Modulation of breast cancer resistance protein (BCRP/ABCG2) by non-basic chalcone analogues

Article abstract of DOI:10.1016/j.ejps.2008.06.001

Chalcones are biosynthetic precursors of flavonoids found to possess cytotoxic and chemopreventive activities. In this study, 17 non-basic chalcone analogues were synthesized and evaluated for their ability to modulate the function of either the human wild-type (482R) or mutant (482T) breast cancer resistance protein (BCRP/ABCG2) stably expressed in breast cancer MDA-MB-231 cells. At 5 μM, chalcones with 2,4-dimethoxy groups or 2,4-dihydroxyl groups on ring A were found to increase mitoxantrone accumulation to a greater extent than an established BCRP inhibitor, fumitremorgin C. At the same time, these chalcones had negligible effect on calcein accumulation in P-glycoprotein overexpressing MDCKII cells, indicating their potential as selective BCRP inhibitors. Functionally, these compounds were able to increase the sensitivity of BCRP-overexpressing cancer cells to mitoxantrone by 2-5-fold. The effect of chalcone compounds on both wild-type and mutant BCRP ATPase activity was also examined and variable effects were observed. A stimulatory effect was mostly observed with chalcones with 2,4-dimethoxy substitution on ring A which were earmarked as good BCRP inhibitors in the MX accumulation and cytotoxicity assays. These findings underscore the potential of methoxylated and hydroxylated chalcones as selective and potent inhibitors of BCRP whose mode of action may not involve the inhibition of ATPase activity.

Full text of DOI:10.1016/j.ejps.2008.06.001

european journal of pharmaceutical sciences 3 5 ( 2 0 0 8 ) 30–41
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/ejps
Modulation of breast cancer resistance protein
(BCRP/ABCG2) by non-basic chalcone analogues
Yi Han, Meliana Riwanto, Mei-Lin Go, Pui Lai Rachel Ee∗
Department of Pharmacy, Faculty of Science, National University of Singapore,
18 Science Drive 4, Singapore 117543, Singapore
a r t i c l e i n f o
a b s t r a c t
Article history:
Chalcones are biosynthetic precursors of flavonoids found to possess cytotoxic and chemo-
preventive activities. In this study, 17 non-basic chalcone analogues were synthesized and
evaluated for their ability to modulate the function of either the human wild-type (482R)
or mutant (482T) breast cancer resistance protein (BCRP/ABCG2) stably expressed in breast
cancer MDA-MB-231 cells. At 5 ␮M, chalcones with 2,4-dimethoxy groups or 2,4-dihydroxyl
groups on ring A were found to increase mitoxantrone accumulation to a greater extent
than an established BCRP inhibitor, fumitremorgin C. At the same time, these chalcones
had negligible effect on calcein accumulation in P-glycoprotein overexpressing MDCKII cells,
indicating their potential as selective BCRP inhibitors. Functionally, these compounds were
able to increase the sensitivity of BCRP-overexpressing cancer cells to mitoxantrone by
2–5-fold. The effect of chalcone compounds on both wild-type and mutant BCRP ATPase
activity was also examined and variable effects were observed. A stimulatory effect was
mostly observed with chalcones with 2,4-dimethoxy substitution on ring A which were ear-
marked as good BCRP inhibitors in the MX accumulation and cytotoxicity assays. These
findings underscore the potential of methoxylated and hydroxylated chalcones as selective
and potent inhibitors of BCRP whose mode of action may not involve the inhibition of ATPase
activity.
Received 15 April 2008
Received in revised form 1 June 2008
Accepted 2 June 2008
Published on line 11 June 2008
Keywords:
Multidrug resistance
Breast cancer resistance protein
Inhibitors
Drug accumulation
Chalcones
© 2008 Elsevier B.V. All rights reserved.
1.
Introduction
is a half-transporter possessing a unique structure of only
one nuclear binding domain and six transmembrane domains.
Multidrug resistance (MDR) remains a major obstacle to suc-
cessful chemotherapy of many malignant diseases. Tumor
cells often acquire resistance to a variety of anticancer agents
with distinct chemical structures or mechanisms of action via
the overexpression of ATP-binding cassette (ABC) transporters
such as P-glycoprotein (Pgp) (MDR1/ABCB1) (Juliano and Ling,
1976), multidrug resistance-related protein 1 (ABCC1/MRP1)
(Cole et al., 1992) and/or breast cancer resistance protein
(BCRP/ABCG2) (Doyle et al., 1998). The latter is among the
latest discovered to be involved in the MDR phenotype and
Like Pgp and MRP1, BCRP displays broad substrate specificity
and transports compounds that include chemotherapeutic
agents such as mitoxantrone, topotecan, irinotecan, SN-38
and doxorubicin (Litman et al., 2000; Yang et al., 2000); flu-
orescent substances such as BODIPY-prazosin (Robey et al.,
2001) and Hoechst 33342 (Kim et al., 2002); as well as chemi-
cal toxicants (Pavek et al., 2005). Substrate specificity has been
found to be crucially dependent on mutations that alter the
amino acid at position 482 of the BCRP protein. In vitro studies
revealed that methotrexate is only transported by wild-type
∗
Corresponding author. Tel.: +65 6516 2653; fax: +65 6779 1554.
E-mail address: phaeplr@nus.edu.sg (P.L. Rachel Ee).
0928-0987/$ – see front matter © 2008 Elsevier B.V. All rights reserved.
doi:10.1016/j.ejps.2008.06.001

european journal of pharmaceutical sciences 3 5 ( 2 0 0 8 ) 30–41
31
ities (Bois et al., 1998, 1999). In an earlier study, we found
that methoxylated chalcones with basic substituents on
either ring A or B were good inhibitors of Pgp but negligible
inhibitors of BCRP (Liu et al., 2008). For example, the basic
chalcone 1-[5-(1-ethylpiperidin-4-yl)-2,4-dimethoxyphenyl]-
3-phenylprop-2-en-1-one (Fig. 1A) increased calcein accumu-
lation in Pgp-overexpressing MDCKII cells and enhanced the
accumulation of the anticancer drug doxorubicin in MCF-7
cells selected for the overexpression of Pgp. However, it did
not increase the uptake of mitoxantrone (MX, a substrate of
BCRP) in MCF-7 cells that overexpressed BCRP. In contrast,
a non-basic chalcone (6, Fig. 1B) showed a reverse profile—6
did not increase calcein accumulation in Pgp-overexpressing
cells but increased MX accumulation significantly in BCRP-
overexpressing MCF-7 cells. We thus concluded that by
introducing a basic group, Pgp inhibition was enhanced at the
expense of BCRP inhibition. Conversely, it follows that omit-
ting the basic functionality resulted in chalcones with more
BCRP than Pgp inhibition. As chalcones have not been rec-
ognized as selective BCRP inhibitors or modulators, it was
of interest to explore their potential in this area. Hence, we
have synthesized several analogues of 6 and evaluated their
effectiveness as specific BCRP inhibitors. The compounds were
investigated for their abilities to increase MX accumulation
in human breast cancer cells (MDA-MB-231) that were trans-
fected with the human wild-type (482R) and mutant (482T)
BCRP cDNA. These BCRP variants were used to shed light on
the inhibitory specificities of the synthesized compounds. The
same compounds were also tested for their influence on MX
cytotoxicity profiles, as well as their effects on BCRP ATPase
activity.
