Methoxy stilbenes as potent, specific, untransported, and noncytotoxic inhibitors of breast cancer resistance protein

Article abstract of DOI:10.1021/cb200435y

The ABCG2 multidrug transporter is known to confer cancer cell multidrug resistance by causing the efflux of anticancer drugs; therefore, selective inhibitors have the potential to improve chemotherapeutic treatments. Here, various methoxy derivatives of resveratrol are shown to be potent inhibitors of mitoxantrone efflux by ABCG2: among a series of 11 derivatives, compound 9 (3,5,3′,4′-tetramethoxy trans-stilbene) had an IC₅₀ of 0.16 μM and showed a maximal inhibition of 75%, as measured by flow cytometry. It was not transported, as shown by HPLC fractionation and mass spectrometry titration and the lack of any cross-resistance in cell survival experiments. Compound 9 had a very low intrinsic cytotoxicity and was able to chemosensitize the growth of resistant ABCG2-transfected HEK293 cells at submicromolar concentrations. Drug-efflux inhibition was specific for ABCG2 since very low effects were observed with ABCB1 and ABCC1. The action mechanism of compound 9 was different from that of GF120918, which produced a complete and partly competitive but not ABCG2-specific inhibition, since ABCB1 was even more strongly inhibited. The two inhibitors also displayed different effects on the ABCG2 vanadate-sensitive ATPase activity, suggesting that they either bound to distinct sites or induced different conformational changes. Mitoxantrone efflux was fully inhibited by combining low concentrations of compound 9 with either GF120918 or a transport substrate such as prazosin or nilotinib. We conclude that methoxy derivatives of stilbene are good candidates for investigating future in vivo modulation of ABCG2 drug-efflux activity.

Full text of DOI:10.1021/cb200435y

Articles
pubs.acs.org/acschemicalbiology
Methoxy Stilbenes as Potent, Specific, Untransported, and
Noncytotoxic Inhibitors of Breast Cancer Resistance Protein
Glaucio Valdameri,†,‡ Luciana Pereira Rangel,†,§ Carmela Spatafora,∥ Jerome Guitton,⊥,#
́
̂
Charlotte Gauthier,† Ophelie Arnaud,† Antonio Ferreira-Pereira,§ Pierre Falson,†
́
Sheila M. B. Winnischofer,‡ Maria E. M. Rocha,‡ Corrado Tringali,∥ and Attilio Di Pietro†,*
†Equipe Labellisee
Lyon, France
‡Department of Biochemistry and Molecular Biology, Federal University of Parana,
§Departament of General Microbiology, Institute of Microbiology, Federal University of Rio de Janeiro, RJ, Brazil
∥Department of Chemical Sciences, University of Catania, Catania, Italy
́ ́ ́
Ligue 2009, Institut de Biologie et Chimie des Proteines FR 3302, BMSSI UMR 5086 CNRS/Universite Lyon 1,
́
Curitiba, PR, Brazil
⊥Hospices Civils de Lyon, Centre Hospitalier Lyon-Sud, Laboratoire de Biochimie-toxicologie, Pierre-Ben
#ISPBL, Faculte
de Pharmacie, Universite Lyon 1, Lyon, France
́
ite, France
́
́
ABSTRACT: The ABCG2 multidrug transporter is known
to confer cancer cell multidrug resistance by causing the efflux
of anticancer drugs; therefore, selective inhibitors have the
potential to improve chemotherapeutic treatments. Here,
various methoxy derivatives of resveratrol are shown to be
potent inhibitors of mitoxantrone efflux by ABCG2: among a
series of 11 derivatives, compound 9 (3,5,3′,4′-tetramethoxy
trans-stilbene) had an IC50 of 0.16 μM and showed a maximal
inhibition of 75%, as measured by flow cytometry. It was not
transported, as shown by HPLC fractionation and mass spectrometry titration and the lack of any cross-resistance in cell survival
experiments. Compound 9 had a very low intrinsic cytotoxicity and was able to chemosensitize the growth of resistant ABCG2-
transfected HEK293 cells at submicromolar concentrations. Drug-efflux inhibition was specific for ABCG2 since very low effects
were observed with ABCB1 and ABCC1. The action mechanism of compound 9 was different from that of GF120918, which
produced a complete and partly competitive but not ABCG2-specific inhibition, since ABCB1 was even more strongly inhibited.
