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Supplementary Table S2 from Involvement of the TGF-β and β-Catenin Pathways in Pelvic Lymph Node Metastasis in Early-Stage Cervical Cancer

Supplementary Figures S1-S2 from Involvement of the TGF-β and β-Catenin Pathways in Pelvic Lymph Node Metastasis in Early-Stage Cervical Cancer

Purpose: Presence of pelvic lymph node metastases is the main prognostic factor in early-stage cervical cancer patients, primarily treated with surgery. Aim of this study was to identify cellular tumor pathways associated with pelvic lymph node metastasis in early-stage cervical cancer.Experimental Design: Gene expression profiles (Affymetrix U133 plus 2.0) of 20 patients with negative (N0) and 19 with positive lymph nodes (N+), were compared with gene sets that represent all 285 presently available pathway signatures. Validation immunostaining of tumors of 274 consecutive early-stage cervical cancer patients was performed for representatives of the identified pathways.Results: Analysis of 285 pathways resulted in identification of five pathways (TGF-β, NFAT, ALK, BAD, and PAR1) that were dysregulated in the N0, and two pathways (β-catenin and Glycosphingolipid Biosynthesis Neo Lactoseries) in the N+ group. Class comparison analysis revealed that five of 149 genes that were most significantly differentially expressed between N0 and N+ tumors (P < 0.001) were involved in β-catenin signaling (TCF4, CTNNAL1, CTNND1/p120, DKK3, and WNT5a). Immunohistochemical validation of two well-known cellular tumor pathways (TGF-β and β-catenin) confirmed that the TGF-β pathway (positivity of Smad4) was related to N0 (OR: 0.20, 95% CI: 0.06–0.66) and the β-catenin pathway (p120 positivity) to N+ (OR: 1.79, 95%CI: 1.05–3.05).Conclusions: Our study provides new, validated insights in the molecular mechanism of lymph node metastasis in cervical cancer. Pathway analysis of the microarray expression profile suggested that the TGF-β and p120-associated noncanonical β-catenin pathways are important in pelvic lymph node metastasis in early-stage cervical cancer. Clin Cancer Res; 17(6); 1317–30. ©2011 AACR.

OBJECTIVE Posttransplantation diabetes mellitus (PTDM) effects up to 30% of all kidney transplant recipients (KTR). Recent studies in mice found that sufficient androgen levels are necessary for β-cell health and adequate insulin secretion. This raises the question whether a similar relationship might be present in KTR. Hence, we hypothesized that dihydrotestosterone and testosterone are associated with the development of PTDM in male KTR. RESEARCH DESIGN AND METHODS We conducted a post hoc analyses of a prospective single-center cohort study including adult male KTR with a functioning graft ≥1 year posttransplantation. Androgen levels were assessed by liquid chromatography–tandem mass spectrometry. Development of PTDM was defined according to the American Diabetes Association’s criteria. RESULTS We included 243 male KTR (aged 51 ± 14 years), with a median dihydrotestosterone 0.9 (0.7–1.3) nmol/L and testosterone of 12.1 (9.4–15.8) nmol/L. During 5.3 (3.7–5.8) years of follow-up, 28 KTR (11.5%) developed PTDM. A clear association was observed, as 15 (19%), 10 (12%), and 3 (4%) male KTR developed PTDM in the respective tertiles of dihydrotestosterone (P = 0.008). In Cox regression analyses, both dihydrotestosterone and testosterone as continuous variables were inversely associated with the risk to development PTDM, independent of glucose and HbA1c (hazard ratio [HR] 0.31 [95% CI 0.16–0.59], P < 0.001; and HR 0.32 [95% CI 0.15–0.68], P = 0.003, respectively). CONCLUSIONS Our results suggest that low androgen levels are a novel potential modifiable risk factor for the development of PTDM in male KTR.

