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1 Division of Molecular Biotherapy, Japanese Foundation for Cancer Research, Tokyo, Japan, and 2 Department of Chemotherapy, Kyoritsu University of Pharmacy, Tokyo, Japan
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the present study, we examined the possible effects of phyto-estrogens and other flavonoids in BCRP-mediated MDR. The chemical structures of isoflavones resemble those of estrogens, and their weak estrogenic activities have been reported previously (10) . Isoflavones constitute a group of flavonoids that are particularly abundant in soybean, and genistein, a member of the isoflavones, revealed stronger BCRP-inhibitory effects than estrone. Naringenin, a member of the flavanones that is contained in grapefruit juice, also showed BCRP-inhibitory effects. In addition, many other flavonoids, especially flavones, were found to strongly reverse BCRP-mediated drug resistance with few growth-inhibitory effects on cells. The BCRP-inhibitory effect of flavonoids might be explained, in part, by competitive inhibition of the BCRP-mediated efflux of anticancer agents because genistein was found to be a natural substrate that is transported by BCRP. The mechanisms by which isoflavones and other flavonoids inhibit drug export by BCRP currently are under investigation.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Establishment of K562/BCRP, LLC/BCRP, K562/MDR, and KB/MRP Cell Lines.
K562 human leukemic cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37°C in 5% CO2. K562/BCRP cells were established by transduction of K562 cells with HaBCRP retrovirus, bearing human BCRP cDNA, and subsequent selection with 20 ng/ml SN-38 for 5 days. LLC-PK1 cells, epithelial cells of the porcine kidney, were cultured in M199 medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum. LLC/BCRP cells were established by the transduction of LLC-PK1 cells with HaBCRP retrovirus and subsequent selection with increasing doses of mitoxantrone (2, 4, and 8 nM) for 17 days. The resulting mixed population of drug-resistant cells was used in this study as described previously (8
, 11)
. K562/MDR cells were established by transduction of K562 cells with HaMDR retrovirus containing human MDR1 cDNA, and this was followed by selection using 4 ng/ml vincristine for 7 days (12)
. KB-3–1 human epidermoid carcinoma cells were cultured in DMEM supplemented with 10% fetal bovine serum at 37°C in 5% CO2. KB/MRP cells were established by introduction of the pJ3U-MRP1 construct bearing human MRP1 cDNA into KB-3–1 cells, followed by selection with increasing concentrations of doxorubicin (13)
. Expression of BCRP in K562/BCRP and LLC/BCRP cells, expression of P-glycoprotein in K562/MDR, and expression of MRP1 in KB/MRP cells were confirmed by Western blot analysis with the anti-BCRP polyclonal antibody 3488, anti-P-glycoprotein monoclonal antibody C219, and anti-MRP1 monoclonal antibody MRPm6, respectively. The Western blot analysis procedure is described elsewhere (11)
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Cell Growth Inhibition Assay.
The effects of specific compounds on the sensitivity of cells to SN-38 and mitoxantrone were evaluated by measuring cell growth inhibition after incubation at 37°C for 5 days in the absence or presence of various concentrations of anticancer drugs in combination with the specific chemicals being examined. Cell numbers were determined using a cell counter (Sysmex, Kobe, Japan). IC50 values (drug dosages that cause 50% inhibition of cell growth) were determined from growth inhibition curves.
Intracellular Topotecan Uptake.
The effects of specific compounds on the cellular accumulation of topotecan were determined by flow cytometry. Cells (5 x 105) were incubated with 20 µM topotecan for 30 min at 37°C in the absence or presence of modifying agents, washed in ice-cold PBS, and subjected to fluorescence analysis using FACSCalibur (Becton-Dickinson, San Jose, CA).
Cellular [3H]Genistein Accumulation in K562/BCRP Cells.
Either K562 or K562/BCRP cells (2 x 106) were incubated with 30 nM [3H]genistein for 0, 1, 2, or 4 h at 37°C. The cells then were washed with ice-cold PBS, dissolved in 100 µl PBS plus 400 µl Soluene-350 (Packard, Downer’s Grove, IL), and mixed with 5 ml ACS II scintillation mixture (Amersham, Piscataway, NJ). Radioactivity levels were measured using a scintillation counter (Beckman, Fullerton, CA).
