Inhibitory Activity of a Green Tea Extract and some of its Constituents on Multidrug Resistance-Associated Protein 2 Functionality

• Abstract
• Introduction
• Materials and Methods
• Materials
• Cell cultures
• Colorimetric cytotoxicity assay
• Real-time polymerase chain reaction (TaqMan assay)
• Autofluorescence and quenching
• Functional assays
• Data analysis
• Results
• Discussion
• Acknowledgements
• References

Abstract

Green tea extracts (GTE) might modulate ABC transporter gene expression or function. This may be relevant in the treatment of cancer or in influencing intestinal drug permeability. To gain more insight on the influence of a GTE on secretory transport proteins we investigated the influence of GTE and several green tea components on the mRNA expression level of P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MRP2) in human gastrointestinal epithelial LS-180 cells. Furthermore, the functional activity of MRP2, using glutathione methylfluorescein (GS-MF) or [³ H]methotrexate (MTX) as substrate, was investigated in canine kidney cells stably overexpressing human MRP2 (MDCK-MRP2). GTE, at a concentration of 0.01 mg/mL, did not increase mRNA expression of P-gp or MRP2 in LS-180 cells. Functional assays in MDCK-MRP2 cells using GS-MF did not show any effect of 0.01 mg/mL GTE on MRP2 activity. In the same cell line the cellular accumulation of MTX (a specific substrate of MRP2) was significantly increased with the MRP-specific inhibitor MK-571 or with 1 mg/mL GTE, but not with 0.1 mg/mL. The green tea components (-)-epigallocatechin gallate, (-)-epigallocatechin, theanine, or caffeine, each in corresponding concentrations to the respective concentration of GTE, did not show any effect on MRP2 function. These data demonstrate that the mRNA expression patterns of P-gp and MRP2 in LS-180 cells are not altered by 0.01 mg/mL of GTE. However, MRP2 function was inhibited by 1 mg/mL GTE, whereas none of the green tea components tested were responsible for this effect.

Introduction

Transmembrane transport proteins play a crucial role for the maintenance of the barrier function of the intestinal epithelium, which is critical for the disposition as well as the cytotoxicity of xenobiotics. Accordingly, enterocyte-derived cell lines, such as LS-180 cells, are provided with different transport systems including the ATP-binding cassette (ABC) transporters P-glycoprotein (P-gp) and the multidrug resistance protein (MRP) family [1], [2], [3], [4]. P-gp, which is encoded by the MDR1 gene, is located in the apical membrane of enterocytes and acts as an efflux pump that extrudes many clinically important drugs thereby limiting their oral bioavailability [5],[6]. Multidrug resistance-associated protein 2 (MRP2) is localized in the apical membrane of polarized cells and transports various glutathione S-conjugates and several anticancer drugs including methotrexate [7], [8], [9], [10], [11].

Green tea is one of the most popular beverages worldwide and several beneficial/protective effects on life-style related diseases, including anticarcinogenic activities, are being attributed to its consumption [11], [12]. Due to the increasing use of green tea preparations as food supplements or drugs, possible influences on drug transporter systems have to be assessed. Recently, some green tea polyphenols, namely (-)-epigallocatechin gallate (EGCG), (-)-epicatechin gallate, and (-)-catechin gallate, have been shown to interact with P-gp and to inhibit its function [13], whereas for (-)-epicatechin a concentration-dependent functional activation of P-gp was reported [14]. Other green tea components are able to inhibit the efflux of the anticancer drug doxorubicin (DOX), a substrate of P-gp and MRP2 [2], [3], [15], [16].

Thus, GTE might modulate ABC transporter gene expression or function. This may be relevant in the treatment of cancer or in influencing intestinal drug permeability. Therefore, this study focused on the effect of GTE on P-gp or MRP2 mRNA expression in cultured intestinal LS-180 cells and on the influence of GTE or different green tea components (Fig. [1]) on MRP2 functional activity.