Background Vitamin B12 deficiency in children may be associated with (severe) neurological manifestations, therefore recognition is important. Diagnosing vitamin B12 deficiency in children is challenging. This study aimed to investigate plasma methylmalonic acid, holotranscobalamin, and total cobalamin in children 0–18 years of age and to estimate age-dependent reference intervals. Methods Plasma vitamin B12 markers were measured in collected plasma samples of 170 children 0–18 years visiting a local primary care laboratory. All had within-reference hemoglobin and MCV values. Pediatric plasma vitamin B12 biomarkers were measured and reference values were derived thereof. Results Plasma methylmalonic acid was higher in young children, in particular between 1 and 6 months of age; total cobalamin and holotranscobalamin were highest from 0.5 to 4 years and decreased till 10 years of age. Plasma holotranscobalamin was highly correlated with plasma total cobalamin; their ratio was independent of age. Plasma methylmalonic acid was slightly more related to total cobalamin than to holotranscobalamin. A large proportion of mainly young children would be misclassified when adult references are applied. Conclusions Pediatric reference values for cobalamin markers are necessary to allow for early recognition and monitoring of children suspect of (clinical) cobalamin deficiency. Impact We analyzed three plasma vitamin B12 status markers, i.e., total cobalamin, holotranscobalamin, and methylmalonic acid, in the plasma of 170 children 0–18 years of age and were able to derive reference intervals thereof. Recognition of vitamin B12 deficiency in children is important but challenging as pediatric reference intervals for plasma vitamin B12 status markers, particularly plasma holotranscobalamin, are not well described. We think that our results may help early recognition and monitoring of children suspect of (clinical) vitamin B12 deficiency.

OBJECTIVE Certain polymorphisms of the DPYD gene encoding for the dihydropyrimidine dehydrogenase (DPD) enzyme are associated with fluoropyrimidine-induced toxicity. Dose reductions of the fluoropyrimidine prodrug capecitabine are recommended for patients carrying these DPYD variants to prevent toxicities. Capecitabine dose escalation after an initial genotype-guided dose reduction is advocated when treatment is well tolerated. However, practical guidelines on how to implement these dose escalations are lacking. We implemented a protocol for tolerance-guided capecitabine dosing in DPYD variant carriers and aimed to explore its effect on toxicity of treatment. PATIENTS AND METHODS Patients receiving capecitabine-based chemotherapy for different types of solid tumors were identified retrospectively. Capecitabine doses were reduced in case of a DPYD variant (DPYD*2A, c.2846A>T, DPYD*13, or c.1236G>A) and subsequently adjusted on the basis of tolerance. Outcome was evaluated by clinical chart review and dosing characteristics from the hospital pharmacy. Results were compared with a cohort of capecitabine-treated DPYD wild-type patients. RESULTS Of 185 patients eligible for analysis, 11 patients were heterozygous for a DPYD variant. A median dose escalation of 8.5% was achieved using the prespecified protocol. One DPYD variant carrier experienced a grade 3 toxicity after a dose escalation. Overall, DPYD variant carriers did not experience more, or more severe toxicities than DPYD wild-type patients. The total prevalence of severe toxicities in the wild-type group was 43.1% and is comparable with the literature. CONCLUSION Tolerance-based capecitabine dose escalation did not lead to more toxicity in DPYD variant carriers compared with wild-type patients. Our results can guide future prospective research.

Background The cause of heart failure with preserved ejection fraction (HFpEF) is poorly understood, and specific therapies are lacking. Previous studies suggested that inflammation plays a role in the development of HFpEF. Herein, we aimed to investigate in community‐dwelling individuals whether a higher plasma interleukin 6 (IL‐6) level is associated with an increased risk of developing new‐onset heart failure (HF) over time, and specifically HFpEF. Methods and Results We performed a case‐cohort study based on the PREVEND (Prevention of Renal and Vascular End‐Stage Disease) study, a prospective general population‐based cohort study. We included 961 participants, comprising 200 participants who developed HF and a random group of 761 controls. HF with reduced ejection fraction or HFpEF was defined on the basis of the left ventricular ejection fraction of ≤40% or >40%, respectively. In Cox proportional hazard regression analyses, IL‐6 levels were statistically significantly associated with the development of HF (hazard ratio [HR], 1.28; 95% CI, 1.02–1.61; P =0.03) after adjustment for key risk factors. Specifically, IL‐6 levels were significantly associated with the development of HFpEF (HR, 1.59; 95% CI, 1.16–2.19; P =0.004), whereas the association with HF with reduced ejection fraction was nonsignificant (HR, 1.05; 95% CI, 0.75–1.47; P =0.77). In sensitivity analyses, defining HFpEF as left ventricular ejection fraction ≥50%, IL‐6 levels were also significantly associated with the development of HFpEF (HR, 1.47; 95% CI, 1.04–2.06; P =0.03) after adjustment for key risk factors. Conclusions IL‐6 is associated with new‐onset HFpEF in community‐dwelling individuals, independent of potential confounders. Our findings warrant further research to investigate whether IL‐6 might be a novel treatment target to prevent HFpEF.

