A synthetic chalcone as a potent inducer of glutathione biosynthesis

Article abstract of DOI:10.1021/jm2016073

Chalcones continue to attract considerable interest due to their anti-inflammatory and antiangiogenic properties. We recently reported the ability of 2′,5′-dihydroxychalcone (2′,5′-DHC) to induce both breast cancer resistance protein-mediated export of glutathione (GSH) and c-Jun N-terminal kinase-mediated increased intracellular GSH levels. Herein, we report a structure-activity relationship study of a series of 30 synthetic chalcone derivatives with hydroxyl, methoxyl, and halogen (F and Cl) substituents and their ability to increase intracellular GSH levels. This effect was drastically improved with one or two electrowithdrawing groups on phenyl ring B and up to three methoxyl and/or hydroxyl groups on phenyl ring A. The optimal structure, 2-chloro-4′,6′-dimethoxy-2′- hydroxychalcone, induced both a potent NF-E2-related factor 2-mediated transcriptional response and an increased formation of glutamate cysteine ligase holoenzyme, as shown using a human breast cancer cell line stably expressing a luciferase reporter gene driven by antioxidant response elements.

Full text of DOI:10.1021/jm2016073

Article
pubs.acs.org/jmc
A Synthetic Chalcone as a Potent Inducer of Glutathione
Biosynthesis
Remy Kachadourian,*,†,‡ Brian J. Day,†,‡,§ Subbiah Pugazhenti,‡,∥ Christopher C. Franklin,§
Estelle Genoux-Bastide,⊥ Gregory Mahaffey,∥ Charlotte Gauthier,# Attilio Di Pietro,#
and Ahcene Boumendjel⊥
̀
†Department of Medicine, National Jewish Health, Denver, Colorado 80206, United States
‡Department of Medicine, University of Colorado, Aurora, Colorado 80045, United States
§Department of Pharmaceutical Sciences, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of Colorado, Anschutz
Medical Campus, Aurora, Colorado 80045, United States
∥Denver VA Medical Center, Denver, Colorado 80220, United States
⊥UJF-Grenoble 1/CNRS, Dep
#Equipe Labellisee
́
artement de Pharmacochimie Molec
́
ulaire UMR 5063, Grenoble, F-38041, France
́
Lyon 1, Institut de Biologie et Chimie des Proteines, 69367
́
Ligue 2009, BMSSI, UMR5086 CNRS-Universite
́
Lyon Cedex 7, France
S
* Supporting Information
ABSTRACT: Chalcones continue to attract considerable interest
due to their anti-inflammatory and antiangiogenic properties. We
recently reported the ability of 2′,5′-dihydroxychalcone (2′,5′-DHC)
to induce both breast cancer resistance protein-mediated export of
glutathione (GSH) and c-Jun N-terminal kinase-mediated increased intracellular GSH levels. Herein, we report a structure−
activity relationship study of a series of 30 synthetic chalcone derivatives with hydroxyl, methoxyl, and halogen (F and Cl)
substituents and their ability to increase intracellular GSH levels. This effect was drastically improved with one or two
electrowithdrawing groups on phenyl ring B and up to three methoxyl and/or hydroxyl groups on phenyl ring A. The optimal
structure, 2-chloro-4′,6′-dimethoxy-2′-hydroxychalcone, induced both a potent NF-E2-related factor 2-mediated transcriptional
response and an increased formation of glutamate cysteine ligase holoenzyme, as shown using a human breast cancer cell line
stably expressing a luciferase reporter gene driven by antioxidant response elements.
associated with the production of reactive oxygen species
(ROS) and mitochondrial dysfunction,14,15 nontoxic concen-
trations have been reported to inhibit the synthesis of the
inducible nitric oxide synthase, induce the expression of HO-1,
and trigger GSH synthesis and export.14,16−20 We recently
reported the ability of the naturally occurring chalcone 2′,5′-
dihydroxychalcone (2′,5′-DHC) to induce breast cancer
resistance protein (BCRP)-mediated export of GSH21 and c-
Jun N-terminal kinase (JNK)-mediated increased levels of
cellular GSH,14 suggesting that chalcones could be used to
modulate cellular levels of GSH.