Materials and Methods

Materials

Standardized green tea special extract EFLAr942 (GTE) was obtained from Frutarom Switzerland Ltd., Wädenswil, Switzerland. Brief manufacturing description: leaves of Camellia sinensis (L.) O. Kuntze are continuously extracted (percolation) with 80 % (m/m) ethanol. After a patented filtration process (US Patent 6,024,998) the crude extract is dried. Finally, 5 % m/m maltodextrin is added as carrier. The drug to extract ratio (DER) is 5.5 : 1. Characteristic components in the extract are polyphenols (47.5 - 52.5 % m/m), caffeine (5.0 - 10.0 % m/m), theobromine (0.30 - 1.20 % m/m), and theanine (1.0 - 3.0 % m/m). (-)-Epigallocatechin gallate (EGCG) was from CHEMOS GmbH, Regenstauf, Germany, (-)-epigallocatechin (EGC) was from Sigma-Aldrich Chemie GmbH, Steinheim, Germany. In the batch used the following concentrations of constituents in 0.01 mg/mL GTE were determined: 4.02 μM EGCG; 2.27 μM EGC; 4.36 μM caffeine; 0.99 μM theanine. MK-571 was from Biomol, Plymouth Meeting, PA, USA. Chloromethylfluorescein diacetate (CMFDA) was from Molecular Probes, Eugene, OR, USA; [³ H]methotrexate (MTX) was from Movarek Biochemicals, CA, USA; [¹⁴ C]sucrose was from Amersham, UK. All other chemicals were obtained from commercial sources in the highest quality available.

Cell cultures

The human colon adenocarcinoma cell line LS-180 was purchased from American Tissue Culture Collection (ATCC, Manassas, USA) and cultured in Dulbecco’s MEM with Glutamax-I, supplemented with 10 % (v/v) fetal calf serum (FCS), 1 % non-essential amino acids, 1 % sodium pyruvate and 50 μg/mL gentamycin; Madin Darby canine kidney (MDCK) cells stably overexpressing human MRP2 (MDCK-MRP2) were a kind gift from Dr. Evers (The Netherlands Cancer Institute, Amsterdam, Netherlands) and were cultured in Dulbecco’s MEM with Glutamax-I, in the presence of 50 μg/mL gentamycin. Per cell culture well, 10⁶ cells were distributed. All cultures were maintained in a humidified 37 °C incubator with 5 % carbon dioxide in the air atmosphere.

Colorimetric cytotoxicity assay

Cytotoxicity of GTE was screened in LS-180 or MDCK-MRP2 cells at confluence. LS-180 cells were incubated for 72 hours in the absence or presence of 0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, and 3 mg/mL GTE, respectively. MDCK-MRP-2 cells were incubated for 30 minutes with or without 0.01, 0.1, and 1 mg/mL GTE, or corresponding concentrations (4.02, 40.2, and 402 μM) of EGCG. Replacement with medium containing the compound of interest was done every 24 h. Only freshly prepared and filtered (0.2 μm) mixtures of GTE with medium were used.

Cultures fixed with trichloroacetic acid were stained for 30 minutes with 0.4 % sulforhodamine B (SRB) dissolved in 1 % acetic acid. Unbound dye was removed by washing four times with 1 % acetic acid and protein-bound dye was extracted with 10 mM Tris buffer. Absorption was measured at 540 nm. (Spectra MAX 250, Microplate Spectrophotometer, Molecular Devices Corporation, California, USA) [17]. SRB absorption intensity correlated linearly with the number of living cells.

Real-time polymerase chain reaction (TaqMan assay)

For mRNA induction experiments LS-180 cells at confluence were incubated either with or without 10 μM rifampicin or with 0.01 mg/mL GTE for 72 h. Replacement with freshly prepared medium containing the compound of interest took place every 24 hours.

After removal of the medium at the end of the culture period, the cells were treated with lysis buffer RLT (Qiagen) and 1 % β-mercaptoethanol (Sigma, St. Louis, USA) was added. The total amount of RNA was extracted by using the RNeasy™ Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. RNA concentration was measured with a Gene Quant photometer (Pharmacia, Uppsala, Sweden). The purity of the RNA preparations was high, as demonstrated by the 260/280 nm ratio (range 1.7 to 2.0). DNA was digested by using the DNA-Digestion Kit with DNase I Amplification Grade, 10 × DNase I Reaction Buffer and 25 mM EDTA (Invitrogen, Life Technologies Basel, Switzerland) according to the manufacturers’ protocol.

2 μg of total RNA were reverse transcribed to cDNA by using SuperScript ^TM II RT, 0.1 M DTT, 5 × Strand Buffer, oligonucleotides (Invitrogen, Life Technologies) and Random Hexamer (Applied Biosystems, Rotkreuz, Switzerland). Reverse transcription was done according to the manufacturers’ protocol.