Table 1 – Structures of chalcones investigated in this
study
Chalcone
Ring A
Ring B
1
2
3
4
5
14
6
7
8
9
10
11
12
13
15
16
17
2-OCH3
2-OCH3
2-OCH3
H
2ꢀ-Cl
4ꢀ-Cl
H
2,4-OCH3
2,4-OCH3
2,4-OCH3
2,4-OCH3
2,4-OCH3
2,3,4-OCH3
2,3,4-OCH3
2,3,4-OCH3
2,3,4-OCH3
2-OH
2ꢀ-Cl
3ꢀ-Cl
4ꢀ-Cl
3ꢀ,4ꢀ-Cl
H
2ꢀ-Cl
4ꢀ-Cl
3ꢀ,4ꢀ-Cl
H
2ꢀ-Cl
2ꢀ-Cl
3ꢀ-Cl
4ꢀ-Cl
2-OH
2,4-OH
2,4-OH
2,4-OH
(482R) BCRP and anthracyclines and rhodamine 123 only by
mutant forms of BCRP (482T or 482G), whereas Hoechst33342
and mitoxantrone are transported by all variants (Yang et al.,
2000; Honjo et al., 2001; Sarkadi et al., 2004).
One strategy to overcome MDR is to co-administer
inhibitors of ABC transporters and anticancer drugs in order
to increase intracellular substrate accumulation by prevent-
ing ATP-dependent drug efflux. In fact, several generations of
compounds have been examined for their potential as Pgp and
MRP1 inhibitors in preclinical and clinical studies, while rela-
tively few are reported for BCRP inhibitors (Ahmed-Belkacem
et al., 2006). Fumitremorgin C (FTC) is the first compound iden-
tified but its intensive neurotoxicity has precluded further
in vivo applications (Nishiyama and Kuga, 1986; Allen et al.,
2002). Some other drugs used clinically such as calcium chan-
nel blockers (Zhang et al., 2005) and corticosteroids (Cooray et
al., 2006) were also found to be effective as BCRP inhibitors.
However, drug–drug interactions may be a potential problem
in patients taking these drugs concomitantly with BCRP sub-
strate drugs. Apart from existing drugs, some natural products
have been evaluated for their effect on BCRP activity in recent
years. Flavonoids are among the most studied and character-
ized BCRP modulators (Cooray et al., 2004; Imai et al., 2004;
Zhang et al., 2004; Ahmed-Belkacem et al., 2005; Katayama et
al., 2007), while some curcuminoids (Chearwae et al., 2006)
and ginsenosides (Jin et al., 2006) have also been shown
recently to interfere with BCRP activity. In addition, chemical
libraries of acridones and benzyopyranones have been tested
for their effectiveness as BCRP inhibitors (Boumendjel et al.,
2005, 2007).
2.
Materials and methods
2.1.
Chemistry
Melting points were determined in open capillary tubes on a
Gallenkamp melting point apparatus and were uncorrected.
1H NMR and 13C NMR spectra were recorded on a Bruker Spec-
trospin 300 Ultrashield spectrometer operating at 300 MHz.
Spectra were analyzed using the Bruker 1D Win NMR software
and chemical shifts (ı, ppm) were reported with tetramethyl-
silane as an internal standard. Mass spectra were collected
on LcQ Finnigan MAT mass spectrometer fitted with an atmo-
spheric chemical ionization (APCI) probe. Analytical thin layer
chromatography was carried out on precoated silica gel 60
F254 (230–400 mesh) supported on aluminium sheets. Silica
gel 60 (0.040–0.063 mm) was used for preparative column chro-
matography. Combustion analyses (C, H) were determined on
a PerkinElmer PE 2400 CHN/CHNS Elemental Analyzer by the
Department of Chemistry, National University of Singapore.
Reagents used in the synthetic procedures were of synthetic or
analytical grade. The syntheses and characterization of chal-
cones 1–11 have been described in earlier reports (Liu et al.,
2001; Liu and Go, 2006) and chalcones 14–17 were synthesized
in a similar manner as reported (Liu et al., 2001). The syntheses
of the remaining chalcones 12–13 are reported in the following
paragraph. The yields of the synthesized compounds, melting
points and spectroscopic data are given in Table 1 (supporting
material).
Chalcones are bioprecursors of flavonoids that bear the
structure of 1,3-diphenylprop-2-en-1-one (Table 1). This class
of compounds has been reported to possess many biolog-
ical activities such as anticancer proliferation (Liu and Go,
2006), antimalarial (Liu et al., 2001) and Pgp inhibitory activ-

32
european journal of pharmaceutical sciences 3 5 ( 2 0 0 8 ) 30–41
Fig. 1 – Structures of (A) the basic chalcone 1-[5-(1-ethylpiperidin-4-yl)-2,4-dimethoxyphenyl]-3-phenylprop-2-en-1-one,
and (B) chalcone 6.
(Netherlands Cancer Institute, Antoni van Leeuwenhoek Hos-
pital, Amsterdam, Netherlands). They were grown in DMEM
supplemented with 10% FBS and 1% penicillin/streptomycin.
Cells were subcultured when they reached 80–90% confluency
and used within 10 passages for all assays.
2.1.1. General method for the synthesis of chalcones 12
and 13
A mixture of 2-hydroxyacetophenone (1.36 g, 10 mmol) and the
benzaldehyde (10 mmol) in ethanolic KOH (20%, w/v, 10 ml)
was heated in an oil bath at 130 ◦C for 3–5 min. On cooling, the
reaction mixture was poured into crushed ice, acidified with
HCl, and kept at low temperatures (2–3 ◦C) overnight for the
separated residue to solidify. The solid was then removed by
filtration, washed several times with water and recrystallized
with methanol.
