effect of flavonoids on mrp1-mediated transport in panc-1 cells

Effect of Flavonoids on MRP1-Mediated Transportin Panc-1 Cells

HANG NGUYEN, SHUZHONG ZHANG, MARILYN E. MORRIS

Department of Pharmaceutical Sciences, School of Pharmacy and Pharmaceutical Sciences, University at Buffalo,The State University of New York, 517 Hochstetter Hall, Amherst, New York 14260-1200

Received 8 January 2002; revised 30 May 2002; accepted 15 July 2002

ABSTRACT: The purpose of this study was to identify the effects of dietary flavonoids,which are present in fruits, vegetables, and plant-derived beverages, on the transport ofdaunomycin (DNM) and vinblastine (VBL) in Panc-1 cells. Panc-1 is a human pancreaticadenocarcinoma cell line, which expresses Multidrug Resistance-Associated Protein1(MRP1). The 2-h accumulation of 3H-DNM and 3H-VBL was determined in the presenceand absence of 22 flavonoids. Biochanin-A, genistein, quercetin, chalcone, silymarin,phloretin, morin, and kaempferol, at 100 mM concentrations, all significantly increasedthe accumulation of bothDNMandVBL in Panc-1 cells, withmorin increasingDNMandVBL accumulation by 546� 50% (mean�SE, n¼ 9) and 553� 37% (n¼ 9), respectively.Fisetin treatment significantly decreased the accumulation of both DNM and VBL.Concentration-dependent studies demonstrated significant effects onVBL accumulationat50mM,butnot at10mMconcentrations, except for chalcone thatwaseffectiveata10mMconcentration. Following a 24-h incubation, there were no changes in MRP1 membraneexpression or glutathione-S-transferase activity in cells. Cellular glutathione (GSH)concentrations were significantly decreased following a 2-h incubation with biochanin A,chalcone, genistein, phloretin, quercetin, and silymarin, and following a 24-h incubationwith biochanin A, chalcone, genistein, and phloretin. These results therefore indicatethat the flavonoidsmorin, chalcone, silymarin, phloretin, genistein, quercetin, biochaninA, and kaempferol can inhibit MRP1-mediated drug transport, effects that may involvebinding interactions with MRP1, as well as modulation of GSH concentrations.� 2003Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 92:250–257, 2003

Keywords: flavonoids; MRP1-mediated transport; Panc-1 cells

INTRODUCTION

The major obstacle for successful chemotherapyin cancer is the development of resistance tochemotherapeutic agents, a phenomenon knownas multidrug resistance (MDR). One mechanismof MDR is the over-expression of the drug effluxmembrane pump, Multidrug-Resistance-Asso-

ciated Protein 1 (MRP1, ABCC1). MRP1 pumpsanticancer drugs out of the cytoplasm, reducingthe intracellular accumulation of these agents incancer cells.1 MRP1 is a 190-kDa protein encodedby the MRP1 gene, which is located on chromo-some 16 (16p 13.1).2,3 MRP1 may be localizedexclusively in the plasma membrane or in themembranes of intracellular organelles, dependingupon the cell type,4 and acts as a glutathioneS-conjugate efflux pump (GS-X pump), by trans-porting drugs that are either conjugated withglutathione or drugs that are cotransported withglutathione (GSH).5,6 Substrates for MRP1 areendogenous and exogenous organic anions thatare conjugated with glutathione, glucuronide, orsulfate,7 including leukotriene C4 (cysteinyl

250 JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 2, FEBRUARY 2003

Hang Nguyen’s present address is Groton Laboratories,Pfizer Inc., Eastern Point Road 8220-2356, Groton, CT 06340.