Herein, we report a structure−activity relationship study of a
series of 30 chalcone derivatives with hydroxyl, methoxyl, and
halogen (chlorine and fluorine) substituents (Figure 1) in their
ability to increase intracellular levels of GSH (iGSH). This
study was carried out using a human breast cancer cell line
stably expressing a luciferase reporter gene driven by
antioxidant response elements [MCF-7/antioxidant response
element (AREc32), referred as AREc32 cells in this study],
INTRODUCTION
■
Glutathione (GSH) is a tripeptide present at high concen-
trations in the cell (5−10 mM) with diverse functions including
modulation of cell proliferation, antioxidant defense, and
detoxification of xenobiotics.1,2 The rate-limiting enzyme for
GSH synthesis is glutamate cysteine ligase (GCL), which is
composed of a catalytic subunit (glutamate cysteine ligase
catalytic subunit, GCLC) and a regulatory subunit (glutamate
cysteine ligase regulatory subunit, GCLM).3 The second step of
GSH synthesis is catalyzed by glutathione synthase (GS). Low
levels of GSH have been associated with a variety of diseases
including diabetes, pulmonary fibrosis, and epilepsy.2,4−6 Along
with a number of phase II antioxidant enzymes such as heme
oxygenase-1 (HO-1), the enzymes involved in GSH synthesis
are tightly regulated by several transcription factors including
NF-E2-related factor 2 (Nrf2), activator protein-1 (AP-1), and
nuclear factor κB (NF-κB).2,3 In the case of GCL, post-
transcriptional factors have also been reported.3,7
Chalcones are both biosynthetic precursors of flavonoids and
end products associated with a variety of biological activities.8,9
Anti-inflammatory and antiangiogenic properties of naturally
occurring and synthetic chalcones continue to attract
considerable interest.10−13 While their toxicity has been
Received: November 28, 2011
Published: January 12, 2012
© 2012 American Chemical Society
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Chalcone 23 Induces GSH Biosynthesis. To determine
whether the 23-induced increase in iGSH was due to increased
GSH synthesis, AREc32 cells were cotreated with 23 (10 μM)
and buthionine sulfoximine (BSO, 20 μM), an inhibitor of
GCL.23 As shown in Figure 2A, 23-induced increased GSH
levels were blocked by BSO. Comparison of the effects of 23 to
those of the known inducers of GSH biosynthesis, sulforaphane
(SFN, mixture of D and L stereoisomers) and tert-butylhy-
droquinone (tBHQ), indicates that 23 is significantly more
potent (Figure 2A).24,25 Moreover, both 3 (2′,5′-DHC) and 23
(20 μM, 6 h of treatment) induced an increase in GCL
holoenzyme formation (Figure 3, bottom panel). Interestingly,
this was not associated with an increase in either GCLC or
GCLM protein levels (Figure 3, top panels). The formation of
GCL holoenzyme using 3 was harder to detect with a longer
treatment time (24 h).14 To examine the signal transduction
pathways mediating 23-induced GSH synthesis, pharmacolog-
ical inhibitors of the JNK and p38 mitogen-activated protein
(MAP) kinase pathways were employed, namely, anthrapyr-
azolone (SP600125, 20 μM) and 4-[4-(4-fluorophenyl)-2-(4-
methylsulfinylphenyl)-1H-imidazol-5-yl]pyridine (SB203580,
20 μM), respectively.14,26−28 Both compounds showed only a
weak inhibitory effect on 23-induced increased iGSH levels
(Figure 2B). Because ROS have also been associated with
increased GSH synthesis, the catalytic antioxidant manganese-
(III) meso-tetrakis(N,N′-diethylimidazolium-2-yl)porphyrin
(MnTDE-1,3-IP5+, 20 μM) was tested as well, showing a
similar weak effect (Figure 2B).14,29
Chalcone 23 Induces Nrf2 Transcriptional Activity.