Transcellular Transport Assay of [3H]Genistein and Silica Gel TLC of Transported Compounds.
Details of the experimental procedure are described previously (8)
. Briefly, exponentially growing LLC-PK1 and/or LLC/BCRP cells were plated on 3-µm pore Transwell 3414 filters (Corning Costar, Cambridge, MA) at a density of 2.4 x 106 cells/well and cultured for 3 days. Culture medium in the upper and lower wells was replaced with 2 ml of serum-free M199 medium 1.5 h before beginning the experiments. When needed, fumitremorgin C was added to the apical and basal side medium at this time (14)
. The medium in either the upper or lower well then was replaced with 2 ml of medium containing 14C-labeled inulin and/or 3H-labeled genistein. The cells were incubated at 37°C in 5% CO2, and 50 µl of the medium from the opposite side were sampled at 1, 2, and 4 h following the addition of radiolabeled compounds. The radioactivity of each sample was measured by liquid scintillation counting and expressed as a percentage fraction of the total radioactivity before incubation. All of the data were presented as mean values with SD of triplicate determinations from three different cultures.
For silica gel TLC, 50 µl of medium in the opposite side of the chamber following incubation were mixed with 100 µl of methanol, spotted, and run on silica gel 60 F254 plates (Merck, Darmstadt, Germany) in chloroform/methanol/acetic acid (8:3:1). Separated zones were excised, and their radioactivities were measured using a liquid scintillation counter. The radioactivities were expressed as a percentage fraction of the total radioactivity before incubation. Each point represents a mean value with SD of triplicate determinations.
Statistical Analysis.
The two-sided unpaired Student’s t test was used to evaluate the statistical significance of the differences between the two sets of data. The differences were considered significant when P < 0.05.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Expression of BCRP, P-glycoprotein, or MRP1 was not detected in parental K562 and KB-3–1 cells.
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. LLC/BCRP cells were five to six times more resistant to SN-38 and mitoxantrone than parental LLC-PK1 cells as described previously (8)
. K562/MDR cells showed significantly higher resistance to vincristine than K562 cells, and KB/MRP cells were significantly more resistant to VP-16 than KB-3–1 cells (Table 1)
. Protein expression and drug-resistance levels in each resistant cell line were stable for at least 2 months.
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. We first examined the effects of representative phytoestrogens, genistein and naringenin, on drug resistance in K562/BCRP cells (Fig. 3, A–C)
. Reversal indexes (ratios of IC50 measurements in the absence of reversing agents divided by levels in the presence of reversing agents) of 3 µM (10 µM) genistein for SN-38 and mitoxantrone were 7.23 ± 0.35 (16.4 ± 0.56) and 6.28 ± 0.51 (11.7 ± 0.40), respectively. In addition, reversal indexes of 3 µM (10 µM) naringenin for SN-38 and mitoxantrone were 5.94 ± 0.26 (15.2 ± 0.92) and 3.42 ± 0.27 (10.6 ± 0.30), respectively. The reversing effects of genistein and naringenin proved to be greater than estrone. Analysis then was extended to other flavonoids, many of which reversed BCRP-mediated SN-38 resistance at a fixed concentration of 3 µM (Fig. 4)
. The flavones acacetin, apigenin, chrysin, diosmetin, and luteolin and the flavonols kaempferide and kaempferol displayed strong reversal effects (Fig. 3, D and E
, and Fig. 4
). Reversal indexes of 1 µM (3 µM) acacetin for SN-38 and mitoxantrone were 15.2 ± 1.10 (21.4 ± 0.34) and 9.89 ± 0.27 (9.71 ± 0.81), respectively. Reversal indexes of 1 µM (3 µM) kaempferol for SN-38 and mitoxantrone were 9.96 ± 0.38 (21.5 ± 0.22) and 10.6 ± 0.99 (14.2 ± 0.95), respectively. However, the flavonoids did not show growth-inhibitory effects on K562 cells under these experimental conditions. K562/BCRP cells treated with estrone or flavonoids, such as genistein, naringenin, and acacetin, for 5 days expressed similar amounts of BCRP as compared with control K562/BCRP cells (Fig. 5)
. This result suggested that flavonoids sensitized K562/BCRP cells to SN-38 and mitoxantrone not by reducing BCRP expression but by inhibiting BCRP function. We then examined the effects of glycosylated flavonoids on the drug-resistance properties of K562/BCRP cells. Although most glycosylated flavonoids had little effect on BCRP-mediated drug resistance, some glycosides, such as naringenin-7-glucoside and luteolin-4'-O-glucoside, displayed moderate reversal activity (Fig. 6)
. Reversal indexes of 3 µM (10 µM) naringenin-7-glucoside for SN-38 and mitoxantrone were 5.70 ± 0.16 (14.7 ± 0.53) and 5.17 ± 0.23 (9.44 ± 0.42), respectively.