Aims/hypothesis Detection and management of gestational diabetes mellitus (GDM) are crucial to reduce the risk of pregnancy-related complications for both mother and child. In 2013, the WHO adopted new diagnostic criteria for GDM to improve pregnancy outcomes. However, the evidence supporting these criteria is limited. Consequently, these new criteria have not yet been endorsed in the Netherlands. The aim of this study was to determine the impact of these criteria on the number of GDM diagnoses and pregnancy outcomes. Methods Data were available from 10,642 women who underwent a 75 g OGTT because of risk factors or signs suggestive of GDM. Women were treated if diagnosed with GDM according to the WHO 1999 criteria. Data on pregnancy outcomes were obtained from extensive chart reviews from 4,431 women and were compared between women with normal glucose tolerance (NGT) and women classified into the following groups: (1) GDM according to WHO 1999 criteria; (2) GDM according to WHO 2013 criteria; (3) GDM according to WHO 2013 fasting glucose threshold, but not WHO 1999 criteria; and (4) GDM according to WHO 1999 2 h plasma glucose threshold (2HG), but not WHO 2013 criteria. Results Applying the new WHO 2013 criteria would have increased the number of diagnoses by 45% (32% vs 22%) in this population of women at higher risk for GDM. In comparison with women with NGT, women classified as having GDM based only on the WHO 2013 threshold for fasting glucose, who were not treated for GDM, were more likely to have been obese (46.1% vs 28.1%, p

BackgroundErythropoietin (EPO) has been reported as a novel determinant of fibroblast growth factor 23 (FGF23) production; however, it is unknown whether FGF23 is stimulated by chronic exposure to EPO or by EPO administration in nonpolycystic chronic kidney disease (CKD) models.MethodsWe analyzed the effects of chronic EPO on FGF23 in murine models with chronically high EPO levels and normal kidney function. We studied the effects of exogenous EPO on FGF23 in wild-type mice, with and without CKD, injected with EPO. Also, in four independent human CKD cohorts, we evaluated associations between FGF23 and serum EPO levels or exogenous EPO dose.ResultsMice with high endogenous EPO have elevated circulating total FGF23, increased disproportionately to intact FGF23, suggesting coupling of increased FGF23 production with increased proteolytic cleavage. Similarly, in wild-type mice with and without CKD, a single exogenous EPO dose acutely increases circulating total FGF23 out of proportion to intact FGF23. In these murine models, the bone marrow is shown to be a novel source of EPO-stimulated FGF23 production. In humans, serum EPO levels and recombinant human EPO dose are positively and independently associated with total FGF23 levels across the spectrum of CKD and after kidney transplantation. In our largest cohort of 680 renal transplant recipients, serum EPO levels are associated with total FGF23, but not intact FGF23, consistent with the effects of EPO on FGF23 production and metabolism observed in our murine models.ConclusionEPO affects FGF23 production and metabolism, which may have important implications for CKD patients.

The present study characterises the effect of phase II metabolism, especially methylation and glucuronidation, of the model flavonoid quercetin on its capacity to inhibit human MRP1 and MRP2 activity in Sf9 inside-out vesicles. The results obtained reveal that 3′-O-methylation does not affect the MRP inhibitory potential of quercetin. However, 4′-O-methylation appeared to reduce the potential to inhibit both MRP1 and MRP2. In contrast, glucuronidation in general, and especially glucuronidation at the 7-hydroxylmoiety, resulting in 7-O-glucuronosyl quercetin, significantly increased the potential of quercetin to inhibit MRP1 and MRP2 mediated calcein transport with inhibition of MRP1 being generally more effective than that of MRP2. Overall, the results of this study reveal that the major phase II metabolites of quercetin are equally potent or even better inhibitors of human MRP1 and MRP2 than quercetin itself. This finding indicates that phase II metabolism of quercetin could enhance the potential use of quercetin- or flavonoids in general-as an inhibitor to overcome MRP-mediated multidrug resistance. © 2007 Elsevier Inc. All rights reserved.