For DNA digestion as well as for reverse transcription the Eppendorf Mastercycler Personal heating cyclometer was used. All RNA isolation procedures and reverse transcriptions were performed under RNase-free conditions (RNeasy Erase Spray, ICN Biochemicals, Inc. Ohio, USA), freshly prepared water from highest quality was used (Milli Q, 18.2 mOhm, Kantonsspital Basel, Switzerland).

25 ng of complementary DNA were used as a template for real-time quantitative PCR analysis. The cDNA was amplified in a 25 μL volume containing Master-Mix (TaqMan® Universal PCR Master Mix, Applied Biosystems, USA), water, forward primer, reverse primer (Eurogentec, Seraing, Belgium) and probe (Invitrogen, Basel, Switzerland) mixed according to the manufacturer’s protocol.

The assay was performed using a Gene Amp 5700 Sequence Detector (Applied Biosystems, Rotkreuz, Switzerland), which combines a thermocycler and a fluorescence detector. Each sample of the reaction mixture was amplified during 40 cycles (15 sec at 90 °C, 1 min at 60 °C). As a negative control not-transcribed total RNA was used. For P-gp detection the following primers and probe were used: 5′-AAGCTGTCAAGGAAGCC-AATGCCTATGACTT-3′ (probe), 5′-CTGTATTGTTTGCCACCACGA-3′ (forward), and 5′-AGGGTGTCAAATTTATGAGGCAGT-3′ (reverse). For MRP2 detection the following primers and probes were used: 5′-CTCAATATCACACAAACCCTGAACTG-GCTG-3′ (probe), 5′-ACTGTTGGCTTTGTT CTGTCCA-3′ (forward), and 5′-CAACA-GCCACAATGTTGGTCTCTA-3′ (reverse).

A relative standard curve was generated by serial dilutions of cDNA. Fragments of cDNA corresponding to MDR1 and MRP2 that covered the TaqMan primer/probe area were obtained by PCR amplification. All of the DNA standards were quantified using the Pico Green reagent (Molecular Probes). For absolute quantification, Ct values of standards were plotted against the log of the respective dilution factors. Slope and y-intercept of the standard curve line were then calculated by linear regression. Each standard curve was generated from a known amount of corresponding cDNA and was then used to calculate the input amount for unknown samples for respective genes. Absolute quantification was done for every experiment.

Autofluorescence and quenching

Changes of fluorescence intensity of the transport buffer, Hank’s balanced salt solution (HBSS, Gibco, Basel, Switzerland) were analyzed without cells in the presence of 0.01, 0.1 or 1 mg/mL GTE or the corresponding concentrations of EGCG, EGC, theanine or caffeine.

Changes in fluorescence of a fixed methylfluorescein (MF) concentration of 50 nM were analyzed in the presence of 0.01, 0.1 or 1 mg/mL GTE or the corresponding concentrations of EGCG, EGC, theanine or caffeine. Experiments were performed at room temperature. Fluorescence was measured with an HTS 7000 Plus Bio Assay Reader (Perkin Elmer Ltd., Buckinghamshire, UK).

Functional assays

Functional assays for MRP2 transport were performed at 37 °C using confluent monolayers of MDCK-MRP2 cells, which were grown in 24-well cell culture plates. Cells were washed twice with HBSS.

In the assays using CMFDA as substrate, cells were preincubated with 10 μmol/L CMFDA for 1 hour at 10 °C. Thereafter cells were washed twice with ice-cold HBSS and kept at 10 °C in HBSS with 10 % sodium pyruvate. CMFDA is metabolized in the cells to glutathione-methylfluorescein (GS-MF). Then, the GS-MF efflux from the cells was measured at 37 °C by incubating with medium containing 10 % sodium pyruvate in the absence or presence of 20 μM MK-571, 0.01 mg/mL GTE, or the corresponding concentrations of EGCG, EGC, theanine, or caffeine in concentrations corresponding to 0.01, 0.1, or 1 mg/mL GTE. After 5, 10, 15, 20, 25 and 30 minutes, 200 μL samples were removed and fluorescence was measured with an HTS 7000 Plus Bio Assay Reader (Perkin Elmer Ltd., Buckinghamshire, UK). Cellular accumulation after 30 minutes was assessed by lysing the cells with 1 % Triton X-100 in PBS and fluorescence determination of the cell homogenate.