2.4.
Western blotting
20 ␮g of cell lysates from MDA-MB-231/V, MDA-MB-231/R
and MDA-MB-231/T cells were subjected to sodium dode-
cyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE)
and transferred to PVDF membranes (Bio-Rad Laboratories
Pte. Ltd., Singapore). Membranes were blocked overnight and
probed with primary antibodies followed by peroxidase conju-
gated anti-mouse secondary antibody. The following primary
antibodies were used: anti-BCRP (BXP-21), anti-Pgp (C219),
anti-MRP1 (MRPr-1) and anti-MRP2 (M2III-6). Proteins were
visualized using the Supersignal West Pico chemilumines-
cence reagent (Pierce Chemicals, Rockford, IL).
2.2.
Materials
All cell culture reagents were purchased from Invitrogen Sin-
gapore Pte. Ltd. (Singapore) unless otherwise stated. Calcein
acetoxymethyl ester (calcein-AM), cyclosporine A, MX and
FTC were purchased from Sigma–Aldrich Chemical Co. (Sin-
gapore). 3-(4,5-Dimethylthiazol-2-il)-2,5-diphenyl tetrazolium
bromide (MTT) was obtained from MP Biomedicals Inc. (Solon,
OH, US). Mouse monoclonal antibodies for Western blot
analyses of BCRP, Pgp, MRP1 and MRP2 were from Signet
Laboratories Inc. (Dedham, MA, US) while the anti-mouse
secondary antibody was from Amersham Biosciences Inc. (Pis-
cataway, NJ, US).
2.5.
Flow cytometry
The effects of the chalcones on the intracellular accumulation
of a fluorescent BCRP substrate, MX, were determined by flow
cytometry as previously described (Zhang et al., 2004). Briefly,
cells cultured in 75 cm2 flasks were trypsinized, washed with
serum-free RPMI 1640 medium and PBS and resuspended in
serum-free medium at a cell density of 106 cells/ml. The cells
were pre-incubated with 5 ␮M of chalcones or vehicle (0.1%
DMSO) for 15 min followed by incubation with MX (3 ␮M) for
40 min. FTC, a specific BCRP inhibitor, was used as a positive
control. The cells were then washed thrice and resuspended
in cold PBS prior to analysis in the CyAnTM Research Flow
Cytometer (Dako, Glostrup, Denmark). Cells were excited at
488 nm with an argon laser and the emission recorded via a
670 nm long-pass filter for MX fluorescence. The raw data were
recorded and analyzed with the Summit Version 4.3 software
(Dako, Glostrup, Denmark). MX cellular levels were normal-
ized to vehicle control levels, which was taken as 100%. Three
or four independent experiments were performed.
2.3.
Cell culture
All the cells were grown at 37 ◦C in a humidified atmosphere
containing 5% CO2. The breast cancer cell line, MDA-MB-
231, stably transfected with expression vectors for wild-type
482R BCRP (MDA-MB-231/R cells), mutant 482T BCRP (MDA-
MB-231/T cells) and pcDNA3.1 (MDA-MB-231/V cells) were
kindly provided by Dr. Douglas D. Ross (University of Mary-
land Greenebaum Cancer Center, Baltimore, US). They were
maintained in RPMI 1640 medium supplemented with 10%
fetal bovine serum (FBS) (HyClone, Logan, UT), 1% peni-
cillin/streptomycin and 1 mg/ml G418. Mardin–Darby canine
kidney cells transfected with expression vectors for human
MDR1, MRP1 and MRP2 (MDCKII/MDR1, MDCKII/MRP1 and
MDCKII/MRP2) were generous gifts from Dr. Anton Berns

european journal of pharmaceutical sciences 3 5 ( 2 0 0 8 ) 30–41
33
vanadate-sensitive ATPase assay was based on a colorimet-
ric method as described previously (Xia et al., 2007). Crude
membranes of MDA-MB-231/R and MDA-MB-231/T cells (10 ␮g
protein) were first incubated for 90 min in assay medium
(5 mM ATP, 50 mM KCl; 2 mM DTT; 50 mM MOPS–Tris; pH 7.0)
in the presence of 1 mM EGTA, 2 mM ouabain and 5 mM NaN3.
Test compounds were then added in various concentrations in
DMSO. DMSO was present at 1% in the final reaction mixture.
The reaction was stopped by adding 30 ␮l of 5% SDS solu-
tion and the released phosphate or phosphate standards were
measured at 800 nm after the reaction mixture was supple-
mented with 160 ␮l of the detection reagent (5 ml of 35 mM
ammonium molybdate in 15 mM zinc acetate, pH 5.0, mixed
with 20 ml of 10% ascorbic acid, pH 5.0) and incubated for
20 min at 37 ◦C. Vanadate-sensitive assays were performed in
the presence of sodium orthovanadate (360 ␮M) and compared
to control assays without the addition of sodium orthovana-
date. BCRP-specific ATPase activity was calculated from the
difference in the readings between these two values. The
amount of inorganic phosphate released from the membrane
was quantified against the phosphate standard KH2PO4 and
was calculated as nmol/(min mg) protein.
2.6.
Calcein-AM uptake assay
accumulation into
Calcein-AM
MDR1-overexpressing
MDCKII/MDR1 cells was performed in the presence and
absence of chalcones as previously described (Liu et al.,
2008). Briefly, MDCKII/MDR1 and MDCKII/WT cells at 80–90%
confluency were trypsinized, seeded in 96-well plates at a
cell density of 5.0 × 104 cells/well, and incubated at 37 ◦C
for 24 h in 5% CO2 atmosphere. The cell monolayer in each
well was carefully washed with PBS, followed by incubation
with test compounds at a concentration of 10 ␮M, prepared in
DMSO–Hank’s buffered saline solution (HBSS) for 30 min. After
30 min incubation, an aliquot of calcein-AM in DMSO–HBSS
was added to each well to give a final concentration of 2 ␮M.
The maximum concentration of DMSO per well was 1% (v/v).
After incubation (10 min), the fluorescence of each well was
measured on a microplate reader with ꢀexcitation of 485 nm
and ꢀemission of 535 nm. Concurrent determinations were
made with positive control, cyclosporine A, at 10 ␮M. The
accumulation of calcein-AM in the presence of chalcones was
calculated as the percentage ratio of the control value (in the
absence of chalcones).