The two inhibitors also displayed different effects on the ABCG2 vanadate-sensitive ATPase activity, suggesting that they either
bound to distinct sites or induced different conformational changes. Mitoxantrone efflux was fully inhibited by combining low
concentrations of compound 9 with either GF120918 or a transport substrate such as prazosin or nilotinib. We conclude that
methoxy derivatives of stilbene are good candidates for investigating future in vivo modulation of ABCG2 drug-efflux activity.
he ability of cancer cells to acquire resistance to anti-
able to catalyze the efflux of a large panel of drugs including
Tcancer drugs, a process termed “multidrug resistance topotecan, irinotecan, mitoxantrone, prazosin, and methotrex-
ate. Other pharmacologically important compounds are also
transported, including the polyphenol resveratrol,12 which has
been reported to provide both antioxidant, chemopreventive,
antiinflammatory, antiaging, cardioprotective, and neuroprotec-
tive activities.13,14
Known inhibitors of ABCG2 belong to various classes of
compounds. Nonselective, dual inhibitors already identified as
ABCB1 inhibitors include GF120918/elacridar,15 taxoids,16 and
XR9576/tariquidar.17 Other inhibitors, such as tyrosine kinase
inhibitors,18 cyclosporin A,19 and curcumin,20 are transported
and also interact with ABCC1. A few selective ABCG2
inhibitors have been identified, including fumitremorgin C
from Aspergillus fumigatus,21 which was however highly
(MDR) phenotype”, is a main obstacle to chemotherapeutic
treatments. The most frequent MDR mechanism is related to
the overexpression of multidrug ABC (“ATP-binding cassette”)
transporters within plasma membranes that alters the efficiency
of chemotherapeutics by lowering their intracellular concen-
tration.1
Among the main ABC transporters found to be overex-
pressed in resistant cancer cells, ABCG2, also called ABCP for
its abundance in placenta,2 BCRP (“breast cancer resistance
protein”),3 or MXR (“mitoxantrone resistance protein”),4 is the
most recently discovered. Earlier studies revealed the existence
of ABCB1/MDR1/P-glycoprotein and ABCC1/MRP1 (“multi-
drug resistance protein 1). The clinical relevance of ABCG2
was demonstrated in acute myeloid leukemia,5,6 and it is clearly
involved in drug bioavailability at protection barriers.7,8
ABCG2, which transports sulfated steroids,9 pheophorbide
a,10 and other porphyrins11 as physiological substrates, is also
Received: May 13, 2011
Accepted: October 31, 2011
Published: October 31, 2011
© 2011 American Chemical Society
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Table 1. Structures of Methoxy trans-Stilbenes
compound
OMe number
C2
C3
C4
C5
C3′
C4′
C5′
a
1
1
or
2
3
3
3
4
4
4
4
5
5
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
OMe
-
a
OMe
-
-
2
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
-
OMe
-
-
-
3
OMe
-
OMe
OMe
OMe
OMe
OMe
-
-
4
-
-
OMe
-
-
5
-
OMe
-
6
OMe
-
OMe
OMe
OMe
OMe
OMe
OMe
-
7
-
OMe
-
8
-
-
OMe
OMe
OMe
OMe
OMe
-
9
-
-
OMe
-
10
11
-
OMe
-
OMe
OMe
OMe
-
a
Because of the symmetry of compound 1 and for reasons of comparability, here are shown two alternative structures; the exact numbering should
be 4.
a
Table 2. Inhibitory Activity of Methoxy Stilbenes on Multidrug ABC Transporters
ABCG2-transfected HEK293 cells
ABCB1-transfected HEK293 cells
ABCC1-transfected HEK293 cells
compound
inhibition (%) 1 μM
inhibition (%) 5 μM
IC50 (μM)
inhibiton (%) 5 μM
b
GF120918
nd
100.0
0.120 0.03
100.0
nd
1
none
5.1 0.1
9.9 3.7
6.6 0.9
36.2 4.6
58.5 21.6
32.5 12.2
55.7 12.1
67.4 8.6
75.6 7.9
76.7 10.5
34.2 8.5
−7.3 0.6
2.5 0.4
−2.2 0.5
4.5 1.6
−2.1 0.1
6.4 7.2
−2.4 1.7
4.6 4.0
3.2 2.9
2.7 1.7
1.1 2.6
2.3 1.4
2
5.5 2.7
none
−12.3 0.5
−14.3 0.4
−15.9 1.0
−19.2 0.3
−23.5 0.4
−19.8 1.5
−12.5 12.6
−14.2 13.6
−9.4 11.0
−16.9 2.3
3
4
13.7 5.4
11.9 0.14
8.8 1.9
20.2 4.9
48.8 3.6
65.2 7.2
57.1 9.7
12.2 3.3
5
6
7
8
0.47 0.08
0.16 0.04
0.29 0.09
9
10
11
a
Inhibition of mitoxantrone efflux in ABCG2-transfected cells (R482 wild-type). For ABCG2-transfected cells, the percent inhibition was determined
using GF120918 as a control (100% inhibition). The values of (%) inhibition and IC50 (half-maximal inhibition) were determined by flow cytometry
as described in Methods. Data are the mean SD of at least three independent experiments. For ABCC1-transfected cells, the percent inhibition was
b
normalized using cells transfected with empty pcDNA3.1 as a control. For ABCB1-transfected cells, the percent inhibition was determined using
GF120918 as a control (100% inhibition); its IC50 value was 0.034 0.01 μM).