Although current cytomorphology-based cervical cancer screening has reduced the incidence of cervical cancer, Papsmears are associated with high false positive and false negative rates. This has spurred the search for new technologies to improve current screening. New methodologies are automation of Pap-smear analysis, addition of new biological or molecular markers to traditional cytology or using these new markers to replace the current screening method. In this overview we will summarize data on cervical cancer epidemiology and etiology and the current cervical cancer screening approach. Available data on new screening approaches, such as quantitative cytochemistry, detection of loss of heterozygosity (LOH) and hypermethylation analysis will be reviewed.We discuss the potential of these approaches to replace or augment current screening. When available, data on cost– effectiveness of certain approaches will be provided. In short, Human Papillomavirus (HPV) DNA detection stands closest to implementation in nation-wide screening programs of all markers reviewed. However, specificity is low in women aged

To study the possible interplay between glutathione metabolism of and MRP inhibition by thiol reactive compounds, the interactions of a series of α,β-unsaturated carbonyl compounds with multidrug resistance proteins 1 and 2 (MRP1/ABCC1 and MRP2/ABCC2) were studied. α,β-Unsaturated carbonyl compounds react with glutathione, and therefore either their parent compound or their intracellularly formed glutathione metabolite(s) can modulate MRP-activity. Inhibition was studied in Madin-Darby canine kidney cells stably expressing MRP1 or MRP2, and isolated Sf9-MRP1 or Sf9-MRP2 membrane vesicles. In the latter model system metabolism is not an issue. Of the series tested, three distinct groups could be discriminated based on differences in interplay of glutathione metabolism with MRP1 inhibition. Curcumin inhibited MRP1 transport only in the vesicle model pointing at inhibition by the parent compound. The glutathione conjugates of curcumin also inhibit MRP1 mediated transport, but to a much lesser extent than the parent compound curcumin. In the cellular model system, it was demonstrated that glutathione conjugation of curcumin leads to inactivation of its inhibitory potential. Demethoxycurcumin and bisdemethoxycurcumin inhibited MRP1 in both the vesicle and cellular model pointing at inhibitory potency of at least the parent compound and possibly their metabolites. A second group, including caffeic acid phenethyl ester inhibited MRP1-mediated calcein transport only in the MDCKII-MRP1 cells, and not in the vesicle model indicating that metabolism appeared a prerequisite to generate the active inhibitor. Finally cinnamaldehyde, crotonaldehyde, trans-2-hexanal, citral, and acrolein did not inhibit MRP1. For MRP2, inhibition was much less in both model systems, with the three curcuminoids being the most effective. The results of this study show the importance to study the complex interplay between MRP-inhibitors and their cellular metabolism, the latter affecting the ultimate potential of a compound for cellular MRP-inhibition. © 2005 Elsevier Inc. All rights reserved.

In the present study, the effects of a large series of flavonoids on multidrug resistance proteins (MRPs) were studied in MRP1 and MRP2 transfected MDCKII cells. The results were used to define the structural requirements of flavonoids necessary for potent inhibition of MRP1- and MRP2-mediated calcein transport in a cellular model. Several of the methoxylated flavonoids are among the best MRP1 inhibitors (IC50 values, ranging between 2.7 and 14.3 μM) followed by robinetin, myricetin and quercetin (IC50 values ranging between 13.6 and 21.8 μM). Regarding inhibition of MRP2 activity especially robinetin and myricetin appeared to be good inhibitors (IC 50 values of 15.0 and 22.2 μM, respectively). Kinetic characterization revealed that the two transporters differ marginally in the apparent Km for the substrate calcein. For one flavonoid, robinetin, the kinetics of inhibition were studied in more detail and revealed competitive inhibition with respect to calcein, with apparent inhibition constants of 5.0 μM for MRP1 and 8.5 μM for MRP2. For inhibition of MRP1, a quantitative structure activity relationship (QSAR) was obtained that indicates three structural characteristics to be of major importance for MRP1 inhibition by flavonoids: the total number of methoxylated moieties, the total number of hydroxyl groups and the dihedral angle between the B- and C-ring. Regarding MRP2 mediated calcein efflux inhibition, only the presence of a flavonol B-ring pyrogallol group seems to be an important structural characteristic. Overall, this study provides insight in the structural characteristics involved in MRP inhibition and explores the differences between inhibitors of these two transporters, MRP1 and MRP2. Ultimately, MRP2 displays higher selectivity for flavonoid type inhibition than MRP1. © 2004 Elsevier Inc. All rights reserved.