Constitutive and inducible GSH biosynthesis has been
associated with Nrf2 transcriptional activity, and recent studies
demonstrate that chalcones with electrowithdrawing groups on
phenyl ring B induce Nrf2 activity.2,10 Selected compounds of
our series, that is, chalcone 23, 28 and 3, were examined at 5
μM for their ability to induce Nrf2-dependent ARE-luciferase
activity in AREc32 cells (18 h of treatment) and compared to
the activities of the known Nrf2 activators SFN and tBHQ (5
μM). The 23-induced luciferase activity was ∼4−5-fold higher
than 3 and SFN and ∼10-fold higher than tBHQ (Figure 4A).
In an attempt to determine the pathways involved in mediating
this Nrf2 transcriptional response, AREc32 cells were
preincubated with 20 μM SP600125 (JNK inhibitor),
SB203580 (p38MAPK inhibitor), or the catalytic antioxidant
MnTDE-1,3-IP5+ before treatment with 23. While SP600125
and MnTDE-1,3-IP5+ showed a slight inhibitory effect (yet not
statistically significant), SB203580 had no effect on 23-induced
ARE-luciferase activity (Figure 4B).
Figure 1. General structure of the chalcones tested.
which also allowed us to examine the chalcone-induced Nrf2
transcriptional activity.22
Chemistry. The chalcones assessed in this study were
synthesized following the classical method shown in Scheme 1.
An acetophenone derivative was condensed with 1 equiv of a
benzaldehyde derivative in the presence of a solution of KOH
in a mixture H2O:MeOH.
RESULTS
■
Chalcone-Induced Increased Levels of iGSH. A series of
chalcone analogues with hydroxyl, methoxyl, and halogen
groups in both phenyl rings A and B were tested at 10 μM in
their ability to elevate iGSH levels in AREc32 cells (6 and 24 h
treatments) (Figure 1 and Table 1). The most active
compound was chalcone 23 with a 3-fold increase in iGSH
levels as compared to control (24 h treatment) followed by
chalcones 28, 24, and 21. The positive role of the chlorine on
ring B can be clearly seen by comparing chalcones 21, 23, 24,
and 28 to their unsubstituted analogues (14, 22, and 26). The
higher activity of chalcone 23 vs 15 and 16 shows the
importance of three methoxyl and/or hydroxyl groups on ring
A. Comparison with 24 shows that one chlorine on ring B has a
higher effect than two chlorines. However, this is not a general
rule since 21 was more active than 16, showing that the optimal
number of electrowithdrawing groups on ring B depends on the
number of substituents on ring A. Adding a third chlorine (29
vs 28) appeared to decrease the effect. The higher activity of 16
(one Cl in position 2) as compared to 19 (one Cl in position 3)
reveals the importance of the position of the chlorine for
optimal activity. To determine whether the electrowithdrawing
effect of the chlorine was responsible for the higher activity, we
synthesized and evaluated chalcones substituted with fluorines
and a nitro group. As shown in Table 1, the introduction of
fluorines (15 vs 16) and a nitro group (27 vs 28) did not
enhance the activity, indicating that the electrowithdrawing
effect is not the only factor mediating the higher activity of the
chlorine analogues. Chlorines on ring A (30) had no increased
effect. It is also worth noting that the chalcone with no
halogens that had the optimal effect was chalcone 7, with two
methoxyl groups in positions 2 and 6 on ring B, and that adding
another methoxyl group in position 4 (8) abolished the effect.