neurotoxic, and Ko143,22 one of several synthetic and less
RESULTS AND DISCUSSION
Methoxy Derivatives of Stilbene As Inhibitors of
ABCG2-Mediated Drug Efflux. Resveratrol, or 3,5,4′-
■
cytotoxic derivatives. Some analogues of XR957623 and of
GF12091924 were found to be ABCG2-specific but they also
rather cytotoxic, and their in vivo activity was limited by their
trihydroxy-trans-stilbene, has been reported to be transported
bioavailability.25 We identified a flavonoid-binding site
interacting with tectochrysin and 6-prenylchrysin, which
generated up to 75% inhibition with good affinity but however
again exhibited significant cytotoxicity.26 Lower-affinity rote-
noids27 and acridone derivatives24 bound to the same site as
flavones and benzo-pyrane/furane derivatives, for which 3D
models exist.28,29
by ABCG212 and to exhibit low affinity inhibition of the efflux
of other drugs.30 Replacing the hydroxyl groups of resveratrol
by methoxy groups led to more potent inhibitors, as shown
by a series of 11 derivatives containing a variable number of
methoxy groups at different positions on the two aromatic
rings (Table 1), when analyzed by flow cytometry using a
mitoxantrone-efflux assay. The results (Table 2) showed that
the increased intracellular accumulation of mitoxantrone,
corresponding to inhibition of mitoxantrone efflux, depended
on the number of methoxy substituents, from 1 to 4, and on
their positions. The highest efficiency of mitoxantrone-efflux
inhibition was obtained with compound 9, characterized by a
low IC50 (concentration giving a half-maximal inhibition) of
The aim of the present study was to identify new, more
potent, and less cytotoxic compounds than 6-prenylchrysin.
Methoxy stilbenes were found effective for inhibition without
inducing cytotoxicity, suggesting that these molecules could be
used in combination with other inhibitors to block ABCG2
drug-efflux activity.
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Figure 1. Inhibition of rhodamine 123 efflux in HEK293 cells transfected by either wild-type ABCB1 or R482T ABCG2 constructs. (A) Effects of
compounds 9 and 10 on ABCB1-mediated efflux. The percent inhibition was determined by flow cytometry, using GF120918 as a control (100%
■
○
inhibition). The data are the mean SD of two independent experiments. (B) Effects of compound 9 ( ) and GF120918 ( ) on R482T-ABCG2-
mediated efflux. The percent inhibition was determined by flow cytometry and normalized using cells transfected with empty pcDNA3.1 as a control.
Data are the mean SD of two independent experiments.
Figure 2. Cytotoxicity of compounds 9 and 10 and sensitization to mitoxantrone. Cell survival was determined by MTT assays as described in
□
●
Methods. (A, B) Cell viability of HEK293-ABCG2 (R482 wild-type) ( ) and HEK293-pcDNA3.1 control cells ( ) upon 72-h treatment with either
compound 9 or compound 10 at increasing concentrations, as indicated. (C, D) Cell viability of HEK293-ABCG2 and HEK293-pcDNA3.1 cells
▲
□
▽
upon co-treatment with compound 9 (C) or compound 10 (D) at either 0.2 μM ( ) or 1 μM ( ) and mitoxantrone (0−0.25 μM) for 72 h.
●
Parallel experiments with only mitoxantrone were performed with ABCG2-transfected ( ) and control ( ) cells. The values represent the mean
SD of percent cell viability with respect to the untreated control. Data correspond to at least three independent experiments performed in triplicate.
0.16 μM, a value comparable to that obtained with the
reference inhibitor GF120918. Methoxy substitution at position
4′ was important since a 3-fold decrease in affinity was observed
when the methoxy group was shifted to position 5′ in
compound 8 (IC50 = 0.47 μM); this may also explain the
lower efficiency of compound 10 (IC50 = 0.29 μM), despite the
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Figure 3. Different mechanism of compounds 9 and 10 in comparison with GF120918. (A) Different types of inhibition produced by compound 9,
■
□
●
○
compound 10, and GF120918 at ( ) 0 μM, ( ) 0.1 μM, ( ) 0.5 μM, and ( ) 2 μM on ABCG2-mediated mitoxantrone efflux, as measured at low
concentrations (upper panels) and high concentrations (lower panels) of the substrate mitoxantrone. (B) Stimulation by compound 9 (white bars)
and compound 10 (black bars) of vanadate-sensitive ABCG2 ATPase activity, by contrast to the lack of GF120918 effects. The data represent three
independent experiments performed in triplicate.
presence of an additional methoxy group at position 4 with an
improved inhibition. By contrast, substitution at position 2 in
compound 11 (and compound 6) was clearly disadvantageous,
compared to substitution at position 4 in compound 10 (and
compound 7). The negative effect of C-2 methoxy substitution
was also confirmed by the poor inhibition of compound 3 (in
comparison with compounds 4 and 5) and of compound 11 (in
comparison with compound 8). In addition to position 4′, the
position 3′ of the right aromatic ring needed to be substituted
since, at 5 μM each, compound 4 was much less active than
compound 9, and compound 7 was less active than compound
10. The advantageous effect of 4- (versus 5-) substitution was
also found in compound 5 by comparison to compound 4, at
5 μM each.