Correspondence to: Marilyn E. Morris (Telephone: 716- 645-2842, ext. 230; Fax: 716-645-3693;E-mail: [email protected])

Journal of Pharmaceutical Sciences, Vol. 92, 250–257 (2003)� 2003 Wiley-Liss, Inc. and the American Pharmaceutical Association

leukotrienes), glutathione disulfide (oxidizedglutathione), and steroid glucuronides (17b-estra-diol 17-b-D-glucuronide)1melphalan-glutathione,8

chlorambucil-glutathione,9 epipodophyllotoxins-16-glucuronide,8 and doxorubicin-glutathione.10

Natural product chemotherapeutic agents thatare not metabolized to a glutathione conjugatesuch as daunomycin, vinca alkaloids, methotrex-ate, fluorouracil, and chlorambucil11 are alsosubstrates for MRP1. These drugs are likelytransported by MRP1 in a GSH-dependent man-ner, which may involve the cotransport of GSHand the chemotherapeutic agent.12,13

MRP1 is expressed inmost tissues in thehumanbody2,14 and in several types of tumors such aslarge-cell lung tumor, small-cell lung carcinoma,colorectal carcinoma, relapsed, and secondaryacute myeloid leukemia, neuroblastoma, andbreast cancer.4,15,16 Identification of MRP1 as anATP-dependent drug efflux pump important forMDR in cancer treatment led to the search foragents that could reverse resistance due to MRP1.Potential mechanisms of MRP1 reversal include:(1) decreasing intracellular concentrations ofglutathione and/or glutathione S-transferase; (2)direct or indirect binding interactions with MRP1at substrate, ATP, or allosteric binding sites; (3)inhibition of tyrosine kinase C that is involved inphosphorylation of MRP1; and (4) downregulationof MRP1 expression. Loe and colleagues havereported that verapamil’s modulation of MRP1-mediated transport is due to the stimulation ofGSH efflux from cells and the subsequent lowerintracellular concentrations of GSH, and not dueto the inhibition of substrate binding to MRP1.17

The objective of this investigation was todetermine the effect of flavonoids on the MRP1-mediated efflux of cancer chemotherapeuticagents, to identify potential reversal agents forMDR in cancer treatment. Flavonoids are poly-phenolic compounds present in fruits, vegetables,and plant-derived beverages such as tea and redwine. Over 4000 naturally occurring flavonoidshave been described.18 Certain plants and spicescontaining flavonoids represent traditional East-ern medicines, including the widely used Ginkgobiloba. Flavonoids exist in foods as b-glycosides,although it appears that the aglycone is respon-sible for the pharmacologic activities of flavonoids.In a recent review, Middleton et al.19 concluded‘‘the flavonoids appear to be remarkably safenutrients with a wide range of biochemical andpharmacologic activities that strongly suggesttheir possible role as health-promoting, disease-

preventing dietary supplements.’’ Current inves-tigations have focused on their antioxidant proper-ties and their role as cancer preventative agents.20

In this study, we investigated the effects of22 dietary flavonoids on the accumulation ofdaunomycin (DNM) and vinblastine (VBL) inPanc-1 cells. Panc-1 is human pancreatic adeno-carcinoma cell line, which overexpresses MRP1but not P-glycoprotein (P-gp).21 Potential molecu-larmechanismsof flavonoid-inducedalterations inMRP1 transportwere also evaluated by examiningthe effect of flavonoids on the protein expression ofMRP1, on intracellular glutathione concentration,and on intracellular glutathione S-transferaseactivity.

MATERIALS AND METHODS

Materials

Verapamil, the flavonoids, daunomycin, vinblas-tine, reduced and oxidized glutathione, andglutathione S-transferase were purchased fromSigma-Aldrich. [3H]-daunomycin (DNM) (14.4 Ci/mmol) was purchased from NEN Life Scienceproducts (Boston, MA) and [3H]-vinblastine (VBL)(7.3 Ci/mmol) from Moravek Biochemicals (Brea,CA). Dulbecco’s Modified Eagle’s medium(DMEM), fetal bovine serum (FBS), and HanksBalanced Salt solution (HBSS) were from GibcoBRL (Buffalo, NY). Human pancreatic adenocar-cinoma (Panc-1) cells were purchased from theAmerican Type Culture Collection (Manassas,VA). Biodegradable counting scintillate waspurchased from Amersham Pharmacia Biotech.(Piscataway, NJ). Commassie blue dye reagentwas from BioRad Laboratory (Hercules, CA).