2.7.
Cytotoxicity assays
2.9.
Statistical analysis
The cytotoxicity profiles of MX in MDA-MB-231/V, MDA-MB-
231/R and MDA-MB-231/T cells in the absence or presence
of chalcones were evaluated using MTT assays. Cells were
seeded into wells of 96-well plates in triplicates and incu-
bated in a humidified atmosphere of 37 ◦C. After 24 h, culture
medium in each well was replaced with fresh medium con-
taining increasing concentrations (0–500 ␮M) of MX or vehicle
(0.1% DMSO). Chalcones were added concomitantly to each
well in a range of concentrations of 0.5, 1 and 2 ␮M. After
72 h of drug exposure, the drug-containing medium was aspi-
rated, followed by the addition of MTT solution (1 mg/ml). The
plates were incubated for 4 h at 37 ◦C prior to analysis. The pur-
ple formazan crystals formed were solubilized in DMSO and
the absorbance measured at 595 nm. 100% cell survival was
defined as the absorbance of cells exposed to control vehicle
only (0.1% DMSO). The percentage survival of tested groups
was calculated by dividing the absorbance values of tested
groups by that of control vehicle. Growth inhibition by MX
(IC50 value) either alone or with the chalcones was obtained
by fitting a nonlinear, sigmoidal regression model to our data
using GraphPad Prism 3.0 (GraphPad Software, San Diego, CA).
The equation used by the software is a four-parameter logistic
Data from control and test groups were analyzed for statistical
significance using one-way ANOVA with Dunnett post hoc test
and Student’s t-test using the SPSS 13.0 software (SPSS Inc.,
Chicago, IL).
3.
Results
3.1.
Synthesis of chalcones
Table 1 lists the structures of the chalcones investigated
in this study. They were synthesized by a base-catalyzed
Claisen Schmidt condensation, starting from appropriately
substituted acetophenones and benzaldehydes. In the case
of the hydroxylated chalcones (15–17), the hydroxyl groups
on 2,4-dihydroxyacetophenone were protected with 2H-3,4-
dihydropyran prior to condensation with the aldehyde. The
tetrahydro-2H-pyran-2-yloxy groups were then removed by
acid-catalyzed hydrolysis to give the final product. No protec-
tion of the hydroxyl group was required for the syntheses of
chalcones 12 and 13, probably due to the anomalous “ortho
effect” of the 2-hydroxyl group in the acetophenone. The reac-
tion scheme is given in Fig. 2. Details of the syntheses and
characterization of chalcones 1–11 have been described ear-
lier (Liu et al., 2001; Liu and Go, 2006) and chalcones 14–17
were synthesized in a similar manner as reported (Liu et al.,
2001).
equation, the Hill equation and given as Y = Bottom + (Top
−
Bottom)/(1 + 10(LogIC -X)HillSlope)), where X is the logarithm of
concentration and Y is the response.
50
2.8.
Crude membrane preparation and
vanadate-sensitive ATPase assay
Since chalcone 6 was found to preferentially inhibit BCRP
activity (Liu et al., 2008), it was chosen as the lead structure for
modification. The structural variations made on 6 were aimed
at understanding the contributions to activity made by the
substituents on the chalcone template. On ring A, the num-
ber of methoxy groups was systematically varied from one
(1–3), two (4–7, 14) and three (8–11) groups. Replacement of
the methoxy groups with hydroxyl groups was also explored
Crude membranes of MDA-MB-231/R and MDA-MB-231/T cells
were prepared as described previously (Ambudkar, 1998).
The protein concentrations were determined using the Bio-
rad protein assay dye reagent and aliquots of the protein
were kept at −80 ◦C until use. Protein concentrations of the
crude membrane preparations were typically 5 mg/ml. The

34
european journal of pharmaceutical sciences 3 5 ( 2 0 0 8 ) 30–41
Fig. 2 – Reaction scheme for the synthesis of chalcones 12 (R = H) and 13 (R = 2ꢀ-Cl): (a) benzaldehyde or
2-chlorobenzaldehyde, ethanolic KOH, 130 ◦C, 3–5 min.
(12, 13 and 15–17). Chalcone 16 is the demethylated analogue
Table 2 – Effects of chalcones on MX accumulation in
MDA-MB-231/R and MDA-MB-231/T cells
of chalcone 6 and its activity would highlight the role of the
methoxy groups on ring A. The chloro substituent on ring B of
6 was retained in most compounds, but additionally, isomeric
2-chloro, 3-chloro and 3,4-dichloro analogues were included
for each class of ring A substituted chalcones.
MX accumulationa
MDA-MB-231/R
MDA-MB-231/T
Control
100 9.2
100 36.0
FTC (10 ␮M)
346.9 18.6*
409.27 15.6*
3.2.
BCRP expression and cytotoxicity profile of stably
Chalcone (5 ␮M)
transfected MDA-MB-231 cells
1
2
3
4
5
14
6
7
8
204.6 20.4
332.7 19.9*
211.3 29.4
295.4 37.4*
315.3 38.6*
323.9 61.7*
370.4 42.0*
315.5 25.4*
231.2 8.9
265.7 42.6*
254.2 19.3*
286.0 50.1*
184.5 31.3
270.4 29.3*
344.1 77.7*
359.0 79.8*
354.4 69.1*
257.9 36.7
432.5 85.7*
247.2 33.8
406.3 92.5
551.9 49.0*
575.9 48.6*
457.3 82.0*
403.1 20.4
273.2 18.8
447.9 67.3*
282.8 57.6
460.1 135.9*
408.1 122.9
432.6 133.8*
745.5 119.7*
665.7 108.2*
774.5 206.7*
To verify the expression levels of BCRP in MDA-MB-231 cells
stably transfected with empty vector (MDA-MB-231/V cells),
wild-type 482R (MDA-MB-231/R cells) and mutant 482T (MDA-
MB-231/T cells) BCRP transporter, western blot analysis was
performed. As shown in Fig. 3A, both MDA-MB-231/R and
MDA-MB-231/T cells displayed high levels of BCRP, whereas no
detectable expression was observed in MDA-MB-231/V cells.