The high inhibition potency of compound 9 against
mitoxantrone efflux has a 2-fold higher affinity than 6-
prenylchrysin (IC50 of 0.29 μM), which was previously
characterized by our group as a good inhibitor lead
compound.26 The fact that the hydroxylated transport substrate
resveratrol was converted into the potent inhibitor compound 9
by complete substitution with methoxy groups was consistent
with our previous observations that methoxylation increases
both inhibition efficiency and binding affinity of flavones, when
comparing tectochrysin to chrysin.26
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Figure 4. Absence of transport of different inhibitors, in contrast to substrates, and bimodulation of mitoxantrone efflux. (A) All compounds were
tested at 2 μM, and their percent intracellular accumulation was determined by HPLC−MS and normalized using pcDNA3.1-transfected cells as a
control (100%). GF120918 (5 μM) was used to inhibit the transport of mitoxantrone, prazosine, and compounds 9 and 10, whereas Ko143
(0.3 μM) was used to inhibit the transport of GF120918. The initial amounts of compounds found in control cells were the following: compound 9
(1.94 ng/mL), compound 10 (2.2 ng/mL), mitoxantrone (107 ng/mL), and GF120918 (101 ng/mL). (B) Bimodulation was assayed by flow
cytometry as above. The compounds were used either alone at the indicated concentration (white bars) or in combination with 2 μM compound 9
(black bars). Data are the mean SD of four independent experiments.
The drug-efflux inhibition by these methoxy derivatives
appears to be quite specific for ABCG2, since no inhibition of
either ABCB1-mediated mitoxantrone efflux or ABCC1-
mediated calcein efflux was observed with any compound, at
least up to a 5 μM concentration. By contrast, the reference
inhibitor GF120918 was even more potent toward ABCB1
(IC50 = 0.034 μM) than toward ABCG2 (IC50 = 0.120 μM).
The specificity of the compounds was also studied with respect
to the efflux of another usual substrate, namely rhodamine 123.
Figure 1A shows that the GF120918-sensitive efflux of
rhodamine 123 by ABCB1 required very high concentrations,
up to 100 μM of compounds 9 and 10, to be partly inhibited
(25% and 10%, respectively), in agreement with other data
published for compound 9.31 Rhodamine 123 efflux could also
be studied using the R482T ABCG2 mutant: a maximum of
75% inhibition was then obtained at 10−20 μM, with an IC50
value of 0.45 μM (Figure 1B). Interestingly, the maximal
inhibition produced by GF120918 was not complete and
GF120918 showed an affinity lower than that of compound 9
(IC50 of 2.7 μM). This contrasts with the inhibition of
mitoxantrone efflux by wild-type ABCG2 reported in Table 2,
suggesting that GF120918 and methoxy stilbenes may use
different mechanisms to inhibit ABCG2-mediated drug efflux.
Figure 2A,B shows the effects of the two most potent
methoxy stilbenes on cell growth, as determined by a cell-
survival MTT assay. Compounds 9 and 10 appeared to have
very low intrinsic cytotoxicity (around 10% and 20%,
respectively, at 10 μM). In both cases, their IG50 (concentration
producing a 50% inhibition of cell growth) value was ≥60 μM,
giving a high therapeutic ratio IG50/IC50 of 200−400 for
cytotoxicity versus inhibition of drug efflux. In addition, the
same curves obtained with either ABCG2-transfected cells or
control cells indicated no apparent cross-resistance, suggesting
that the compounds were not transported by ABCG2. By
contrast, GF120918 was more cytotoxic on control cells (IG50
around 20 μM, not shown here) and produced cross-resistance
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at concentrations higher than 5 μM, suggesting that GF120918
might be transported at high concentrations. Both methoxy
stilbenes sensitized the growth of ABCG2-transfected cells to
mitoxantrone (Figure 2C,D), with a stronger effect produced
by compound 9, in comparison to compound 10 at
submicromolar concentration, in agreement with its higher
affinity to inhibit mitoxantrone efflux (cf. Table 2).
Absence of Transport of ABCG2 Inhibitors, and
Combination toward a Complete Modulation of
Mitoxantrone Efflux. The two methoxy stilbenes, com-
pounds 9 and 10, were not transported in ABCG2-transfected
cells at 2 μM, a saturating inhibition concentration, when
assayed by HPLC separation and mass spectrometry titration
(Figure 4A). By contrast, the positive control with mitoxan-
trone showed an 80% efflux, which was largely blocked in the
presence of GF120918 acting as an ABCG2 inhibitor. As
expected, addition of GF120918 did not modify accumulation
of compound 9, although it produced some decrease in that of
compound 10. Transport was also observed with the other
substrate prazosin (not shown here). GF120918 itself was not
transported when assayed at 2 μM by the same procedure, and
no transport was observed with 6-prenylchrysin (not shown
here), another ABCG2-specific inhibitor that we previously
characterized.26 The lack of transport of methoxy stilbenes
agrees with their noncompetitive inhibition. In contrast, the
dual inhibitor GF120918, which fully inhibits mitoxantrone
efflux with high affinity, appears able to be transported at high
(but not low) concentrations, in agreement with its partially
competitive inhibition. This suggests that its binding site partly
overlaps with the mitoxantrone binding/transport site.