In the present study, the inhibition of human glutathione S-transferase P1-1 (GSTP1-1) by the flavonoid quercetin has been investigated. The results show a time- and concentration-dependent inhibition of GSTP1-1 by quercetin. GSTP1-1 activity is completely inhibited upon I h incubation with 100 muM quercetin or 2 h incubation with 25 muM quercetin, whereas 1 and 10 muM quercetin inhibit GSTP1-1 activity to a significant extent reaching a maximum of 25 and 42% inhibition respectively after 2 h. Co-incubation with tyrosinase greatly enhances the rate of inactivation, whereas co-incubation with ascorbic acid or glutathione prevents this inhibition. Addition of glutathione upon complete inactivation of GSTP1-1 partially restores the activity. Inhibition studies with the GSTP1-1 mutants C47S, C101S and the double mutant C47S/C101S showed that cysteine 47 is the key residue in the interaction between quercetin and GSTP1-1. HPLC and LGMS analysis of trypsin digested GSTP1-1 inhibited by quercetin did not show formation of a covalent bond between Cys 47 residue of the peptide fragment 45-54 and quercetin. It was demonstrated that the inability to detect the covalent quercetin-peptide adduct using LGMS is due to the reversible nature of the adduct-formation in combination with rapid and preferential dimerization of the peptide fragment once liberated from the protein. Nevertheless, the results of the present study indicate that quinone-type oxidation products of quercetin likely act as specific active site inhibitors of GSTP1-1 by binding to cysteine 47. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

In a recent article in Clinical Chemistry , Piehler et al. (1) presented a screening algorithm for detecting monoclonal gammopathies that, in our opinion, is a complicated diagnostic route since many of the included parameters for analysis are not readily available on request. As pointed out in the editorial by Katzmann and Dispenzieri (2), by current standards serum protein electrophoresis (SPE)1 and immunofixation electrophoresis (IFE) analysis both need to be performed for optimal detection of monoclonal bands; however, IFE was not performed in Piehler’s study. Clearly, combining SPE and IFE for detecting/excluding monoclonal bands is rather laborious. This problem might be overcome by the more rapid and analytically more sensitive light chain analysis, which also allows earlier detection in cases of light chain disease, nonsecretory myeloma, and amyloidosis. Therefore, we were interested in the study of Piehler et al., but would like to raise the issue that adequate interpretation of free light chain results can …

Purpose: Presence of pelvic lymph node metastases is the main prognostic factor in early-stage cervical cancer patients, primarily treated with surgery. Aim of this study was to identify cellular tumor pathways associated with pelvic lymph node metastasis in early-stage cervical cancer. Experimental Design: Gene expression profiles (Affymetrix U133 plus 2.0) of 20 patients with negative (N0) and 19 with positive lymph nodes (N+), were compared with gene sets that represent all 285 presently available pathway signatures. Validation immunostaining of tumors of 274 consecutive early-stage cervical cancer patients was performed for representatives of the identified pathways. Results: Analysis of 285 pathways resulted in identification of five pathways (TGF-β, NFAT, ALK, BAD, and PAR1) that were dysregulated in the N0, and two pathways (β-catenin and Glycosphingolipid Biosynthesis Neo Lactoseries) in the N+ group. Class comparison analysis revealed that five of 149 genes that were most significantly differentially expressed between N0 and N+ tumors (P < 0.001) were involved in β-catenin signaling (TCF4, CTNNAL1, CTNND1/p120, DKK3, and WNT5a). Immunohistochemical validation of two well-known cellular tumor pathways (TGF-β and β-catenin) confirmed that the TGF-β pathway (positivity of Smad4) was related to N0 (OR: 0.20, 95% CI: 0.06–0.66) and the β-catenin pathway (p120 positivity) to N+ (OR: 1.79, 95%CI: 1.05–3.05). Conclusions: Our study provides new, validated insights in the molecular mechanism of lymph node metastasis in cervical cancer. Pathway analysis of the microarray expression profile suggested that the TGF-β and p120-associated noncanonical β-catenin pathways are important in pelvic lymph node metastasis in early-stage cervical cancer. Clin Cancer Res; 17(6); 1317–30. ©2011 AACR.