Using MTX as substrate, cells were washed twice with HBSS and preincubated without tracers in the presence or absence of 20 μM MK-571, 0.1 or 1 mg/mL GTE, or the corresponding concentrations of EGCG for 10 minutes at 37 °C. Then cells were incubated under the same conditions in the presence of 11.8 μM MTX (0.3 μCi) and 488 μM [¹⁴ C]sucrose (0.3 μCi) for 30 minutes at 37 °C. For quantification of the cellular accumulation of MTX and [¹⁴C]sucrose the cells were lysed with trypsin-EDTA and transferred to a vial containing 3 mL of scintillation cocktail (Insta-Gel, Packard Instrument B.V., NL). Finally, the radioactivity was measured.

Data analysis

For statistical comparison, data of groups were compared by analysis of variance (ANOVA). The level of significance was P = 0.05. If this analysis revealed significant differences, pairwise comparisons within groups were performed by two-sided unpaired t tests. P values were adjusted by Bonferroni’s correction for multiple comparisons.

Results

The cytotoxicity assay with LS-180 cells was performed to determine the concentration range of GTE that was without toxic effects (Fig. [2] A). Significant toxic effects were observed at 0.3 mg/mL GTE. Since toxicity itself might influence the expression level of transport proteins, it is important that the GTE concentrations used in mRNA expression studies exhibit no cellular toxicity. None of the GTE or EGCG concentrations exhibited toxicity on MDCK-MRP2 cells after incubation for 30 minutes (Fig. [2] B).

At confluence, LS-180 cells were incubated for 72 h with medium only, 10 μM rifampicin or with 0.01 mg/mL GTE. Quantitative real-time PCR was performed to determine the mRNA expression of MDR1 and MRP2. Rifampicin showed a significant induction of MDR1 mRNA expression, whereas GTE did not alter MDR1 mRNA levels. MRP2 mRNA expression was not influenced by GTE. Unfortunately, rifampicin did not serve as a positive control and elevated MRP2 mRNA expression only slightly but not significantly (Figs. [3] A and B).

To investigate the influence of GTE or the green tea components EGCG, EGC, theanine, or caffeine on the transport activity of MRP2, kinetic assays were performed in MDCK-MRP2 cells overexpressing MRP2. The cells were loaded with CMFDA, which is metabolized to glutathione-methylfluorescein (GS-MF), a specific substrate of MRP2. MK-571, a specific MRP-inhibitor, significantly increased the cellular accumulation of GS-MF, indicating a blockage of MRP2 function by MK-571. The cellular accumulation of GS-MF was not altered by 0.01 mg/mL GTE or the corresponding concentration of EGCG (Figs. [4] A and B). These results were confirmed by measurement of the extrusion of GS-MF, which was significantly decreased after treatment with MK-571, whereas 0.01 mg/mL GTE or the corresponding concentration of EGCG did not alter the extrusion of GS-MF in a relevant manner (Fig. [4] C and D). Concentrations of EGC, theanine or caffeine corresponding to 0.01, 0.1 and 1 mg/mL GTE showed no effect on GS-MF accumulation (Figs. [5] A - C).

Due to significant autofluorescent activity of GTE at concentrations of 0.1 and 1 mg/mL as well as significant quenching activities of 0.1 and 1 mg/mL GTE or the corresponding EGCG concentrations with GS-MF (data not shown), functional assays in MDCK-MRP2 cells with these concentrations were performed with MTX as substrate (Fig. [6]). Therefore, cells were incubated with MTX, which is transported out of the cell via MRP2 but not via P-gp. MK-571 significantly decreased the efflux of MTX. GTE, at a concentration of 1 mg/mL, significantly increased the cellular accumulation of MTX by a factor of 1.7, suggesting a functional inhibition of MRP-2. Neither the corresponding concentration of EGCG nor the lower concentration of GTE or EGCG exerted an influence on MRP2 activity. The integrity of the monolayer barrier was not affected in these experiments as demonstrated by the measurement of [¹⁴ C]sucrose transport.