To rule out any possible contribution by other transporters
in this cell model, cellular protein extracts were also sub-
jected to western blot analysis for the expression of Pgp, MRP1
and MRP2 using cell lysates from transfected MDCKII/MDR1,
MDCKII/MRP1 and MDCKII/MRP2 cells as positive controls. As
expected, no detectable levels of these proteins were found
in all three cell lines (Fig. 3A). MTT assays using MX, a typ-
ical BCRP substrate, were then performed to evaluate the
cytotoxicity profiles of these cells. As shown in Fig. 3B, the
forced expression of both wild-type 482R and mutant 482T
BCRP transporters in MDA-MB-231 cells conferred a 34–36-fold
increase in resistance to MX, as compared to that transfected
with an empty vector. We thus confirmed that this cell model
is suitable for evaluating chalcone interactions with the BCRP
transporter in the present study.
9
10
11
12
13
15
16
17
*p < 0.01.
a
MX accumulation was expressed as percentage of control.
Data from three independent experiments were expressed as
mean S.E.M.
DMSO vehicle control in Fig. 4. In the presence of FTC, the MX
accumulation amount was 300% of the control levels in both
MDA-MB-231/R and MDA-MB-231/T cells (Table 2), indicating
that FTC was active in inhibiting both wild-type (482R) and
mutant (482T) BCRP, thus preventing the efflux of MX. Com-
pared to untreated MDA-MB-231/R or MDA-MB-231/T cells, all
compounds except 1, 3, 8, 10 and 12 increased MX accumula-
tion in these cells to a significant degree (p < 0.01), indicating
that they were also inhibitors of the 482R and 482T BCRP pro-
tein. Among these “active” compounds, chalcones 2, 5–7 and
14–17 elicited comparable or higher levels of MX accumula-
tion compared to 10 ␮M FTC. These inhibitory effects were
observed at low micromolar concentrations of the test chal-
cones, suggesting that these compounds worked effectively
as BCRP inhibitors. MX accumulation assays were concur-
rently investigated on MDA-MB-231/V cells and neither FTC
nor chalcones markedly increased MX accumulation levels in
these cells (data not shown). This result further confirmed
that the enhanced MX uptake in MDA-MB-231/R and MDA-
3.3.
Chalcones inhibited the efflux of mitoxantrone
from both wild-type 482R and mutant 482T
BCRP-expressing MDA-MB-231 cells
To investigate the effect of chalcones on the accumulation of
BCRP substrates, MX accumulation assays were carried out
in the cell lines. MDA-MB-231/V, MDA-MB-231/R and MDA-
MB-231/T cells were pre-incubated with DMSO vehicle, 5 ␮M
chalcones or 10 ␮M FTC for 15 min before incubation with MX
at 37 ◦C for 40 min. FTC, used as a positive control here, is a
well-established specific BCRP inhibitor, whose use as a mod-
ulator in animal and human studies was precluded by serious
neurotoxic effects (Nishiyama and Kuga, 1989). The accumu-
lation of MX in the cells was measured as described in Section
2 by flow cytometry and shown as percentages relative to

european journal of pharmaceutical sciences 3 5 ( 2 0 0 8 ) 30–41
35
with FTC (300–350%) in MDA-MB-231/R cells (Table 2). In
MDA-MB-231/T cells, however, the same compounds induced
a
much higher increase in MX accumulation levels of
up to 600–700%, which was twice that of FTC. Looking
closely, we observed in particular that chalcones 5, 9 and
14–16 caused significantly higher levels of MX accumula-
tion (p < 0.01) in MDA-MB-231/T cells than MDA-MB-231/R cells
(Fig. 4).
A cursory examination of the “inactive” compounds that
did not significantly increased MX accumulation showed
that these were chalcones with 2-methoxy, 2,3,4-trimethoxy
or 2-hydroxy groups on ring A. No chalcone with 2,4-
dimethoxy or 2,4-dihydroxy groups was represented among
the “inactive” compounds. In addition, 2,4-dimethoxy and
2,4-dihydroxychalcones which had 2-chloro or 3-chloro on
ring B (5, 14–16) showed a greater potency in MDA-MB-
231/T cells than MDA-MB-231/R cells. The 4-chloro analogues
(6, 17) from each class did not show such
a differ-
ence.
3.4.
Chalcones do not affect transport activity of
calcein-AM by Pgp
Our recent report showed that various basic chalcones acted
as Pgp inhibitors whereas a non-basic member (6) was a
more potent inhibitor of BCRP than Pgp (Liu et al., 2008).
Since this observation was made for only one chalcone (6),
it was of interest to investigate this effect in other chalcones
that are structurally related to 6. Hence, we tested our chal-
cone library for their effects on Pgp-mediated uptake using
the calcein-AM assay. Calcein-AM is a Pgp substrate, which
upon intracellular deesterification is converted to calcein, a
florescent substance (Litman et al., 2000). Consequently, fluo-
rescence detected will be markedly increased in the presence
of inhibitors of Pgp-mediated efflux of calcein-AM. As shown
in Fig. 5, only cyclosporine (positive control) increased calcein-
AM uptake to a significant degree (p < 0.01). The test chalcones
had no significant effect on Pgp-mediated uptake, indicating
that this series of compounds do not have the dual func-
tionality reported earlier for the other chalcones (Liu et al.,
2008).
Fig. 3 – (A) Western blot analyses of BCRP, Pgp, MRP1 and
MRP2 expressions and (B) IC50 of MX in MDA-MB-231/V,
MDA-MB-231/R and MDA-MB-231/T cells. In (A), 20 ␮g of
cell lysates from each cell type were subjected to SDS-PAGE
and probed with anti-BCRP (BXP-21), anti-Pgp (C219),
anti-MRP1 (MRPr-1) and anti-MRP2 (M2III-6) antibodies. Cell
lysates derived from MDCKII/MDR1, MDCKII/MRP1 and
MDCKII/MRP2 cells, overexpressing Pgp, MRP1 and MRP2,
respectively, were used as positive controls for the
corresponding transporters. ␤-Actin was used as loading
control. In (B), cells in 96-well plates were treated with
increasing concentrations of MX for 72 h, after which cell
survival was determined via the MTT assay. Results were
obtained from three independent experiments with at least
triplicate measurements.
3.5.
Reversal of mitoxantrone resistance by chalcone
treatment
To determine the functional effect of chalcone inhibition
of the BCRP transporter, MX toxicity profile in MDA-MB-
231/R and MDA-MB-231/T cells were evaluated using MTT
assays. Initial experiments performed with chalcones alone
in these cells yielded IC50 values of above 5 ␮M (data not
shown). As such, various concentrations at 0.5, 1 and 2 ␮M
were chosen for the following experiments. Table 3 shows
the IC50 values of MX determined in the absence and pres-
ence of the chalcones on both cell lines. In the absence
of the chalcones, the IC50 values of MX in MDA-MB-231/R
and MDA-MB-231/T cells were 3.40 and 3.55 ␮M, respectively.