Chalcone 23-Induced Cytotoxicity. Because 23 has a
markedly higher ability to induce GSH synthesis than 3, we
compared their relative toxicity in AREc32 cells (0−60 μM, 48
h of treatment). As shown in Figure 5, 23 had a slight higher
toxicity than 3 (IC50 around 25 and 33 μM, respectively),
suggesting a correlation between toxicity at high concentrations
Scheme 1. Synthetic Procedure of Chalcones
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a
Table 1. Induction of iGSH Increased Levels in AREc32 Cells
substituents on phenyl A
substituents on phenyl B
iGSH (%)
chalcone
2′
3′
4′
5′
6′
2
3
4
5
6
6 h
24 h
1
2
103
111
82
115
189
177
174
160
153
231
104
180
193
183
232
173
158
195
211
204
171
165
182
256
191
299
250
226
209
223
261
246
120
88
OCH3
OCH3
3
OH
OH
4
OH
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
Cl
OCH3
100
114
100
90
5
OH
OCH3
OCH3
6
OH
OCH3
7
OH
OCH3
OCH3
Cl
8
OH
91
9
OH
108
107
88
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
OH
OCH3
Cl
Cl
OH
OCH3
Cl
Cl
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OH
OCH3
OCH3
Cl
109
78
F
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
Cl
87
F
103
105
106
82
Cl
NO2
F
Cl
88
F
F
104
122
101
107
119
135
91
Cl
Cl
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OCH3
OH
Cl
Cl
F
OH
Cl
F
OH
OCH3
OCH3
OCH3
OCH3
Cl
F
F
118
122
119
93
Cl
Cl
Cl
Cl
Cl
OCH3
OCH3
OCH3
OCH3
Cl
Cl
OCH3
98
a
Cells were exposed to chalcones (10 μM) tested in their ability to increase cellular levels of GSH (iGSH as a percentage as compared to control, 6
and 24 h of treatment, margin error 10%, n = 3).
and the ability to induce both the Nrf2 transcriptional activity
and the GSH synthesis at nontoxic concentrations.
precise mechanism by which 23 promotes increased GCL
holoenzyme formation.
We have previously reported that chalcones induce a bimodal
response in iGSH levels overtime.14 The initial drop in iGSH
levels at 6 h of treatment is due to chalcone-induced efflux of
GSH, and we recently demonstrated that BCRP mediates this
phenomenon.21 The most active compounds in inducing GSH
biosynthesis showed no decrease in iGSH levels at 6 h of
treatment, suggesting that the synthesis of GSH compensates
for the loss of GSH early in treatment. On the other hand,
some chalcones have been found to inhibit BCRP-mediated
drug efflux.31−33 The fact that the combination of 23 and BSO
resulted in lower iGSH levels than BSO alone (Figure 2A)
suggests that 23 is able to induce GSH efflux as well. It is worth
noting that BCRP expression has been associated with Nrf2
activation.34 It remains to be determined whether there is a
correlation between the abilities of chalcones to inhibit BCRP
and to induce GSH transport.
New compounds that are able to induce GSH biosynthesis
might have beneficial effects in a number of diseases that have
been associated with low levels of GSH.4−6 The chemo-
protective effects of sulforaphane have been studied in a
number of models, including cancer and neuroprotec-
tion.24,30,35 Some triterpenoids have also raised interest as
potent Nrf2 activators with antioxidant and anti-inflammatory
DISCUSSION
■
Among the chalcones tested, chalcone 23 showed the highest
activity in inducing GSH biosynthesis. It also showed a strong
ability to trigger the Nrf2 transcriptional response. These
effects were significantly higher than those of sulforaphane, a
well-known inducer of Nrf2-trancriptional activity and GSH
biosynthesis.24,30 While the JNK pathway seemed to play a
significant role in mediating 2′,5′-DHC-induced Nrf2 activation
and GSH synthesis,14 this role appeared to be rather weak in
the case of 23. Also, results from studies employing 23 in
combination with the catalytic antioxidant MnTDE-1,3-IP5+
suggest an insignificant role for ROS in triggering this Nrf2
response. Similar to 3, 23 enhanced GCL holoenzyme
formation. Intriguingly, this was not the result of increased
GCL subunit expression since 23 did not increase either GCLC
or GCLM protein expression. This and previous results suggest
a post-transcriptional and post-translational effect of 23 and 3
on GCL holoenzyme formation.14 Taken together, our results
show that chalcone-induced GSH synthesis does not rely
exclusively on Nrf2 activation, despite an apparent cause−effect
relationship. Further studies are needed to determine the
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Figure 3. Chalcone 23-induced formation of GCL holoenzyme in
AREc32 cells. As shown by immunoblotting protein extracts of treated
AREc32 cells (6 h treatment), both 3 and 23 (20 μM, respectively)
increased the formation of GCL holoenzyme as compared to control
(native PAGE), whereas no increase in GCLC nor GCLM was
detected (SDS PAGE). α-GAPDH was used as an internal standard.