Cell Culture

Panc-1 cells (passages number 60–75) were cul-tured in 75-cm2 flasks with DMEM culture mediasupplemented with 10% fetal bovine serum at378C in a humidified atmosphere with 10% CO2/90% air. A solution of 0.25% trypsin-EDTA wasused to detach the cells. For the accumulationstudies, the Panc-1 cells were seeded onto 35-mm2

dishes at a density of approximately 106 cells perdish. Confluence was reached after 2 days.

3H-DNM and 3H-VBL Accumulation Studies

The accumulation studies were performed whenthe Panc-1 cells were confluent in 35-mm2 dishes.

EFFECT OF FLAVONOIDS ON MRP1-MEDIATED TRANSPORT 251

JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 92, NO. 2, FEBRUARY 2003

Cells were washed twice using assay buffer(137 mM NaCl, 54 mM KCl, 2.8 mM CaCl2,1.2 mM MgCl2, 10 mM HEPES) with pH¼ 7.4.The cells were then incubated with [3H]-DNM or[3H]-VBL at a final concentration of 0.05 mMeither alone or with flavonoids (100 mM dissolvedin DMSO) for 2 h. Verapamil (100 mM was addedas a positive control. Preliminary studies demon-strated that equilibrium conditions were achievedby 2 h. The reaction was stopped by rinsing thecells five times with ice-cold buffer (137 mMNaCl,and 14 mM Trisbase, pH¼ 7.4). Cells were thensolubilized using a solution of 0.3N NaOH and 1%SDS, and aliquots (150 mL) were used to deter-mine radioactivity by liquid scintillation counting(1900 CA, Tri-Carb Liquid Scintillation Analyzer,Packard) and protein content using the Bioradmethod with bovine g-globulin as a standard(Hercules, CA). Results were normalized for theprotein content of the cells in each dish and ac-cumulation was expressed as pmol/mg protein oras percent control. The accumulations of 0.05 mMof [3H]-DNM and [3H]-VBL at 2 h in the absence offlavonoids (solvent control) were compared to thecellular accumulation observed in the presence offlavonoids using at least six determinations foreach treatment.

Western Analysis of MRP1 and P-gp

Cells grown in 75-cm2 plastic flasks were washedwith PBS, and then detached by scraping. Totalcell lysates were prepared by lysing harvestedcells in 10 mM KCl, 1.5 mM MgCl2, and 10 mMTris-HCl (pH 7.4) and 0.2% (w/v) SDS sup-plemented with 1 mM phenylmethylsulfonyl fluo-ride, leupeptide (2 mg/mL), pepstatin (1 mg/mL),and aprotinin (2 mg/mL),22 followed by sonifica-tion. Protein concentration was determined witha Bio-Rad protein assay using bovine g-globulin asa standard. Proteins were electrophoresed on7.5% SDS-acrylaminde gels and electroblottedon nitrocellulose membrane. The membrane wasblocked overnight at 48C in PBS containing 0.2%Tween-20 and 1% BSA, incubated with theprimary antibody MRPr1 (Kamiya BiomedicalCo., Seattle, WA) 1:30 in blocking buffer, or withC219 (1 mg/mL) for P-gp detection, for 2 h at roomtemperature. The membrane was then rinsedwith washing buffer (PBS pH 7.4 with 0.05%Tween-20) and incubated for 2 h with secondaryantibody, antirat IgG (Zymed, San Francisco,CA), 1:1000 in blocking buffer(for MRPr1) or1:1500 (v/v) antimouse IgG HRP secondary anti-

body (Amersham) (for C219). After washing,the protein was detected using the ECL detec-tion reagent (Amersham RPN 2106). Kodak 1Dimage analysis software was used to analyze theWestern blot results.