Since the methoxy stilbenes were not transported, in
common with GF120918 at low concentration, we checked
the possibility of using these compounds together since they
apparently act with different mechanisms and probably bind to
distinct sites, in order to reach a complete inhibition at low
inhibitor concentrations. Figure 4B shows the effects of different
compounds in combination with compound 9. In agreement
with the data of Table 2, compound 9 alone at 2 μM produced a
maximal inhibition of 75%, significantly lower than the 100%
inhibition produced by 5 μM GF120918. Interestingly, the
combination of compound 9 with either 0.4 μM of compound
10 or 2 μM 6-prenylchrysin (a saturating inhibition concen-
tration) did not further increase the maximal inhibition of
compound 9 alone, suggesting that the three compounds might
bind to the same site. In contrast, combination with non-
saturating concentrations of either GF120918 (0.12 μM) (a
partly competitive inhibitor that appeared to be transported at
high concentrations) or one of the two transported substrates
prazosin and nilotinib32 led to complete inhibition, consistent
with the fact that both GF120918 and nilotinib alone at
saturation (5 μM and 1 μM, respectively) produced a complete
inhibition. An intermediate situation was observed with another
ABCG2-specific inhibitor, fumitremorgin C (FTC), which partly
increased the inhibition.
Different Molecular Mechanisms of ABCG2-Specific
Methoxy Stilbenes and the Dual Inhibitor GF120918. The
difference in maximal inhibition of both types of inhibitors
on mitoxantrone efflux (around 75% versus 100% at 10 μM
mitoxantrone, cf. Table 2) justified the further investigation of
the inhibition mechanism. When the mitoxantrone concentration
varied from 0.5 to 10 μM, Lineweaver−Burk double-reciprocal
plots showed a downward curvature, indicating a negative
cooperativity. A Hill number of 0.7 was calculated, suggesting
the involvement of at least two interacting mitoxantrone-
transporting sites within a functional ABCG2 oligomer (not
shown here). The type of inhibition was therefore studied at
both low and high mitoxantrone concentrations (Figure 3A).
For compounds 9 and 10, the inhibition always appeared to be
noncompetitive, since the degree of inhibition only depended
on the inhibitor concentration and not on the substrate con-
centration (at either low or high concentrations). By
contrast, the GF120918 inhibition appeared to be partly
competitive at low mitoxantrone concentrations, for each
inhibitor concentration, since the extent of inhibition was
stronger at low versus high substrate concentrations (between
0.5 and 2 μM); however, at higher substrate concentrations
(2.5−10 μM) the inhibition appeared to be noncompetitive.
The partially competitive inhibition of GF120918 at low
mitoxantrone concentrations was confirmed by the fact that the
IC50 for GF120918 was clearly lower at low substrate con-
centrations. This was not the case for either compounds 9 or
10. This suggests that the slight concentration-dependence
observed for compound 9 at low concentrations is not
significant and that both methoxy trans-stilbenes were non-
competitive inhibitors.
Figure 3B shows that the effects produced by the different
compounds on ABCG2 ATPase activity were also quite
distinct. Indeed, while GF120918 at 5 μM did not display
any effect, both methoxy stilbenes produced a strong
stimulation of vanadate-sensitive ATP hydrolysis, up to 200%
for compound 9, and 150% for compound 10, at the same, low,
concentrations that inhibit mitoxantrone efflux.
The specificity of compounds 9 and 10 for ABCG2 is
consistent with their noncompetitive inhibition indicating a
binding site distinct from the transport site. This transport site
is expected to have many common features with ABCB1 since
both transporters efflux a number of common substrates
including mitoxantrone, Hoechst 33342, and prazosine. The
interaction at this transport site was more sensitive to the
R482T mutation than the interaction at the ABCG2-specific
site, as monitored here by the relative loss in efficiency against
rhodamine 123 and mitoxantrone efflux.26 Methoxy stilbenes
probably bind to the same site as 6-prenylchrysin, since both
types of ABCG2-specific inhibitors display the same partial
inhibition (around 75%), possibly due to their rather small size.
Also the combined action of the two compounds does not
Methoxy stilbenes thus appear to be useful for modulating
ABCG2 drug-efflux activity in two alternative ways: either
alone, if 75% inhibition is sufficient to increase anticancer drug
bioavailability and toxicity (with the advantage of not fully
inhibiting the physiological transport activity in normal cells),
or in combination with other inhibitors, such as GF120918 or a
transport substrate such as prazosin or nilotinib32 (as shown here,
but probably as well with other substrates such as curcumin33)
with the advantage of yielding complete inhibition at low
concentrations and thus limiting side effects.