These values were 34–36-fold higher than that of MDA-MB-
231/V cells (IC50 0.10 ␮M). In the presence of chalcones, the
IC50 values of MX in MDA-MB-231/R and MDA-MB-231/T cells
were significantly decreased in a concentration-dependent
MB-231/T cells was due to the specific inhibition of BCRP
transporter.
Interestingly, we found that the chalcone analogues dis-
played different modulation profiles on MX accumulation
in the MDA-MB-231/R and MDA-MB-231/T cells (Fig. 4 and
Table 2), with MDA-MB-231/T cells being more sensitive
than MDA-MB-231/R cells. For the “active” compounds, the
increase in MX accumulation was comparable to that observed

36
european journal of pharmaceutical sciences 3 5 ( 2 0 0 8 ) 30–41
Fig. 4 – Effect of chalcones 1–17 on intracellular MX accumulation in MDA-MB-231/R and MDA-MB-231/T cells as measured
by flow cytometry. Cells were pre-incubated with 10 ␮M FTC (positive control) or 5 ␮M chalcones or vehicle (0.1% DMSO) for
15 min, followed by incubation with 3 ␮M MX for 40 min. The level of intracellular fluorescence was then analyzed by flow
cytometry and normalized with control fluorescence as percentage values after corrections for cell autofluorescence. Data
were representative of 3–4 independent experiments and expressed as mean S.E.M. Ctrl, control; 1–17, chalcones 1–17.
Open bar, MDA-MB-231/R cells; closed bar, MDA-MB-231/T cells. Data from vehicle control group and chalcone-treated groups
were analyzed using one-way ANOVA for statistical significance. *p < 0.01. Statistical difference was also compared between
MDA-MB-231/R and MDA-MB-231/T cells for each effective compound using Student’s t-test. ˆp < 0.01.
Fig. 5 – Effect of chalcones 1–17 on calcein-AM accumulation in Pgp-overexpressing MDCKII/MDR1 cells. Cells were
incubated with 10 ␮M cyclosporine A (positive control) or 10 ␮M chalcones or vehicle control (1% DMSO) for 30 min, after
which an aliquot of calcein-AM was added to each well to give a final concentration of 2 ␮M. After 10 min, the fluorescence
of each well was measured on a microplate reader with ꢀexcitation of 485 nm and ꢀemission of 535 nm. Ctrl, control; Cyc,
cyclosporine A; 1–17, chalcones 1–17. Data were obtained from four independent experiments and were expressed as
mean S.E.M. Data from control group and chalcone-treated groups were analyzed using one-way ANOVA for statistical
significance. *p < 0.01.
manner (Table 3), showing that the chalcones potentiated
MX cytotoxicity in the cells. Although such a modulation
effect was observed for most of the chalcones evaluated,
compounds 5 and 14–16 stood out for their potency on
both MDA-MB-231/R and MDA-MB-231/T cells. In contrast,
such modulation effect was not observed in the BCRP-
deficient MDA-MB-231/V cells (data not shown). Our results
clearly demonstrated the reversal of BCRP mediated-MDR
to MX by the co-administration of chalcones. In correla-
tion with the results obtained in MX accumulation studies,
chalcones with 2,4-dimethoxy or 2,4-dihydroxyl groups on
ring A (4–6, 14–17) exhibited higher reversal activities than
other chalcones (Table 3). However, no significant differ-
ence in the reversal of MX resistance by these compounds
was observed between MDA-MB-231/R and MDA-MB-231/T
cells.
3.6.
Effect of chalcones on BCRP ATPase activity
BCRP transports substrates by utilizing ATP hydrolysis
as energy source. As sodium orthovanadate inhibits
only BCRP-specific ATPase activity (Xia et al., 2007), the
vanadate-sensitive ATPase assay allows us to exclude
contributions by other ATPases and quantify BCRP-
specific ATPase activity. The difference between values
obtained in the presence and absence of sodium ortho-
vanadate will represent net ATPase activity contributed
by BCRP. As shown in Fig. 6, the ATPase activities of
the wild-type 482R and mutant 482T BCRP transporters
were 7.1 and 8.2 nmol ATP hydrolyzed/(min mg) protein,
respectively. The presence of 10 ␮M of FTC led to a signif-
icant reduction of BCRP ATPase activity to 4–5 nmol ATP
hydrolyzed/(min mg) protein in T cell membranes (p = 0.011).

european journal of pharmaceutical sciences 3 5 ( 2 0 0 8 ) 30–41
37
Table 3 – IC50 values of MX in MDA-MB-231/R and MDA-MB-231/T cells in the absence and presence of chalcones (0.5, 1
and 2 ␮M)
Chalconea
IC50 values of MXb
MDA-MB-231/R (controlc: 3.40 0.16)
MDA-MB-231/T (controlc: 3.55 0.84)
0.5 ␮M
1 ␮M
2 ␮M
1.24 0.49*
0.5 ␮M
1 ␮M
2 ␮M
1.20 0.26*
1
2
3
4
5
14
6
7
8
9
10
11
12
13
15
16
17
1.52 0.15*
1.83 0.70
3.10 1.08
2.12 0.50
1.64 0.27*
1.33 0.65*
2.72 0.37
2.25 0.40
2.80 0.54
2.25 0.40*
3.01 0.29
2.61 0.19*
2.33 0.34*
2.50 0.32*
1.10 0.30*
0.85 0.27*
2.22 0.31*
1.51 0.11*
1.46 0.19*
1.97 0.30*
1.14 0.26*
1.16 0.34*
0.82 0.25*
2.72 0.38
2.14 0.39*
2.04 0.33*
1.64 0.26*
2.60 0.19*
3.12 0.67
2.56 0.51
1.73 0.17*
1.13 0.06*
0.64 0.16*
1.42 0.30*
2.92 0.13
1.59 0.63*
3.19 0.13
2.49 0.50
1.89 0.37
1.52 0.34
2.03 0.15*
2.97 0.38
3.18 0.70
2.78 0.06
3.21 0.74
2.86 0.17
2.82 0.44
1.70 0.13
2.86 0.16
1.93 0.27
1.72 0.35
1.94 0.20
1.08 0.43*
2.41 0.81
1.16 0.15*
0.88 0.21*
0.99 0.29*
1.57 0.67*
1.58 0.44*
1.88 0.26*
1.80 0.40*
2.03 0.50
1.74 0.41
1.87 0.02
1.47 0.30
0.81 0.11*
1.02 0.18*
1.00 0.22*
0.86 0.21*
1.67 0.28*
0.87 0.19*
0.78 0.27*
0.45 0.12*
1.00 0.24*
1.96 0.52*
1.53 0.44*
1.67 0.33*
1.99 0.23*
2.63 0.54
1.72 0.29*
1.31 0.10*
0.70 0.28*
0.30 0.07*
1.03 0.29*
0.77 0.26*
2.02 0.40
1.06 0.30*
0.60 0.06*
0.66 0.29*
1.28 0.25*
1.11 0.25*
1.88 0.29*
1.23 0.22*
1.63 0.62
1.28 0.10*
1.22 0.39
0.67 0.15*
0.55 0.10*
0.64 0.25*
0.61 0.16*
*p < 0.01.