GST Activity Studies

The cell supernatant was prepared as describedby Vanhoefer et al.23 Briefly, cells were grown in75-cm2 plastic flasks until confluent. Rhoifolin,morin, biochanin-A, chalcone, phloretin, genis-tein, quercetin, or silymarin were preincubatedfor 24 h in protein-free media. After this 24-hpreincubation period, cells were washed twicewith PBS, and then detached by scraping. Cellswere lysed by sonication in a 5 mM dipotassiumhydrogen phosphate buffer and ultracentrifugedat 140,000� g at 48C for 45 min. GST activity wasdetermined by measuring the conjugation ofCDNB (1-chloro-2, 4-dinitrobenzene) with GSHusing a U-2000 spectrophotometer at 340 nm(Hitachi Ltd, Tokyo, Japan). A unit of enzymaticactivity is defined as that forming 1 mmol ofproduct per minute at 258C. The molar extinctioncoefficient of the CDNB-GSH adduct is 9.6 mM�1

cm�1.24 Total protein concentrations were mea-sured using a BioRad assay kit. The results wereexpressed as nmol �min�1/mg protein.

GSH Concentration Studies

Cells were grown in 35-mm2 plastic dishes untilconfluent. Rhoifolin, morin, biochanin-A, chal-cone, phloretin, genistein, quercetin, or silymarinwere preincubated for 2 h or 24 h in protein-freemedia. After these preincubation periods, cellswere washed three times with D-PBS, and thendetached by scraping in 5% sulfosalicylic acid.Cells were lysed by sonication for 1 min in 5%sulfosalicylic acid and centrifuged at 5000� g at48C for 10 min. The supernatant was used todetermine the cellular GSH concentration usingan enzymatic recycling procedure.25 Control ex-periments examined the effect of the flavonoidsthemselves on known concentrations of GSHdetermined by this assay, and found no effect.

Statistical Analysis

The differences between the mean values wereanalyzed for significance using an ANOVA testfollowed by a Dunnett’s post hoc test. p-values

252 NGUYEN, ZHANG, AND MORRIS


<0.05 were considered statistically significant.Data are presented as mean�SE, n¼ 6–9.

RESULTS

Panc-1 Cells

Western analysis of Panc-1 cells demonstratedthat the presence of MRP1 at 190 kDa. We alsoconfirmed the absence of P-gp in Panc-1 cells(Figure 1), as previously reported byMiller et al.21

Effect of Flavonoids on DNM Accumulation

To examine whether various dietary flavonoidshad any effect on the MRP1-mediated efflux ofDNM, the 2-h accumulation of DNM was mea-sured in Panc-1 cells in the presence and absenceof flavonoids. Preliminary studies examined theuptake of 0.05 mM of VBL into Panc-1 cells fortimes up to 3 h, and demonstrated that equili-brium was reached by 2 h. Verapamil (100 mM)was used as a positive control and increased theaccumulation of DNM in all studies. All theflavonoids were used at a concentration of100 mM. The accumulation of DNM was signifi-cantly increased when cells were treated with100 mM of morin, biochanin-A, chalcone, sily-marin, phloretin, genistein, quercetin, chrysin, orkaempferol, and significantly decreased by api-genin, fisetin, galangin luteolin, myricetin, andrhoifolin (Table 1, Figure 2). The other flavonoids

Figure 1. Western blots of P-gp and MRP1 in Panc-1cells, using the antibodies C219 and MRPr1, respec-tively (as described under Methods). The human breastcancer cell lineMCF-7/ADR (which expresses P-gp andasmall amount ofMRP1) is used for comparison purposes.