The experiment was repeated once.
purchased from Biomol (Plymouth Meeting, PA) and SP600125 from
Calbiochem (San Diego, CA). MnTDE-1,3-IP5+, prepared as
previously described in U.S. patent #6,544,975B1, was a kind gift
from Aeolus Pharmaceuticals (Mission Viejo, CA).
Synthesis of Chalcones: Typical Procedure. To a stirred
solution of acetophenone derivative (1 mmol) and a benzaldehyde
derivative (1 mmol) in MeOH (10 mL) was added KOH (50%
aqueous solution, 1 mL). The solution was heated at 70 °C for 3−5 h,
MeOH was evaporated, and the residue was dissolved in CH2Cl2/H2O
(50 mL, 4:1). The organic layer was washed with brine and
evaporated, and then, column chromatography was carried out over
silica gel (AcOEt/hexane 1:2). In the case of fluorinated chalcones,
KOH (25%) was used. The structural analysis and characterization are
provided in the Supplementary Information.
Figure 2. Chalcone-induced increases in iGSH levels in AREc32 cells.
(A) Chalcone 23-induced increase in iGSH (10 μM) was higher when
compared to 3 (10 μM), 28 (10 μM), sulforaphane (SFN, 10 μM),
and tert-butylhydroquinone (BHQ, 10 μM) (24 h treatment) and was
inhibited by BSO (20 μM), an inhibitor of GSH synthesis. (B) The
SP600125 and SB203580 inhibitors of JNK and p38MAPK,
respectively, and the catalytic antioxidant MnTDE-1,3-IP5+ (SP, SB,
and MnP, 20 μM, respectively) (24 h of treatment) had a weak
inhibitory effect on 23-induced increase in iGSH. Bars with different
letters were statistically different from one another (n = 3, P < 0.05).
Analytical Methods. 1H NMR spectra were recorded on a Bruker
̈
AC-400 instrument (400 MHz). Chemical shifts (δ) are reported in
ppm relative to Me4Si (internal standard). Electrospray ionization ESI
mass spectra were acquired by the Analytical Department of Grenoble
University on an Esquire 300 Plus Bruker Daltonis instrument with a
nanospray inlet. Combustion analyses were performed at the
Analytical Department of Grenoble University, and all tested
compounds had a purity of at least 95%. Thin-layer chromatography
(TLC) used Merck silica gel F-254 plates (thickness 0.25 mm) and
flash chromatography used Merck silica gel 60, 200−400 mesh.
Cell Line and Culture Conditions. Transformed human breast
cancer cells stably expressing a luciferase reporter gene driven by AREs
(MCF-7/AREc32) were obtained from Dr. Joe M. McCord
(University of Colorado, Aurora, CO) and were grown in DMEM
(low glucose) supplemented with 10% fetal bovine serum (FBS), 1%
pen/strep (10000 unit, Cellgro), and Geneticin (400 mg/500 mL) at
37 °C and 5% CO2-supplemented air atmosphere.22
activities.36 Mounting data support that, rather than being
“antioxidants” per se, flavonoids and other polyphenols induce
an antioxidant adaptive response, most likely by triggering cell-
signaling pathways.26,37 In this regard, chalcones in particular
appear to be very interesting.14 Another salient aspect of this
study is our finding that chalcones post-translationally enhance
GCL holoenzyme formation. Although 23 was slightly more
toxic than 3, 23 was markedly more efficient in inducing both
the Nrf2 transcriptional response and the GSH synthesis at
nontoxic concentrations. To conclude, 23 is a potent new
activator of Nrf2 transcriptional activity that has the ability to
modulate intracellular levels of GSH.