Background: The objective of this study was to evaluate the efficiency of free light chain (FLC) analysis in comparison to serum protein electrophoresis (SPE) for detecting M-proteinemia. Methods: A total of 553 consecutive patients for whom evaluation of M-proteinemia was requested were included in this study. For all patients, serum FLC analysis and SPE followed by pentavalent immunofixation analysis was performed. Identification of monoclonal bands was performed using specific antisera. FLC analysis was performed using the Modular P analyzer in accordance with the manufacturer’s recommendations. Local reference ranges for FLCs on this platform were established based on samples from patients with a normal electrophoretic pattern wno monoclonal bands, no hypo- or hypergammaglobulinemia, no acute phase pattern and normal kidney function, i.e., estimated glomerular filtration rate (eGFR) )60 mL/minx. Results: Local reference ranges (95%) were established (ns243): k: 8.01–28.26 mg/L; l: 8.07–23.58 mg/L and k/l ratio: 0.74–1.66. Negative and positive predictive values were 98.6% and 49.5%, respectively, for screening for M-proteinemia by SPE alone, 94.3% and 21.7% for FLC concentration and 95.1% and 21.4% for FLC with the k/l-ratio included. Combining protein electrophoresis and FLCs resulted in a negative predictive value of 99.0% and a positive predictive value of 23.4%. Conclusions: Serum FLC analysis alone is not suitable for screening for M-proteinemia. Clin Chem Lab Med 2009;47:1507–11.

The transcellular transport of ingested food ingredients across the intestinal epithelial barrier is an important factor determining bioavailability upon oral intake. This transcellular transport of many chemicals, food ingredients, drugs or toxic compounds over the intestinal epithelium can be highly dependent on the activity of membrane bound ATP binding cassette (ABC) transport proteins, able to export the compounds from the intestinal cells. The present review describes the ABC transporters involved in the efflux of bioactive compounds from the intestinal cells, either to the basolateral blood side, facilitating absorption, or back into the intestinal lumen, reducing bioavailability. The role of the ABC transporters in intestinal transcellular uptake also implies a role for inhibitors of these transporters in modulation of the bioavailability upon oral uptake. The present paper focuses on the role of flavonoids as important modulators or substrates of intestinal ABC transport proteins. Several examples of such an effect of flavonoids are presented. It can be concluded that flavonoid-mediated inhibition of ABC transporters may affect the bioavailability of drugs, bioactive food ingredients and/or food-borne toxic compounds upon oral uptake. All together it appears that the flavonoid-mediated interactions at the level of the intestinal ABC transport proteins may be an important mechanism for unexpected food-drug, food-toxin or food-food interactions. The overview also indicates that future studies should focus on i) in vivo validation of the flavonoid-mediated effects on bioavailability of drugs, toxins and beneficial bioactive food ingredients detected in in vitro models, and on ii) the role of flavonoid phase II metabolism in modulating the activity of the flavonoids to act as ABC transporter inhibitors and/or substrates. © 2006 Elsevier Masson SAS. All rights reserved.