These new inhibitors are characterized by a very low cytotoxicity,
in contrast with 6-prenylchrysin,26 and provide a 10-fold higher
therapeutic ratio (200−400 versus 20−40). In addition, at low
concentrations these compounds chemosensitize cell growth to
mitoxantrone, indicating that no major intracellular metabolization
appears to occur. This is not the case with curcumin, which is
increase this maximal inhibition. Such a specific site, which is
27
able to bind other natural products such as rotenoids
and
acridones,24 was characterized in detail and allowed the
elaboration of a molecular model for typical inhibitors.28,29
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easily metabolized.34 Methoxy stilbenes therefore appear to be
good candidates for in vivo experiments and are expected to be
more efficient than previously used compounds.24,25,35,36
(DMEM high glucose), supplemented with 10% fetal bovine serum
(FBS), 1% penicilin/streptomycin, and drug supplemented in some
cases with either 0.75 mg mL−1 G418 (HEK293-pcDNA3.1, HEK293-
R482 or R482T), 2 mg mL−1 G418 (HEK293-MDR1), or 5 μM
etoposide (HEK293-MRP1).
METHODS
ABCG2- and ABCB1-Mediated Drug Transport. HEK293 cells
were seeded at a density of 1 × 105 cells/well into 24-well culture
plates and, after 48 h, exposed to 10 μM mitoxantrone (HEK293-R482
and HEK293-MDR1 cells) or 10 μM rhodamine 123 (HEK293-R482T
and HEK293-MDR1 cells) for 30 min at 37 °C, in the absence or
presence of compounds at various concentrations. After cell washing
with phosphate buffered saline (PBS) and incubation for 1 h in
substrate-free medium with compounds at the same concentrations,
the intracellular drug fluorescence was monitored with a FACS Calibur
cytometer (Becton Dickinson). At least 10,000 events were collected,
for which the maximal fluorescence (taken as 100%) was the difference
between the geometric mean fluorescence of cells incubated with
5 μM GF120918 and cells without inhibitor. The Hill number of
mitoxantrone efflux in HEK293 cells transfected by either wild-type
ABCG2 or R482T ABCG2 contructs was determined from the curves
(sigmoidal -3 parameters) fitted in the SigmaPlot 11.0 software and
calculated by the equation f = axb/(cb + xb), where b is the Hill
number.
MRP1-Mediated Transport. HEK293 cells transfected with either
MRP1 or the empty vector were seeded at a density of 1 × 105 cells/
well into 24-well culture plates. After 48-h incubation, the cells were
exposed to 0.2 μM calcein-AM and analyzed by flow cytometry as
described above. The maximal fluorescence (taken as 100%) was the
difference between the geometric mean fluorescences of control cells
(HEK293-pcDNA3.1) and MRP1-transfected cells, incubated with
calcein-AM but without inhibitor.
Cytotoxicity Assays. HEK293-R482 and HEK293-pcDNA3.1 cells
were seeded into 96-well culture plates at a 1 × 104 cells/well density.
After overnight incubation, the cells were treated with various
concentrations of compounds 9 and 10 for 72 h at 37 °C under 5%
CO2. For the sensitization experiments, after overnight incubation, the
cells were concomitantly treated with compound 9 or compound 10
and increasing concentrations of mitoxantrone for 72 h at 37 °C under
5% CO2. In both cases, cell viability was evaluated with a 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) col-
orimetric assay. Control experiments were performed with high
glucose DMEM containing 0.1% of DMSO (v/v). The results were
expressed as percentage of viable cells versus control cells taken as
100%.
■
Materials. Mitoxantrone, rhodamine 123, calcein-AM, and 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) were
purchased from Sigma Aldrich (France). All other reagents were
commercial products of the highest available purity grade.
1
The H spectra were run in CDCl3 using a Varian Unity Inova
spectrometer operating at 500 MHz. Mass spectra were recorded
in ESI positive mode on a Micromass ZQ2000 spectrometer (Waters).
All reactions were monitored by TLC on commercially avail-
able precoated plates (silica gel 60 F254), and the products were
visualized with cerium sulfate solution; silica gel 60 (25−40 μm)
was employed as stationary phase for preparative column flash
chromatography.