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, whereas these levels were not altered in K562 cells (data not shown). The results indicate that these flavonoids reverse anticancer drug resistance by increasing the cellular levels of anticancer drugs in BCRP-expressing cells.
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. However, in the presence of 3 µM fumitremorgin C, secretion and reabsorption of [3H]genistein were at similar levels between LLC-PK1 and LLC/BCRP cells.
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, indicating that genistein itself is directly transported by BCRP. In a separate experiment, when [3H]genistein was incubated with LLC-PK1 cells for 24 h, >80% of the radioactivity shifted to Rf = 0.12–0.21 (segment 3–4) and approximately 10% shifted to Rf = 0.38–0.47 (segment 6–7), in which metabolites of genistein were expected to exist (data not shown). However, only small amounts of such metabolites were found in the 4-h transport assay (Fig. 11)
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Isoflavones derived from soybean, such as genistein and daidzein, constitute a subset of flavonoids that have been reported to have weak estrogenic activity (10)
. In addition, chemical structures of isoflavones resemble those of estrone and 17ß-estradiol (Fig. 2)
. Naringenin, a flavanone contained in grapefruit juice, also resembles 17ß-estradiol in chemical structure, albeit to a lesser extent than isoflavone (Fig. 2)
. Flavonoids are remarkably safe nutrients, being the most abundant polyphenolic compounds present in the human diet in fruits, vegetables, and plant-derived beverages such as tea and red wine (15)
. Some flavonoids also have been reported to interact with ABC transporters, such as P-glycoprotein, MRP1, MRP2, and cystic fibrosis transmembrane conductance regulator (16, 17, 18, 19, 20, 21)
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Genistein and naringenin displayed stronger interaction with BCRP than estrone (Fig. 3, A–C)
, and many flavones, such as acacetin, apigenin, chrysin, diosmetin, and luteolin, and some flavonols, such as kaempferide and kaempferol, demonstrated strong reversing activity of BCRP-mediated drug resistances (Fig. 3, D and E
, and Fig. 4
). Flavanol, flavanonol, and one-half of flavonols tested did not show BCRP-reversing activity (Fig. 4)
. Two glycosylated flavonoids, naringenin-7-glucoside and luteolin-4'-O-glucoside, did show anti-BCRP activity, whereas six other glycosylated flavonoids did not (Fig. 6)
. From these results, we speculated that the 3-hydroxyl group of the C ring might be important for BCRP-inhibitory activity, although some exceptions do exist. The flavonoids did not show growth-inhibitory effects in K562 and KB-3–1 cells at the highest concentrations used in our experiments, suggesting that they might be safely used for circumventing BCRP-mediated drug resistance in clinical practice. Glycosylated flavonoids with anti-BCRP activity also may be useful because of their water solubility. In our preliminary animal experiments, some flavonoid aglycones were insoluble to either water or hydrophilic solvents and therefore would be difficult to administer i.v. Therefore, use of either glycosylated flavonoids or water-soluble derivatives of flavonoids would be an alternative way to develop BCRP inhibitors.
GF120918 was first developed as a P-glycoprotein inhibitor but also was shown to be a BCRP inhibitor (22
, 23)
. TAG-139, which we identified as a tamoxifen-derived BCRP inhibitor, also, like tamoxifen, inhibited P-glycoprotein (9)
. Genistein was reported to be a substrate/inhibitor of MRP1 because genistein inhibited daunorubicin transport out of cells overexpressing MRP1 at a concentration of 50 µM (20)
. In the present study, genistein and naringenin effectively inhibited BCRP at a concentration of 3 µM (Fig. 3)
but showed little effect on vincristine resistance in K562/MDR cells or on VP-16 resistance in KB/MRP cells even at a concentration of 10 µM (Fig. 7)
. Therefore, these flavonoids could be specific inhibitors of BCRP.