Membrane transport proteins belonging to the ATP-binding cassette (ABC) family of transport proteins play a central role in the defence of organisms against toxic compounds, including anticancer drugs. However, for compounds that are designed to display a toxic effect, this defence system diminishes their effectiveness. This is typically the case in the development of cellular resistance to anticancer drugs. Inhibitors of these transporters are thus potentially useful tools to reverse this transporter-mediated cellular resistance to anticancer drugs and, eventually, to enhance the effectiveness of the treatment of patients with drug-resistant cancer. This review highlights the various types of inhibitors of several multidrug resistance-related ABC proteins, and demonstrates that the metabolism of inhibitors, as illustrated by recent data obtained for various natural compound inhibitors, may have considerable implications for their effect on drug transport and their potential for treatment of drug resistance.

In the present study, the effects of myricetin on either MRP1 or MRP2 mediated vincristine resistance in transfected MDCKII cells were examined. The results obtained show that myricetin can inhibit both MRP1 and MRP2 mediated vincristine efflux in a concentration dependent manner. The IC50 values for cellular vincristine transport inhibition by myricetin were 30.5 ± 1.7 μM for MRP1 and 24.6 ± 1.3 μM for MRP2 containing MDCKII cells. Cell proliferation analysis showed that the MDCKII control cells are very sensitive towards vincristine toxicity with an IC50 value of 1.1 ± 0.1 μM. The MDCKII MRP1 and MRP2 cells are less sensitive towards vincristine toxicity with IC50 values of 33.1 ± 1.9 and 22.2 ± 1.4 μM, respectively. In both the MRP1 and MRP2 cells, exposure to 25 μM myricetin enhances the sensitivity of the cells towards vincristine toxicity to IC50 values of 7.6 ± 0.5 and 5.8 ± 0.5 μM, respectively. The increase of sensitivity represents a reversal of the resistance towards vincristine as a result of MRP1 and MRP2 inhibition. Thus, the present study demonstrates the ability of the flavonoid myricetin to modulate MRP1 and MRP2 mediated resistance to the anticancer drug vincristine in transfected cells, indicating that flavonoids might be a valuable adjunct to chemotherapy to block MRP mediated resistance. © 2005 Elsevier Inc. All rights reserved.

The objective of this study was to investigate the structural requirements necessary for inhibition of glutathione S-transferase P1-1 (GSTP1-1) and GS-X pump (MRP1 and MRP2) activity by structurally related flavonoids, in GSTP1-1 transfected MCF7 cells (pMTG5). The results reveal that GSTP1-1 activity in MCF7 pMTG5 cells can be inhibited by some flavonoids. Especially galangin was able to inhibit almost all cellular GSTP1-1 activity upon exposure of the cells to a concentration of 25 muM. Other flavonoids like kaempferol, eriodictyol and quercetin showed a moderate GSTP1-1 inhibitory potential. For GSTP1-1 inhibition, no specific structural requirements necessary for potent inhibition could be defined. Most flavonoids appeared to be potent GS-X transport inhibitors with IC50 values ranging between 0.8 and 8 muM. Luteolin and quercetin were the strongest inhibitors with IC50 values of 0.8 and 1.3 muM, respectively. Flavonoids without a C2-C3 double bond like eriodictyol, taxifolin and catechin did not inhibit GS-X pump activity. The results of this study demonstrate that the structural features necessary for high potency GS-X pump inhibition by flavonoids are (1) the presence of hydroxyl groups, especially two of them generating the 3',4-catechol moiety; and (2) a planar molecule due to the presence of a C2-C3 double bond. Other factors, like lipophilicity and the total number of hydroxyl groups do not seem to be dominating the flavonoid-mediated GS-X pump inhibition. To identify the GS-X pump responsible for the DNP-SG efflux in MCF7 cells, the effects of three characteristic flavonoids quercetin, flavone and taxifolin on MRP1 and MRP2 activity were studied using transfected MDCKII cells. All three flavonoids as well as the typical MRP inhibitor (MK571) affected MRP1-mediated transport activity in a similar way as observed in the MCF7 cells. In addition, the most potent GS-X pump inhibitor in the MCF7 cells, quercetin, did not affect MRP2-mediated transport activity. These observations clearly indicate that the GS-X pump activity in the MCF7 cells is likely to be the result of flavonoid-mediated inhibition of MRP1 and not MRP2. Altogether, the present study reveals that a major site for flavonoid interaction with GSH-dependent toxicokinetics is the GS-X pump MRP1 rather than the conjugating GSTP1-1 activity itself. Of the flavonoids shown to be most active especially quercetin is frequently marketed in functional food supplements. Given the physiological levels expected to be reached upon supplement intake, the IC50 values of the present study point at possible flavonoid-drug and/or flavonoid-xenobiotic interactions especially regarding transport processes involved in toxicokinetics. (C) 2004 Elsevier Inc. All rights reserved.