The previously reported trans-4-methoxystilbene (compound 1),37
trans-3,5-dimethoxystilbene (compound 2),38 trans-2,3,4′-trimethox-
ystilbene (compound 3),39 trans-3,5,4′-trimethoxystilbene (compound
4),40 trans-3,4,4′-trimethoxystilbene (compound 5),41 trans-3,4,5,4′-
tetramethoxystilbene (compound 7),42 trans-3,5,3′,5′-tetramethoxys-
tilbene (compound 8),40 trans-3,5,3′,4′-tetramethoxystilbene (com-
pound 9),40 and trans-3,4,5,3′,5′-pentamethoxystilbene (compound
10)43 were synthesized according to a general protocol based on an
Arbuzov rearrangement followed by the Horner−Emmons−Wads-
worth reaction; the spectral data of the isolated products were in
perfect agreement with the literature; the 1H NMR data of compound
1
10, which were not previously reported, are the following: H NMR
(CDCl3, 500 MHz) δ 3.84 (s, 6H, -OCH3), 3.88 (s, 3H, -OCH3), 3.92
(s, 6H, 3-OCH3), 6.41 (t, J = 2.0, 1H, H-4′), 6.67 (d, J = 2.0, 2H, H-2′
and H-6′), 6.74 (s, 2H, H-2 and H-6), 6.95 (d, J = 16.5, 1H, H-7), 7.02
(d, J = 16.5, 1H, H-8).
The previously unreported compounds trans-2,3,5,4′-tetramethox-
ystilbene (compound 6) and trans-2,3,5,3′,5′-pentamethoxystilbene
(compound 11) were synthesized in two steps: (i) a mild m-CPBA
direct aromatic hydroxylation, according to a previous work of some of
us, which afforded the intermediates 2-hydroxy-3,5,4′-trimethoxystil-
bene and 2-hydroxy-3,5,3′,5′-tetramethoxystilbene; (ii) subsequent
methylation with dimethyl sulfate to obtain the final products. A
solution of m-CPBA in CH2Cl2 (0.150 mmol/mL) was added to a
stirred solution of the substrate in CH2Cl2 (0.105 mmol/mL) at RT.
The reaction mixture was then washed with a NaHSO3 solution and
subsequently with saturated aqueous NaHCO3. The organic layer was
dried (Na2SO4), filtered, and concentrated in vacuo; the residue was
submitted to flash chromatography on a 3 cm × 25 cm silica gel
column, eluted with EtOAc in n-hexane (from 0% to 30%) to give the
hydroxylated stilbenoids; these intermediates (0.107 mmol) were
submitted to methylation using dimethyl sulfate (15 μL) in acetone
(20 mL) in presence of anhydrous potassium carbonate (0.107 mmol).
trans-2,3,5,4′-Tetramethoxystilbene (6). 1H NMR (CDCl3,
500 MHz) δ 3.78 (s, 3H, OCH3), 3.84 (s, 6H, OCH3), 3.86 (s, 3H,
-OCH3), 6.43 (d, J = 3.0, 1H, H-4) 6.69 (d, J = 3.0, 1H, H-6), 6.91 (d,
J = 8.5, 2H, H-3′ and H-5′), 7.11 (d, J = 16.5, 1H, H-a), 7.32 (d, J =
16.5, 1H, H-8), 7.50 (d, J = 8.5, 2H, H-2′ and H-6′).
ATPase Activity. ATPase activity was determined as previously44
by quantifying the release of inorganic phosphate from ATP by a
colorimetric assay. Membranes from HEK293-R482 cells (10 μg
proteins) were incubated for 30 min at 37 °C in 50 mM NaCl, 50 mM
Tris pH 7.5 buffer supplemented with 5 mM ATP, 5 mM NaN3, 7 mM
MgCl2, 0.1 mM EGTA, 2 mM oubain, the effectors (compound 9,
compound 10 and GF120918) with or without vanadate 0.6 mM, at a
final volume of 60 μL. After incubation, the reaction was stopped by
adding 30 μL of 10% SDS for 10 min at 4 °C and then 180 μL of the
solution B: 5 mL of solution A (2.16 g ammonium molybdate
dissolved in 35 mL of 15 mM zinc acetate) dissolved in 20 mL of
freshly prepared 20 μM ascorbic acid pH 5.0. After 20-min incubation
at 37 °C, the released phosphate was colorimetrically quantified at
620 nm.
Transport by HPLC and Mass Spectrometry. HEK293-R482
and HEK293-pcDNA3.1 cells were seeded at a density of 5 × 105 cells/
well into 6-well culture plates. After 48-h incubation, cells were treated
with the compounds for 30 min at 37 °C under 5% CO2, then washed
with PBS, and trypsinized. They were suspended in 1 mL of PBS and
centrifuged at 1,000 × g for 5 min. The pellet was suspended in 1 mL
of PBS, and 50 μL was removed for protein quantification
by the BCA method. Cells were again centrifuged and suspended in
50 μL of methanol and stored at −80 °C until further analysis.
Intracellular quantification of the different compounds was
performed using a triple quadrupole tandem mass spectrometer with
an electrospray source coupled to a liquid chromatography (LC−ESI-
trans-2,3,5,3′,5′-Pentamethoxystilbene (11). 1H NMR
(CDCl3, 500 MHz) δ 3.79 (s, 3H, -OCH3), 3.84 (s, 9H, -OCH3),
3.86 (s, 3H, -OCH3), 6.41 (t, J = 2.0, 1H, H-4′), 6.45 (d, J = 2.0, 1H,
H-4), 6.69 (d, J = 2.0, 1H, H-6), 6.70 (d, J = 2.0, 2H, H-2′ and H-6′),
7.02 (d, J = 16.5, 1H, H-7), 7.41 (d, J = 16.5, 1H, H-8).