Discussion

Intestinal cellular drug availability might be strongly modulated by P-gp and MRP2 function. Both are abundantly expressed in intestinal epithelial cells and show partly overlapping substrate specificities [18], [19]. The antibiotic rifampicin has been shown to significantly increase intestinal MDR1 and MRP2 expression [6], [20]. To investigate the induction of MDR1 mRNA expression, the LS-180 cell line has been shown to be a suitable model [21], [22]. However, the induction of MRP2 has not been demonstrated yet. Recently, inhibitory effects on P-gp function by green tea components have been reported in the literature [13], [16], whereas no data about the influence of green tea on MRP2 are available yet. Therefore, this study was designed to investigate the influence of GTE on the expression pattern of MDR1 and MRP2 as well as to examine the potential effect of GTE and several green tea components on the functional activity of MRP2.

The cytotoxicity assay demonstrated that 0.01 mg/mL GTE was in the non-toxic range for LS-180 cells after an incubation for 72 h. Thus, artifacts of mRNA induction experiments due to cellular detoxification activities were prevented. No significant induction of MDR1 or MRP2 mRNA expression by GTE was observed in LS-180 cells. Regarding the influence on MRP2 protein functionality in MDCK-MRP2, neither 0.01 to 0.1 mg/mL GTE nor the green tea components EGCG, EGC, caffeine, or theanine, each in corresponding concentrations to GTE, exerted any appreciable influence on the MRP2-mediated export in comparison to the MRP-specific inhibitor MK-571. At a concentration of 1 mg/mL, GTE significantly inhibited MRP2 activity, whereas the green tea components used above did not show any effect. All GTE concentrations used for MDCK-MRP2 cells were in the non-toxic range.

In brief, 0.01 mg/mL GTE did not modulate mRNA expression of MRP2 or MDR1 in intestinal epithelial LS-180 cells. Therefore, our results indicate that neither the intestinal absorption through MRP2 nor through P-gp is altered by a change in gene transcription by 0.01 mg/mL GTE. The efflux of MRP2-substrates including GS-MF and MTX was inhibited only at a high concentration of 1 mg/mL GTE. EGCG is the principal catechin in green tea and most of the effects associated with green tea might be mediated by this catechin. It is tempting to speculate that other green tea components besides EGCG, EGC, caffeine, or theanine are responsible for the demonstrated inhibition of MRP2 function.

Acknowledgements

We are grateful to U. Behrens for excellent technical assistance. This work was supported by an unconditional research grant of Frutarom Switzerland Ltd., 8820 Waedenswil, Switzerland.