In previous studies, we showed that estrone inhibits BCRP function but was not transported by BCRP in its native form (7
, 8)
. In contrast, progesterone is known to inhibit the function of P-glycoprotein but is not transported by P-glycoprotein (24)
. In the case of genistein, K562/BCRP cells accumulated smaller amounts of [3H]genistein than parental K562 cells (Fig. 9)
. Secretion of [3H]genistein from LLC/BCRP cells was greater than from LLC-PK1 cells in transcellular transport assays (Fig. 10)
. TLC analysis of transported [3H]genistein suggested that there was increased transport of genistein aglycone in LLC/BCRP cells compared with LLC-PK1 cells (Fig. 11)
. Intracellular accumulation of [3H]genistein also was decreased in LLC/BCRP cells compared with levels in parental cells (data not shown). These results suggest that inhibition of BCRP-mediated drug resistance by genistein is caused by the competitive transport of genistein by BCRP. Unlike estrone and 17ß-estradiol, genistein would be transported in its native form but not in either sulfated or glucuronated forms (25
, 26)
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Another possible mechanism of BCRP inhibition by flavonoids is the interaction with the nucleotide-binding domain of BCRP because some flavonoids, including genistein, have been shown previously to interact with nucleotide-binding domain of P-glycoprotein, which was predicted to suppress ATP-hydrolysis and energy-dependent drug transport (16, 17, 18) . In the case of BCRP, modulation of ATPase activity by the flavonoids should be investigated further to clarify this possibility.
The data presented here might have clinically important implications because some flavonoids effectively inhibited BCRP-mediated drug resistance at relatively low concentrations. For instance, we showed that 3 µM of genistein effectively circumvented BCRP-mediated drug resistance. Soybean (100 g) contains 100–200 mg isoflavones consisting of genistein, daidzein, glycitein, and their corresponding glycosides. Several groups have investigated the pharmacokinetics of soy isoflavone. A single bolus ingestion of 50 mg genistein in healthy premenopausal women was shown to result in a peak plasma concentration of 1.26 ± 0.27 µM at 9.33 ± 1.33 h (25) . The rate of unconjugated genistein was only approximately 2–4%. However, in another report of 6-consecutive-day feeding study of 25 g soymilk powder in young adult women, the percentage of plasma aglycone genistein sampled on days 5 and 6 was 26 ± 7% of total genistein (26) . In addition, glucuronide was the main metabolite in that study. Therefore, the concentration of active genistein in cancer tissues may reach sufficient level for BCRP inhibition via oral ingestion because ß-glucuronidase activity is elevated in cancer (27) . Parenteral administration may validate genistein as BCRP inhibitor. Subcutaneous injection of genistein was shown previously to enable high concentration of active genistein in mice (28) .
The findings that glycosylated flavonoids naringenin-7-glucoside and luteolin-4'-O-glucoside effectively inhibited BCRP-mediated drug resistance might prove to be of great importance. These glycosylated flavonoids are well soluble in water. Because deglycosidation by intestinal enzymes precedes glucuronidation via oral routes, the i.v. injection of glycosylated flavonoids would bypass deglycosidation and retain their native structures, which are effective as BCRP inhibitors. In fact, i.v. naringenin-7-glucoside was excreted in urine mostly as its native glucoside form in a rat model (29) .
Despite the promising results using flavonoids in the reversal of BCRP-mediated drug resistance, we must bear in mind that that coadministration or intake of flavonoids with BCRP-substrate antitumor agents may result in the alteration of their pharmacokinetics and may increase the toxicity of the antitumor drugs in the recipient patients. In this regard, orally administered GF120918 has been reported to increase oral bioavailability of topotecan (30) .
In summary, phytoestrogens/flavonoids reverse BCRP-mediated drug resistance effectively, and these findings may bring direct and immediate clinical benefits via more effective and safer cancer chemotherapy treatments.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Requests for reprints: Yoshikazu Sugimoto, Division of Molecular Biotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, 1-37-1 Kami-Ikebukuro, Toshima-ku, Tokyo 170-8455, Japan. Fax: 81-3-3918-3716; E-mail: ysugimot{at}jfcr.or.jp
Received 1/ 9/04. Revised 3/28/04. Accepted 4/ 2/04.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-tubulin was presented as an internal control.