All compounds were dissolved in DMSO and then diluted in DMEM
high glucose medium. The stock solution was stored at −20 °C and
warmed to 25 °C just before use.
Cell Cultures. Human fibroblast HEK293 cell lines transfected
with either wild-type or mutant ABCG2 (HEK293-R482 cells) or the
empty vector (HEK293-pcDNA3.1 cells) were obtained as previ-
ously.25 HEK293 cell lines transfected with either MDR1 or MRP1
were kindly provided by Dr. S. E. Bates (NCI, NIH, Bethesda, MD).
All cells were maintained in Dulbecco’s modified Eagle’s medium
328
dx.doi.org/10.1021/cb200435y | ACS Chem. Biol. 2012, 7, 322−330

ACS Chemical Biology
Articles
MS/MS) from ThermoFisher. The data were acquired using the
positive ion mode. The SRM transitions were m/z 384.3 → 247.2 for
prazosin, 445.5 → 88.0 for mitoxantrone, 323.0 → 267.0 for 6-
phenylchrysin, 564.0 → 252.0 for GF120918, 331.0 → 177.2 for
compound 10, 301.1 →146.2 for compound 9, and 349.0 → 305.0 for
camptothecin used as internal standard. The chromatographic
separation was achieved on HypersilGold 100 mm × 2.1 mm column
(ThermoFisher, USA). A first method was used for HPLC analysis of
compounds 9 and 10, using a mobile phase constituted by ammonium
acetate buffer (pH 6, 50 mM), propanol-2, and acetonitrile. A second
method was performed for the other compounds with a mobile phase
constituted by water and acetonitrile both with 0.1% formic acid. For
both methods a gradient elution mode was used. The I.S. was added
and the mixture was then submitted to vortex (for 30 s) and to
centrifugation (for 5 min at 13,000 × g). Calibration curves and quality
control of different compounds were prepared by spiking blank cells
with appropriate standard solutions. The organic layer was removed
and evaporated to dryness under a stream of nitrogen. The residues
were suspended in 200 μL of mobile phase, and 10 μL was injected in
the HPLC device.
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AUTHOR INFORMATION
■
(12) Breedveld, P., Pluim, D., Cipriani, G., Dahlaus, F., van
Eijndhoven, M. A. J., de Wolf, C. J. F., Kuil, A., Beijnen, J. H.,
Scheffer, G. L., Jansen, G., Borst, P., and Schellens, J. H. (2007) The
effect of low pH on breast cancer resistance protein (ABCG2)-
mediated transport of methotrexate, 7-hydroxymethotrexate, metho-
trexate diglutamate, folic acid, mitoxantrone, topotecan and resveratrol
in in vitro drug transport models. Mol. Pharmacol. 71, 240−249.
(13) Shukla, Y., and Singh, R. (2011) Resveratrol and cellular
mechanisms of cancer prevention. Ann. N.Y. Acad. Sci. 1215, 1−8.
(14) Szekeres, T., Saiko, P., Fritzer-Szekeres, M., Djavan, B., and
Corresponding Author
*E-mail: a.dipietro@ibcp.fr.
ACKNOWLEDGMENTS
■
We thank R. W. Robey and S. E. Bates, NCI-NIH Bethesda, for
providing the transfected HEK293 cell lines, V. M.
Bhusainahalli for help in preparing some methoxy stilbenes,
and R. Lavery for rereading the manuscript. The following grant
providers are acknowledged: French National League against
Jager, W. (2011) Chemopreventive effects of resveratrol and
̈
̂
Cancer (Labellisation 2009); Rhone-Alpes Region (CIBLE
2010); International ANR-NKTH (2010-INTB-1101-01) to
A.D.P.; CNRS and University Lyon 1 (UMR 5086); Ministero
resveratrol derivatives. Ann. N.Y. Acad. Sci. 1215, 89−95.
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Ojima, I., Baer, M. R., and Bernacki, R. J. (2003) Taxane-based reversal
agents modulate drug resistance mediated by P-glycoprotein, multi-
drug resistance protein, and breast cancer resistance protein. Mol.
Cancer Ther. 2, 1195−1205.
̀
della Pubblica Istruzione, Universita e Ricerca Scientifica
(PRIN, Rome, Italy) and University of Catania (Progetti di
Ricerca di Ateneo, Catania, Italy). G.V. and L.R. were
supported by sandwich Ph.D. fellowships from the Brazilian
Federal Agency for the Support and Evaluation of Graduate
Education (CAPES) (process numbers 1203-10-8 and 1842-08-
0, respectively).
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is a broad-spectrum multidrug resistance modulator. Clin. Cancer Res.
11, 2320−2326.
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