The multidrug resistance proteins MRP1 and MRP2 are efflux transporters with broad substrate specificity, including glutathione, glucuronide, and sulfate conjugates. In the present study, the interaction of the dietary polyphenol curcumin with MRP1 and MRP2 and the interplay between curcumin-dependent MRP inhibition and its glutathione-dependent metabolism were investigated using two transport model systems. In isolated membrane vesicles of MRP1- and MRP2-expressing Sf9 cells, curcumin clearly inhibited both MRP1- and MRP2-mediated transport with IC(50) values of 15 and 5 microM, respectively. In intact monolayers of MRP1 overexpressing Madin-Darby canine kidney (MDCKII-MRP1) cells, curcumin also inhibited MRP1-mediated activity, although with a 3-fold higher IC(50) value than the one observed in the vesicle model. Interestingly, MRP2-mediated activity was hardly inhibited in intact monolayers of MRP2-overexpressing MDCKII (MDCKII-MRP2) cells upon exposure to curcumin, whereas the IC(50) value in the vesicle incubations was 5 microM. The difference in extent of inhibition of the MRPs by curcumin in isolated vesicles as compared to intact cells, observed especially for MRP2, was shown to be due to a swift metabolism of curcumin to two glutathione conjugates in the MDCKII cells. Formation of both glutathione conjugates was about six times higher in the MDCKII-MRP2 cells as compared with the MDCKII-MRP1 cells, a phenomenon that could be ascribed to the significantly lower glutathione levels in the cell line. The efflux of both conjugates, identified in the present study as monoglutathionyl curcumin conjugates, was demonstrated to be mediated by both MRP1 and MRP2. From dose-response curves with Sf9 membrane vesicles, glutathionylcurcumin conjugates appeared to be less potent inhibitors of MRP1 and MRP2 than their parent compound curcumin. In conclusion, curcumin clearly inhibits both MRP1- and MRP2-mediated transport, but the glutathione-dependent metabolism of curcumin plays a crucial role in the ultimate level of inhibition of MRP-mediated transport that can be achieved in a cellular system. This complex interplay between MRP inhibition and metabolism of MRP inhibitors, the latter affecting the ultimate potential of a compound for cellular MRP inhibition, may exist not only for a compound like curcumin but also for many other MRP inhibitors presently or previously developed on the basis of vesicle studies.

Formation of quercetin quinone/quinone methide metabolites, reflected by formation of the glutathionyl quercetin adducts as authentic metabolites, was investigated in an in vitro cell model (B16F-10 melanoma cells). Results of the present study clearly indicate the formation of glutathionyl quercetin adducts in a tyrosinase-containing melanoma cell line, expected to be representative also for peroxidase-containing mammalian cells and tissues. The data obtained also support that the adducts are formed intracellular and subsequently excreted into the incubation medium and reveal for the first time evidence for the pro-oxidative metabolism of quercetin in a cellular in vitro model.

Natural antioxidants like vitamin C, vitamin E, carotenoids, and polyphenols like flavonoids, are at present generally considered to be beneficial components from fruit and vegetables. The anti-oxidative properties of these compounds are often claimed to be responsible for various beneficial health effects of these food ingredients. Together these studies provide the basis for the present rapidly increasing interest for the use of natural antioxidants as functional food ingredients and/or as food supplements. However, at higher doses or under certain conditions antioxidant-type functional food ingredients may exert toxic pro-oxidant activities. The present manuscript gives an overview of especially this pro-oxidative chemistry and toxicity of well-known natural antioxidants including vitamin C, vitamin E, carotenoids and flavonoids. © 2002 Elsevier Science B.V. All rights reserved.