Intracellular Levels of GSH. Intracellular GSH levels were
determined by HPLC with electrochemical detection (HPLC-EC).38
Cultured AREc32 cells from 24-well plates were washed once with 1
mL of PBS, resuspended in 0.5 mL of PBS, and sonicated. Ten percent
meta-phosphoric acid (25 μL) was then added to the samples (1% v/v
final concentration), the samples were centrifuged at 20000g for 10
min, and the supernatants were used for HPLC analysis. The HPLC
column used was a Synergi 4u Hydro-RP 80A (150 mm × 4.6 mm)
from Phenomenex (Torrance, CA), and the mobile phase was sodium
EXPERIMENTAL SECTION
■
Chemicals and Reagents. Chemicals and reagents for chalcone
synthesis were obtained from either Sigma-Aldrich (St. Louis, MO) or
Acros (Geel, Belgium). Chalcone (1), 2′,5′-DHC (3), and 2′,4′,6′-
trimethoxychalcone (26) were purchased from Indofine Chemicals
Company, Inc. (Hillsborough, NJ). BSO, D,L-sulforaphane (SFN), tert-
butylhydroquinone (tBHQ), L-GSH, pyruvate, NADH, and meta-
phosphoric acid were purchased from Sigma-Aldrich. SB203580 was
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The remaining 0.1 mL sample was used to measure protein content
using Coomassie plus protein assay reagent (Thermoscientific,
Rockford, IL).
Immunoblotting of GCLC, GCLM, and GCL Holoenzyme.
AREc32 cells were grown in six-well plates treated and washed with
PBS. Cells were then sonicated in 200 μL of PBS. Cell debris was spun
down, and the supernatant volume was reduced to approximately 30
μL by evaporation using a speed vacuum system. GCLC, GCLM, and
GCL holoenzyme levels were detected as described previously.7 α-
Glyceraldehyde-3-phosphate dehydrogenase (α-GAPDH) was used as
an internal standard.
Luciferase Activity. AREc32 cells were cultured in 12-well plates
to ∼60% confluence and incubated in the presence of 5−10 μM
compound for 18 h. In some experiments, the cells were preincubated
with the catalytic antioxidant MnTDE-1,3-IP5+, the SP600125 JNK
inhibitor, or the SB203580 p38MAPK inhibitor for 30 min before
exposure to 23. The treated cells were washed with PBS and lysed with
cell lysis buffer (BD Biosciences, San Jose, CA). The cell lysate was
centrifuged at 20000g for 15 min, and the supernatant was used for the
firefly luciferase assay. Luciferase assays were carried out using
enhanced luciferase assay kit (BD Biosciences) on a Moonlight 2010
luminometer.39
Assessment of Cytotoxicity. The 3-[4,5-dimethylthiazol-2-yl]-
2,5-diphenyl-tetrazolium bromide (MTT) assay is commonly used to
measure cancer cell survival; yet, it has revealed artifacts when
measuring the cytotoxicity of pro-oxidant agents.40 Another simple
method to evaluate drug-induced cytotoxicity is using membrane
integrity as an index, which can be assessed by monitoring the release
of cytosolic lactate dehydrogenase (LDH). AREc32 cells were grown
in 24-well plates, and LDH activity was measured after 48 h of
treatment in both culture medium and cell lysates (50 mM HEPES,
0.5% Triton X-100, pH 7) using a plate reader format as previously
described.41 Briefly, 5 μL of either cell culture supernatant or lysates
were incubated with 0.24 mM NADH in a Tris/NaCl, pH 7.2, buffer
in 96-well plates for 5 min at 25 °C. The reaction was started by the
addition of 9.8 mM pyruvate, and the consumption of NADH was
followed at 340 nm for 5 min at 30 °C. The percent LDH release was
calculated by the following equation: supernatant LDH/(supernatant
LDH + lysate LDH) × 100.