Table 1. Effect of Flavonoids on 3H-DNM and 3H-VBL Accumulation in Panc-1 Cells

Flavonoids

3H-DNMAccumulation(% Control,Mean�SE,n¼ 6–9)

3H-VBLAccumulation(% Control,Mean�SE,n¼ 6–9)

Apigenin 62.3� 5.3b 184� 4.9b

Biochanin A 178� 11.1b 330� 20.3b

Chalcone 241� 8.4b 342� 19.3b

Chrysin 198� 26.0b 180� 19.4b

Daidzein 102� 5.5 126� 8.9Diosmin 116� 8.4 106� 6.9Epigallocatechin 108� 6.8 164� 7.9b

Fisetin 59.6� 8.8b 53.6� 3.1b

Galangin 67.9� 14.4a 102� 14.8Genistein 156� 9.7a 191� 11.2b

Hesperetin 126� 2.4 132� 8.8Kaempferol 280� 17.1b 196� 6.3b

Luteolin 65.3� 5.6b 128� 11.5Morin 546� 50.1b 553� 36.7b

Myricetin 76.8� 2.8b 87.7� 9.5Naringenin 112.9� 4.7 89.5� 2.0Naringin 94.5� 3.4 109� 5.1Phloretin 295� 49.6b 251� 9.8b

Quercetin 199� 11.6b 268� 14.5b

Rhoifolin 58.2� 3.5b 120� 6.8Rutin 107� 2.2 71.8� 3.5b

Silymarin 305� 12.6b 329� 27.4b

ap< 0.05.bp<0.01.Equilibrium concentrations of DNM and VBL were 1–

3 pmol/mg protein.

Figure 2. Effect of flavonoids on 3H-DNM accumula-tion in Panc-1 cells. Those flavonoids that significantlyincreased DNM accumulation are shown in this figure.Verapamil is a positive control. Cells were incubatedwith 3H-DNM alone and in the presence of 100 mM offlavonoids and the accumulation of 3H-DNM wasmeasured after 2 h of incubation. All are significantlydifferent from control. Data are mean�SE, n¼ 6–9.



tested did not significantly alter DNM accumula-tion in Panc-1 cells compared with the control.

Effect of Flavonoids on VBL Accumulation

The effect of flavonoids on the accumulation ofVBL was determined in similar manner as forDNM, with verapamil (100 mM) used as a positivecontrol. The accumulation of VBL in Panc-1 cellswas greater than that observed with DNM.Morin,biochanin-A, chalcone, silymarin, phloretin, gen-istein, quercetin, apigenin, epigallocatechin, andkaempferol all significantly increased the accu-mulation of VBL (Table 1, Figure 3) in Panc-1cells. Morin produced the greatest degree of VBLaccumulation (554� 50.1% of control, mean�SE,n¼ 6). Fisetin and rutin significantly decreasedthe accumulation of VBL in Panc-1 cells. For otherflavonoids, the effect on VBL accumulation wasnot significant compared with the control. Therewas a significant correlation seen between inhibi-tion of DNM transport and inhibition of VBLproduced by the various flavonoids (r2¼ 0.803,p< 0.001) (Figure 4 ).

The concentration-dependent effects of morin,biochanin-A, chalcone, silymarin, andphloretin onthe 2-h accumulation of 3H-VBL in Panc-1 cellswere examined at concentrations of 0.1, 1, 10, 50,

and 100 mM.All compounds had a significant effectat 50 mM and above, but were ineffective at aconcentration of 10 mM or below (data not shown),except for chalcone that showed significant inhibi-tion at a concentration of 10 mM.

Effect of Flavonoids on Protein Expression

MRP1 protein expression was determined follow-ing a 24-h preincubation period. There were nosignificant effects on protein expression for bio-chanin A, chalcone, phloretin, quercetin, morin,genistein, rhoifolin, and silymarin (data notshown).

