prostate Tissue

Scope: Observational studies have associated consumption of cruciferous vegetables with reduced risk of prostate cancer. This effect has been associated with the degradation products of glucosinolates—thioglycosides that accumulate within crucifers. The possible role of S-methyl cysteine sulfoxide, a metabolite that also accumulates in cruciferous vegetables, and its derivatives, in cancer prevention is relatively unexplored compared to glucosinolate derivatives. The hypothesis that consuming a broccoli soup results in the accumulation of sulfate (a SMCSO derivative) and other broccoli-derived metabolites in prostate tissue is tested. Methods and results: Eighteen men scheduled for transperineal prostate biopsy were recruited into a 4-week parallel single blinded diet supplementation study (NCT02821728). Nine men supplemented their diet with three 300 mL portions of a broccoli soup each week for four weeks prior to surgery. Analyses of prostate biopsy tissues reveal no detectable levels of glucosinolates and derivatives. In contrast, SMCSO is detected in prostate tissues of the participants, with significantly higher levels in tissue of men in the supplementation arm. SMCSO was also found in blood and urine samples from a previous intervention study with the identical broccoli soup. Conclusion: The consequences of SMCSO accumulation in prostate tissues and its potential role in prevention of prostate cancer remains to be investigated

Transcriptional changes in prostate of men on active surveillance after a 12-mo glucoraphanin-rich broccoli intervention—results from the Effect of Sulforaphane on prostate CAncer PrEvention (ESCAPE) randomized controlled trial

Background Epidemiological evidence suggests that consumption of cruciferous vegetables is associated with reduced risk of prostate cancer progression, largely attributed to the biological activity of glucosinolate degradation products, such as sulforaphane derived from glucoraphanin. Because there are few therapeutic interventions for men on active surveillance for prostate cancer to reduce the risk of cancer progression, dietary approaches are an appealing option for patients. Objective We evaluated whether consumption of a glucoraphanin-rich broccoli soup for 1 y leads to changes in gene expression in prostate tissue of men with localized prostate cancer. Methods Forty-nine men on active surveillance completed a 3-arm parallel randomized double-blinded intervention study for 12 mo and underwent transperineal template biopsy procedures and dietary assessment at the start and end of the study. Patients received a weekly 300 mL portion of soup made from a standard broccoli (control) or from 1 of 2 experimental broccoli genotypes with enhanced concentrations of glucoraphanin, delivering 3 and 7 times that of the control, respectively. Gene expression in tissues from each patient obtained before and after the dietary intervention was quantified by RNA sequencing followed by gene set enrichment analyses. Results In the control arm, there were several hundred changes in gene expression in nonneoplastic tissue during the 12 mo. These were associated with an increase in expression of potentially oncogenic pathways including inflammation processes and epithelial–mesenchymal transition. Changes in gene expression and associated oncogenic pathways were attenuated in men on the glucoraphanin-rich broccoli soup in a dose-dependent manner. Although the study was not powered to assess clinical progression, an inverse association between consumption of cruciferous vegetables and cancer progression was observed. Conclusion Consuming glucoraphanin-rich broccoli soup affected gene expression in the prostate of men on active surveillance, consistent with a reduction in the risk of cancer progression. This trial was registered at clinicaltrials.gov as NCT01950143