Figure 4. Chalcone 23-induced Nrf2 transcriptional response in
AREc32 cells. (A) Using 5 μM concentrations, 23-induced luciferase
activity was ∼4−5-fold higher than 3 and SFN (18 h treatment). (B) A
10 μM concentration of 23-induced luciferase activity was slightly
decreased (statistically insignificantly) by the SP600125 inhibitor of
JNK (SP, 20 μM) and by the catalytic antioxidant MnTDE-1,3-IP5+
(MnP, 20 μM). The SB203580 inhibitor of p38MAPK (SB, 20 μM)
had no inhibitory effect. Bars with different letters were statistically
different from one another (n = 4, P < 0.05).
Statistical Analysis. Data are presented as means
standard
errors. Each experimental group consisted of 3−4 wells, and the results
were repeated at least once. Data were subsequently analyzed for
significant differences using ANOVA analysis coupled with a Tukey's
range test where significance was preset at P < 0.05 (Prizm v.4,
GraphPad, San Diego, CA).
ASSOCIATED CONTENT
* Supporting Information
Characterization of chemical compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
AUTHOR INFORMATION
Corresponding Author
*Tel: 303-398-1295. Fax: 303-270-2168. E-mail: remyka@
gmail.com.
■
Notes
Figure 5. Cytotoxicity of chalcones 23 and 3 in AREc32 cells.
Chalcone 23 was more toxic than 3 (IC50 of 25 and 33 μM,
respectively) using the percentage of LDH release as an index of
cytotoxicity (48 h of treatment, n = 3).
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We are grateful to Madeleine Blanc and Chantal Beney for
technical assistance in the synthesis of the chalcones. We also
thank Dr. Joe M. McCord (University of Colorado, Aurora) for
providing the MCF-7/AREc32 cell line generated by Dr. C.
Roland Wolf (University of Dundee, United Kingdom). This
work was supported by NIH Grants HL755223, HL84469,
ES015678, and ES017582 to B.J.D. and VA Merit Review
NEUD-004-07F to S.P. C.G. is the recipient of a doctoral
phosphate buffer (125 mM sodium phosphate monobasic, pH
adjusted to 3 with phosphoric acid) and 0.9% methanol. The flow
rate was 0.5 mL/min. The retention time for GSH under these
conditions was 7.0 min. The HPLC instrument was from ESA, Inc.
(Chelmsford, MA) and was equipped with an autosampler (model
540) and a Coul array detector (model 5600A). The potential applied
was +0.75 V vs H/Pd electrode, and the injection volume was 5 μL.
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drial outer membrane determined by fluorescence spectroscopy. J.
Biochem. Biophys. Methods 2006, 69, 143−150.
fellowship from the Ligue Nationale Contre le Cancer (Equipe
́
Labelisee Ligue 2009). B.J.D. is a consultant for and holds
equity in Aeolus Pharmaceuticals that is commercially
developing metalloporphyrins as human therapeutic agents.
(16) Ban, H. S.; Suzuki, K.; Lim, S. S.; Jung, S. H.; Lee, S.; Ji, J.; Lee,
H. S.; Lee, Y. S.; Shin, K. H.; Ohuchi, K. Inhibition of
lipopolysaccharide-induced expression of inducible nitric oxide
synthase and tumor necrosis factor-alpha by 2′-hydroxychalcone
derivatives in RAW 264.7 cells. Biochem. Pharmacol. 2004, 67,
1549−1557.
ABBREVIATIONS USED
■
AP-1, activator protein-1; ARE, antioxidant response element;
BCRP, breast cancer resistance protein; BSO, L-buthionine
sulfoximine; 2′,5′-DHC, 2′,5′-dihydroxychalcone; α-GAPDH, α-
glyceraldehyde-3-phosphate dehydrogenase; GCLC, glutamate
cysteine ligase catalytic subunit; GCLM, glutamate cysteine
ligase regulatory subunit; GSH, reduced glutathione; HO-1,
heme oxygenase-1; HPLC-EC, HPLC with electrochemical
detection; JNK, c-Jun N-terminal kinase; LDH, lactate
dehydrogenase; MAP, mitogen-activated protein; MnTDE-
1,3-IP5+, manganese(III) meso-tetrakis(N,N′-diethylimidazo-
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