Effect of Flavonoids on CellularGlutathione Concentration

Following a 2-h preincubation of Panc-1 cellswith various flavonoids, biochanin A, chalcone,genistein, phloretin, quercetin, and silymarin allsignificantly decreased cellular GSH concentra-tions; morin had no effect. Following a 24-hpreincubation the findings were similar withbiochanin-A, chalcone, genistein, phloretin, andquercetin all decreasing cellular GSH concentra-tions. Only morin increased the GSH con-centration to 131% of the control value at 24 h(Table 2).

Effect of Flavonoids on GlutathioneS-Transferase Activity

The cellular GST activity was determined aftercells were preincubated for 24 h with flavonoids.There were no statistically significant effects onGST activity (data not shown).

Figure 3. Effect of flavonoids on 3H-VBL accumula-tion in Panc-1 cells. Those flavonoids that significantlyincreased VBL accumulation are shown in this figure.Verapamil is a positive control. Cells were incubatedwith 3H-VBL alone and in the presence of 100 mM offlavonoids and the accumulation of 3H-VBL was mea-sured after 2 h of incubation. All are significantlydifferent from control. Data are mean�SE, n¼ 6–9.

Figure 4. Correlation between 3H-DNM accumula-tion and 3H-VBL accumulation in the presence ofvarious flavonoids (r2¼ 0.803, p< 0.001).



DISCUSSION

MRP1 is overexpressed in some tumor cells4,16

and its ATP-dependent efflux of drugs from cellsresults in resistance to many chemotherapeuticagents. MRP1 expression in breast cancer is animportant predictor of poor prognosis.26 Com-pounds that alter the expression and/or functionalactivity of MRP1 will produce changes in thecellular accumulation of chemotherapeutic agentsthat are substrates for MRP1. In this study, weexamined 22 flavonoids, which are from fivedifferent flavonoid subclasses: flavonols, flavanol,isoflavonoids, flavone, and flavanones, and deter-mined their effects on the MRP1-mediated effluxof DNM and VBL in Panc-1 cells.

Little is known regarding the effects of flavo-noids on MRP1-mediated efflux, but it might besurmised that these plant-derived polyphenoliccompounds may represent natural substrates forMRP1. In this investigation, following a 2-hincubation period, a number of flavonoids increas-ed the accumulation of both DNM and VBL inPanc-1 cells, while one flavonoid (fisetin) decreas-ed the accumulation of both DNM and VBL. Asignificant correlation was noted for the percentinhibition of these two substrates (r2¼ 0.80).Significant increases in the accumulation of DNMoccurred following treatment with nine flavonoidsand for VBL with 10 flavonoids. The greatesteffects were seen with morin; morin increased theaccumulation of both DNM and VBL to greaterthan 500% of the control value. The flavonoideffects were concentration-dependent, with signi-

ficant inhibition seen at 50 mM concentrations formorin, biochanin A, and silymarin, and at 10 mMconcentrations for chalcone. Our results are con-sistent with those of Zhang and Wong27 whoreported that quercetin, 20-hydroxychalcone, 2-hydroxychalcone and morin have IC50s in the mMrange for the inhibition of dinitrophenylglutath-ione efflux in human colon carcinoma cells thatexhibit MRP1-mediated efflux. Recently, Leslieet al.28 reported that the flavonoids kaempferol,apigenin, quercetin, myricetin, and naringeninare effective inhibitors of leukotriene C4 (LTC4)transport in membrane vesicles isolated fromMRP1-transfected HeLa cells. Isoflavonoids havebeen reported to interact with MRP1 increasingATPase activity and daunorubicin accumulation,effects that can be inhibited by a specific antibody(MIB6) to MRP1.29

Following a 2-h incubation with biochanin A,chalcone, genistein, quercetin, silymarin, orphloretin, cellularGSH concentrationswere signi-ficantly decreased; GSH concentrations were un-changed after morin treatment. Because GSH is acosubstrate for the transport of chemotherapeuticagents, decreased GSH concentrations mightbe associated with decreased MRP1-mediatedefflux and increased accumulation of substrates.The effect of GSH on MRP1-mediated transportis concentration-dependent and inhibited byMRP1-specific antibodies.7 Buthionine sulfoxi-mine (BSO) treatment can reverse the MRP1-mediated efflux of some natural product drugs bydepleting cellularGSHconcentrations.Thealtera-tions in GSH accumulation found in this studyfollowing a 2-h flavonoid treatment suggest thatalterations in cellular GSH may represent onemechanism involved in the MRP1-mediated inhi-bition of some flavonoids. Morin treatment had noeffect on cellularGSH concentrations, so its effectsare likely mediated by different mechanisms.