References

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Juergen Drewe, MD, MSc

Department of Clinical Pharmacology and Toxicology

University Clinic Basel

Kantonsspital

Petersgraben 4

4031 Basel

Switzerland

Phone: +41-61-265-3848

Fax: +41-61-265 8581

Email: juergen.drewe@unibas.ch

References

• 1 Gottesman M M, Pastan I. Biochemistry of multidrug resistance mediated by the multidrug transporter.  Annu Rev Biochem. 1993;  62 385-427
  CrossrefPubMedSearch in Google Scholar
• 2 Ambudkar S V, Dey S, Hrycyna C A, Ramachandra M, Pastan I, Gottesman M M. Biochemical, cellular, and pharmacological aspects of the multidrug transporter.  Annu Rev Pharmacol Toxicol. 1999;  39 361-98
  CrossrefPubMedSearch in Google Scholar
• 3 Borst P, Evers R, Kool M, Wijnholds J. A family of drug transporters: the multidrug resistance-associated proteins.  J Natl Cancer Inst. 2000;  92 1295-302
  CrossrefPubMedSearch in Google Scholar
• 4 Keppler D, Leier I, Jedlitschky G. Transport of glutathione conjugates and glucuronides by the multidrug resistance proteins MRP1 and MRP2.  Biol Chem. 1997;  378 787-91
  PubMedSearch in Google Scholar
• 5 Kim R B, Fromm M F, Wandel C, Leake B, Wood A J, Roden D M. et al . The drug transporter P-glycoprotein limits oral absorption and brain entry of HIV-1 protease inhibitors.  J Clin Invest. 1998;  101 289-94
  CrossrefPubMedSearch in Google Scholar
• 6 Greiner B, Eichelbaum M, Fritz P, Kreichgauer H P, von Richter O, Zundler J. et al . The role of intestinal P-glycoprotein in the interaction of digoxin and rifampin.  J Clin Invest. 1999;  104 147-153
  PubMedSearch in Google Scholar
• 7 König J, Nies A T, Cui Y, Leier I, Keppler D. Conjugate export pumps of the multidrug resistance protein (MRP) family: localization, substrate specificity, and MRP2-mediated drug resistance.  Biochim Biophys Acta. 1999;  1461 377-94
  PubMedSearch in Google Scholar
• 8 Evers R, Kool M, van Deemter L, Janssen H, Calafat J, Oomen L C. et al . Drug export activity of the human canalicular multispecific organic anion transporter in polarized kidney MDCK cells expressing cMOAT (MRP2) cDNA.  J Clin Invest. 1998;  101 1310-9
  PubMedSearch in Google Scholar
• 9 Cui Y, König J, Buchholz J K, Spring H, Leier I, Keppler D. Drug resistance and ATP-dependent conjugate transport mediated by the apical multidrug resistance protein, MRP2, permanently expressed in human and canine cells.  Mol Pharmacol. 1999;  55 929-37
  PubMedSearch in Google Scholar
• 10 Hooijberg J H, Broxterman H J, Kool M, Assaraf Y G, Peters G J, Noordhuis P. et al . Antifolate resistance mediated by the multidrug resistance proteins MRP1 and MRP2.  Cancer Res. 1999;  59 2532-5
  PubMedSearch in Google Scholar
• 11 Fujiki H. Two stages of cancer prevention with green tea.  J Cancer Res Clin Oncol. 1999;  125 589-97
  PubMedSearch in Google Scholar
• 12 Kostyuk V A, Potapovich A I, Vladykovskaya E N, Hiramatsu M. Protective effects of green tea catechins against asbestos-induced cell injury.  Planta Medica. 2000;  66 762-4
  Thieme ConnectPubMedSearch in Google Scholar
• 13 Jodoin J, Demeule M, Beliveau R. Inhibition of the multidrug resistance P-glycoprotein activity by green tea polyphenols.  Biochim Biophys Acta. 2002;  1542 149-59
  CrossrefPubMedSearch in Google Scholar
• 14 Wang E J, Barecki-Roach M, Johnson W W. Elevation of P-glycoprotein function by a catechin in green tea.  Biochem Biophys Res Commun. 2002;  297 412-8
  CrossrefPubMedSearch in Google Scholar
• 15 Sadzuka Y, Sugiyama T, Hirota S. Modulation of cancer chemotherapy by green tea.  Clin Cancer Res. 1998;  4 153-56
  PubMedSearch in Google Scholar
• 16 Sadzuka Y, Sugiyama T, Sonobe T. Efficacies of tea components on doxorubicin induced antitumor activity and reversal of multidrug resistance.  Toxicol Lett. 2000;  114 155-62
  CrossrefPubMedSearch in Google Scholar
• 17 Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D. et al . New colorimetric cytotoxicity assay for anticancer-drug screening.  J Natl Cancer Inst. 1990;  82 1107-12
  CrossrefPubMedSearch in Google Scholar
• 18 Luo F R, Paranjpe P V, Guo A, Rubin E, Sinko P. Intestinal transport of irinotecan in Caco-2 cells and MDCK II cells overexpressing efflux transporters Pgp, cMOAT, and MRP1.  Drug Metab Dispos. 2002;  30 763-70
  CrossrefPubMedSearch in Google Scholar
• 19 Marbeuf-Gueye C, Broxterman H J, Dubru F, Priebe W, Garnier-Suillerot A. Kinetics of anthracycline efflux from multidrug resistance protein-expressing cancer cells compared with P-glycoprotein-expressing cancer cells.  Mol Pharmacol. 1998;  53 141-7
  PubMedSearch in Google Scholar
• 20 Fromm M F, Kauffmann H M, Fritz P, Burk O, Kroemer H K, Warzok R W. et al . The effect of rifampin treatment on intestinal expression of human MRP transporters.  Am J Pathol. 2000;  157 1575-80
  CrossrefPubMedSearch in Google Scholar
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Juergen Drewe, MD, MSc

Department of Clinical Pharmacology and Toxicology

University Clinic Basel

Kantonsspital

Petersgraben 4

4031 Basel

Switzerland

Phone: +41-61-265-3848

Fax: +41-61-265 8581

Email: juergen.drewe@unibas.ch