a
Chalcone, in the presence of each compound.
Data from three independent experiments were expressed as mean S.E.M. in ␮M.
Control, in the absence of chalcones.
b
c
Fig. 6 – Effect of chalcones 1–17 on vanadate-sensitive BCRP ATPase activity in crude membranes taken from MDA-MB-231/R
and MDA-MB-231/T cells. Crude membrane protein (5 mg protein/ml) from cells was incubated at 37 ◦C with 10 ␮M MX or
FTC or chalcones in the presence and absence of sodium orthovanadate (360 ␮M) in ATPase assay buffer for 20 min. The
reaction was stopped by adding 30 ␮l of 5% SDS solution and the released phosphate or phosphate standards were
measured as described in Section 2. Open bar, MDA-MB-231/R cells; shaded bar, MDA-MB-231/T cells. Data were obtained
from three independent experiments and expressed as mean S.E.M. Data from control group and chalcone-treated groups
were analyzed using one-way ANOVA for statistical significance. *p < 0.05.
Although a similar reduction was observed in R cell mem-
branes, there was no statistical significance (p = 0.166).
MX, on the other hand, did not produce any significant
effect.
Several 2,4-dimethoxy chalcones (5, 6 and 14) stimulated
BCRP ATP hydrolysis, with the rank order of 6 < 14 < 5, although
only the effect of compound 5 was statistically significant in
MDA-MB-231/R cells (p = 0.002). These stimulatory effects on
BCRP ATPase activity were not compatible with their inhibitory
effects on BCRP activity in MX accumulation and toxicity stud-
ies, as reported in Sections 3.3 and 3.5. The conflicting findings
may be related to the different concentrations of chalcones
used for the various assays and this led us to determine if
the effect of chalcones on BCRP ATPase activity showed a
dose response relationship. Hence, we selected compounds
5 and 14 and evaluated their effects on BCRP ATPase activ-
ity on both MDA-MB-231/R and MDA-MB-231/T membranes
at varying concentrations of 1, 2, 5, 10 and 20 ␮M. As seen
from Fig. 7, a dose response effect was evident with these
compounds. The stimulatory effects of compounds 5 and 14

38
european journal of pharmaceutical sciences 3 5 ( 2 0 0 8 ) 30–41
Fig. 7 – Concentration-dependent effects of FTC and selected chalcones (5 and 14) on vanadate-sensitive BCRP ATPase
activity in crude membranes taken from MDA-MB-231/R (A and C) and MDA-MB-231/T (B and D) cells. The
vanadate-sensitive BCRP ATPase assays were performed with the incubation of crude membrane protein (5 mg protein/ml)
from cells with increasing concentrations of FTC or chalcones in the presence and absence of sodium orthovanadate
(360 ␮M) in ATPase assay buffer at 37 ◦C for 20 min. The reaction was stopped by adding 30 ␮l of 5% SDS solution and the
released phosphate or phosphate standards were measured as described in Section 2. Data were obtained from three
independent experiments and expressed as mean S.E.M. Data from control group and treated groups were analyzed using
one-way ANOVA for statistical significance. *p < 0.05.
tease inhibitors on wild-type 482R BCRP than mutant 482T
BCRP. In order to compare the inhibition specificity of the
chalcones on wild-type and mutant BCRP, MX accumulation
and toxicity assays were performed in the presence of chal-
cones at 5 ␮M on both MDA-MB-231/R and MDA-MB-231/T
cells. A 3- or 6-fold induction was observed in MDA-MB-231/R
and MDA-MB-231/T cells in the MX accumulation studies,
respectively, showing that chalcones displayed a stronger
BCRP inhibitory activity on MDA-MB-231/T cells than MDA-
MB-231/R cells. In contrast, MX IC50 data obtained using
lower concentrations of chalcones (0.5–2 ␮M) did not reveal
significant differences in the ability of chalcones to reverse
MX resistance in both cells. This was especially evident for
compounds 5, 9 and 14–16, where there was a significant
difference in MX accumulation (p < 0.01) between MDA-MB-
231/R and MDA-MB-231/T cells, but not in the reversal of MX
resistance. Furthermore, even though the sensitivity of the
cells to MX was increased, it was not restored to levels sim-
ilar to that of MDA-MB-231/V cells. This suggests that the
specificity in inhibition occurred only in an acute manner but
was not sustained throughout the 72 h of incubation in the
cytotoxicity studies. In this case, we cannot rule out the pos-
sibility of rapid cellular metabolism of the chalcones which
may have limited their effects over a longer period of time.
Moreover, the concentrations used in the cytotoxicity stud-
ies were lower to ensure that the toxicity contributed by the
chalcones alone did not interfere with the studies and higher
concentrations may be used in future studies. In general, it
increased with concentration (more gradually in the case of
the MDA-MB-231/T membranes) and reached a plateau at
10 ␮M. Similar experiments were performed using membranes
prepared from MDA-MB-231/V cells but no detectable readings
were obtained (data not shown), suggesting that endogenous
ATPase activities did not interfere with our assays.
4.
Discussion
In this study, we investigated the ability of a small library of
methoxylated and hydroxylated chalcones to modulate BCRP
activity. The chalcones may be conveniently classified as 2-
methoxy, 2,4-dimethoxy, 2,3,4-trimethoxy, 2-hydroxy and 2,4-
dihydroxy chalcones, based on the substitution of the A ring.