Following a 24-h incubation period and exten-sive washing to remove any remaining flavonoid,membraneMRP1 protein expression and intracel-lular GSH and GST concentrations were examin-ed. No significant changes in MRP1 expression inmembrane preparations were found. Additionally,no effects on cellular GST concentrations werefound; this conflicts with the findings of Zhanget al.,30 who reported that a number of flavonoidsinhibit the activity of GST. However, GST is notimportant in the metabolism of DNM or VBL,and it is not a glutathione conjugate of these drugsthat is transported byMRP1.31 On the other hand,GSH concentrations were significantly decreased

Table 2. Effects of Flavonoids on Intracellular GSHConcentrations

Flavonoids (100 mM)

Relative Intracellular GSH Level

2 h 24 h

Control 1.00� 0.09 1.00� 0.05Biochanin A 0.69� 0.01b 0.24� 0.07c

Chalcone 0.70� 0.06b 0.53� 0.03c

Genistein 0.53� 0.09c 0.47� 0.07c

Morin 0.72� 0.18 1.31� 0.18a

Phloretin 0.53� 0.06c 0.46� 0.04c

Quercetin 0.40� 0.40c 0.66� 0.09b

Rhoifolin ND 0.79� 0.14Silymarin 0.620� 0.08b 0.96� 0.06

Mean�SE, n¼3 separate cell preparations.ap<0.05.bp<0.01.cp<0.001.ND: Not determined.



following biochanin A, chalcone, genistein, andphloretin treatment, and increased after morintreatment.

In conclusion, the results in the present studyprovide evidence that flavonoids such as morin,chalcone, silymarin, phloretin, genistein, querce-tin, rhoifolin, fisetin, and kaempferol are capableof modulating MRP1-mediated drug transport ofDNM and VBL. We found that flavonoids couldincrease, decrease, or have no effect on the ac-cumulation of DNM and VBL in Panc-1 cells. Theconcentrations necessary for inhibition are higherthan the concentrations of flavonoids reported inplasma (1 mM or less) achieved after dietaryintake,20 although intestinal concentrations arelikely higher. However, the inhibitory concentra-tions will vary with the MRP1 substrate used inthe investigations, and Leslie et al.28 reportedIC50s of 2.4–21 mM for inhibition of LTC4 trans-port. We identified nine flavonoids, with represen-tatives from all chemical classes of flavonoids,that inhibited MRP1-mediated efflux after acuteexposure; all of these flavonoids, except morin,produced significant decreases in cellular GSHconcentrations following a 2-h exposure. Themechanisms underlying the inhibition of MRP1-mediated efflux by flavonoids are unknown,but likely involve direct binding interactions, aswell as indirect effects including altered GSHconcentrations.

ACKNOWLEDGMENTS

This work was previously presented as a posterpresentation at the American Assoc. of Pharma-ceutical Scientists Annual Meeting November2–6, 2000, and published in Pharm. Res. Suppl.2000. Financial support for this study wasprovided by grants from the New York StateHealth Research Science Board (EMPIRE grant),Susan G. Komen Breast Cancer Foundation, andU.S. Army Breast Cancer Research ProgramContract DAMD17-00-1-0376. We thank Nghi U.Duong, Jianying Xiao and Elizabeth Raybon fortechnical assistance.

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effect of flavonoids on mrp1-mediated transport in panc-1 cells

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