Ring B in the chalcone is either substituted with chloro groups
(at 2ꢀ, 3ꢀ, 4ꢀ or 3ꢀ,4ꢀ positions) or left unsubstituted. We found
a good representation of chalcones from the 2,4-dimethoxy
and 2,4-dihydroxy families that significantly modulated BCRP
activity as revealed by MX accumulation and toxicity assays.
There was also a preference for a mono-halogenated ring B,
with good inhibitory activity associated with 2ꢀ and 3ꢀ chloro
groups.
Amino acid at position 482 has been shown to be criti-
cal in determining the substrate specificity of BCRP (Honjo
et al., 2001). Recent reports also revealed inhibitor speci-
ficity on wild-type 482R and mutant 482T BCRP. Gupta et al.
(2004) demonstrated a stronger inhibitory potential of HIV pro-

european journal of pharmaceutical sciences 3 5 ( 2 0 0 8 ) 30–41
39
is thus unclear how the amino acid position 482 on BCRP
may affect the recognition and binding of the chalcones to
BCRP and further studies are required to elucidate its exact
role.
result in an increase in specificity in the inhibition of ABC
transporters. It is also worth noting that besides BCRP and
Pgp, several flavonoids have recently been shown to inhibit
MRP1 and MRP2 (Van Zanden et al., 2005). Compounds such
as diosmetin, chrysoeriol and tamarixetin were observed to
inhibit MRP1 while myricetin and robinetin were observed to
inhibit MRP2 with IC50 values in the micromolar range (Van
Zanden et al., 2005). Given their structural similarities, we
cannot exclude the possible effect of our chalones on these
transporters. Further experiments need to be performed to
evaluate their impact on other transporters other than BCRP
and Pgp.
In conclusion, our present in vitro studies provide strong
evidence of chalcones as effective BCRP-reversing agents and
suggest that they may be potential candidates for develop-
ment as BCRP inhibitors. MDR, as conferred by BCRP, has been
a difficult barrier to overcome in the treatment of solid tumors
and hematological malignancies. A specific BCRP inhibitor
which would not interfere with other transporters and cause
unwanted drug–drug interactions is highly desired in clin-
ical use. Although our chalcone compounds do not inhibit
Pgp, pharmacokinetic interactions with drugs that are BCRP
substrates may still occur, resulting in alterations in bioavail-
ability and clearance during concomitant use. In addition,
mutant BCRP protein such as 482T BCRP has only been found
in drug-induced cell lines, but we cannot exclude the possible
expression of such proteins in cancer tissues due to exten-
sive exposure to chemotherapeutic agents. If these conditions
are encountered, chalcones may offer an advantage since they
inhibit both the wild-type and one mutant form of BCRP.
BCRP is energized by ATP hydrolysis and the ATPase assay
has been widely validated as one of the major methods to
determine the binding affinity and inhibitory mechanism
of BCRP inhibitors. Data in the literature suggested diverse
effects and different mechanisms of interaction between
BCRP inhibitors and the BCRP protein. Of the compounds
observed to inhibit BCRP activity, FTC is used widely as an
ATPase inhibitor (Rabindran et al., 2000); curcuminoids sig-
nificantly stimulated wild-type BCRP ATPase activity with
EC50 of 7.5–18 nM (Chearwae et al., 2006); 6-prenylchrysin and
tectochrysin (belonging to the flavonoid family) showed a
stimulation on mutant (482T) BCRP ATPase at a concentration
of 5 ␮M but not on wild-type BCRP ATPase (Ahmed-Belkacem
et al., 2005); and dietary phenethyl isothiocyanate was shown
to inhibit BCRP ATPase activity (Ji and Morris, 2005). There
are contradictory reports, however, on the effects of MX on
BCRP ATPase activity in several cellular systems. A stimula-
tory effect was reported using MX resistant MCF/MX100 cells,
which correlated well to its nature of being a BCRP substrate
(Ji and Morris, 2005), whereas no significant changes in wild-
type BCRP ATPase activity was observed in BCRP-transfected
HEK293 cells with MX (Vethanayagam et al., 2005). Our data
showed a correlation with the latter in that MX did not produce
significant effect in both MDA-MB-231/R and MDA-MB-231/T
membranes.
Surprisingly, although all our chalcone compounds inhib-
ited BCRP-mediated drug accumulation and toxicity, they
displayed diverse effects on BCRP ATPase activity. A stim-
ulatory effect was mostly observed with chalcones with
2,4-dimethoxy substitution on ring A (5, 6 and 14) which
were earmarked as good BCRP inhibitors in the MX accumu-
lation and cytotoxicity assays. A significant effect, however,
was observed only in compound 5 although the other two
members (6 and 14) have also shown high potency in MX accu-
mulation and cytotoxicity. This discrepancy may be a result of
low basal ATPase levels and relatively huge variations among
the samples. Besides, similar stimulatory effects on BCRP
ATPase activity have been observed with newly characterized
BCRP inhibitors. Chearwae et al. has reported a concentra-
tion dependent stimulatory effect on BCRP ATPase activity
with curcumin (Chearwae et al., 2006). Instead of exerting
their inhibitory effect directly by inhibiting ATP hydrolysis,
the authors proposed that curcumin might have bound to the
drug binding site (ATP hydrolysis site) of the BCRP protein,
leading to the stimulation of ATPase activity but the impair-
ment of transporter efflux activity of substrate drugs. In our
study, although the interactions between chalcones and BCRP
remain to be elucidated, their mode of BCRP inhibition may
not have involved BCRP ATPase inhibition.
Acknowledgements
We would like to thank Drs. Douglas Ross and Takeo
Nakanishi (University of Maryland Greenebaum Cancer
Center, Baltimore, US) for providing the MDA-MB-231/V,
MDA-MB-231/R and MDA-MB-231/T cells and Dr. Anton
Berns (Netherlands Cancer Institute, Antoni van Leeuwen-
hoek Hospital, Amsterdam, Netherlands) for providing the
MDCKII/MDR1, MDCKII/MRP1 and MDCKII/MRP2 cells. This
study was supported by the research grants R148050078101
and R148050078133 from the National University of Singapore
(PLRE).
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at doi:10.1016/j.ejps.2008.06.001.
r e f e r e n c e s
Lastly, in good agreement with our previous study (Liu et
al., 2008), we demonstrated that our extended series of non-
basic chalcones increased BCRP-mediated drug accumulation
and toxicity, with little or no effect on Pgp inhibition. This is
further evidence that the removal of basic functionalities from
chalcones caused a shift in the inhibitory profile from Pgp to
BCRP. Such a finding shows that structural modifications can
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