Semisynthetic flavonoid 7-O-galloylquercetin activates Nrf2 and?induces Nrf2-dependent gene expression in RAW264.7 and?Hepa1c1c7 cells

Article abstract of DOI:10.1016/j.cbi.2016.10.015

The natural flavonoid quercetin is known to activate the transcription factor Nrf2, which regulates the expression of cytoprotective enzymes such as heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxidoreductase 1 (NQO1). In this study, a novel semisynthetic

Full text of DOI:10.1016/j.cbi.2016.10.015

Chemico-Biological Interactions 260 (2016) 58e66
Contents lists available at ScienceDirect
Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint
Semisynthetic flavonoid 7-O-galloylquercetin activates Nrf2
and induces Nrf2-dependent gene expression in RAW264.7
and Hepa1c1c7 cells
a
,
b
c
d
a
Lenka Roubalov aꢀ , David Biedermann , Barbora Papou ꢁs kov aꢀ , Jan Vacek ,
c
c
a,
b
e
Marek Kuzma , Vladimír K ꢁr en , Jitka Ulrichov aꢀ , Albena T. Dinkova-Kostova ,
a, b, *
Ji ꢁr í Vrba
a
Department of Medical Chemistry and Biochemistry, Faculty of Medicine and Dentistry, Palacký University, Hn ꢁe votínsk aꢀ 3, Olomouc 77515, Czech Republic
Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacký University, Hn ꢁe votínsk aꢀ 3, Olomouc 77515, Czech Republic
Institute of Microbiology, Laboratory of Biotransformation, Czech Academy of Sciences, Víde nꢁ sk aꢀ 1083, Prague 14220, Czech Republic
Regional Centre of Advanced Technologies and Materials, Department of Analytical Chemistry, Faculty of Science, Palacký University, 17. listopadu 12,
b
c
d
Olomouc 77146, Czech Republic
e
Jacqui Wood Cancer Centre, Division of Cancer Research, School of Medicine, University of Dundee, Dundee DD1 9SY, Scotland, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 August 2016
Received in revised form
The natural flavonoid quercetin is known to activate the transcription factor Nrf2, which regulates the
expression of cytoprotective enzymes such as heme oxygenase-1 (HO-1) and NAD(P)H:quinone oxido-
reductase 1 (NQO1). In this study, a novel semisynthetic flavonoid 7-O-galloylquercetin (or quercetin-7-
gallate, 3) was prepared by direct galloylation of quercetin, and its effect on the Nrf2 pathway was
examined. A luciferase reporter assay showed that 7-O-galloylquercetin, like quercetin, significantly
activated transcription via the antioxidant response element in a stably transfected human AREc32 re-
porter cell line. In addition, 7-O-galloylquercetin caused the accumulation of Nrf2 and induced the
expression of HO-1 at both the mRNA and protein levels in murine macrophage RAW264.7 cells. The
6
October 2016
Accepted 19 October 2016
Available online 21 October 2016
Keywords:
Quercetin
induction of HO-1 by 7-O-galloylquercetin was significantly suppressed by N-acetyl-L-cysteine and
Quercetin-7-gallate
Methyl gallate
Nrf2
Heme oxygenase-1
Metabolism
SB203580, indicating the involvement of reactive oxygen species and p38 mitogen-activated protein
kinase activity, respectively. HPLC/MS analyses also showed that 7-O-galloylquercetin was not degal-
loylated to quercetin, but it was conjugated with glucuronic acid and/or methylated in RAW264.7 cells.
Furthermore, 7-O-galloylquercetin was found to increase the protein levels of Nrf2 and HO-1, and also
the activity of NQO1 in murine hepatoma Hepa1c1c7 cells. Taken together, we conclude that 7-O-gal-
loylquercetin increases Nrf2 activity and induces Nrf2-dependent gene expression in RAW264.7 and
Hepa1c1c7 cells.
©
2016 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
Natural flavonoids are a large group of extensively studied plant
Abbreviations: ARE, antioxidant response element; DMSO, dimethyl sulfoxide;
ERK, extracellular signal-regulated kinase; Gapdh, glyceraldehyde-3-phosphate
dehydrogenase; GCLC, glutamate-cysteine ligase catalytic subunit; GCLM, gluta-
mate-cysteine ligase modifier subunit; HO-1/Hmox1, heme oxygenase-1; JNK, c-Jun
N-terminal kinase; Keap1, Kelch-like ECH-associated protein 1; MAPK, mitogen-
phenolic compounds. The basic flavonoid structure consists of two
benzene rings (A and B) connected by three carbons that form an
oxygenated heterocycle (ring C). The flavonoid skeletons such as
flavone and flavanone may bear different numbers of hydroxyl
groups, modifications of which can yield a multitude of compounds
[1]. The flavonol quercetin (3,5,7,3 ,4 -pentahydroxyflavone; Fig. 1),
arguably the most abundant and most studied bioflavonoid, is
predominantly found in plants in the form of its glycosides and
activated
protein
kinase;
MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-
-cysteine; NQO1, NAD(P)H:quinone
0
0
diphenyltetrazolium bromide; NAC, N-acetyl-
L
oxidoreductase 1; Nrf2, NF-E2 p45-related factor 2; p38 MAPK, p38 mitogen-
activated protein kinase; ROS, reactive oxygen species.
*
Corresponding author. Department of Medical Chemistry and Biochemistry,
methyl ethers, such as rutin (quercetin-3-O-rutinoside), iso-
Faculty of Medicine and Dentistry, Hn eꢁ votínsk aꢀ 3, Olomouc 77515, Czech Republic.
0
E-mail address: vrbambv@seznam.cz (J. Vrba).
quercitrin (quercetin-3-O-
b
-D-glucoside) and isorhamnetin (3 -O-
http://dx.doi.org/10.1016/j.cbi.2016.10.015
0
009-2797/© 2016 Elsevier Ireland Ltd. All rights reserved.

L. Roubalov aꢀ et al. / Chemico-Biological Interactions 260 (2016) 58e66
59
Fig. 1. Chemical structures of tested compounds.
1
13
methylquercetin) [2]. Naturally occurring conjugated forms of
quercetin also include those produced by the biotransformation
processes in the intestine and liver of animals and humans. The
main quercetin conjugates found in human plasma are quercetin-3-
spectra, i.e. H NMR and C NMR spectra, were zero-filled to four-
1
fold data points prior to the Fourier transformation. Moreover, H
NMR data were multiplied by the two-parameter double-expo-
nential Lorentz-Gauss function to improve resolution, and line
0
13
O-glucuronide, 3 -O-methylquercetin-3-O-glucuronide and quer-
broadening (1 Hz) was applied to the C NMR data to improve the
0
cetin-3 -O-sulfate [3]. In addition, a number of quercetin de-
signal-to-noise ratio. Chemical shifts are listed in
coupling constants in Hz. The digital resolution enabled us to
declare values to three ( ) or two ( ) decimal places. ESI-MS
spectra were measured with a Micromass LC-MS Platform in
MeOH with the addition of HCO H; HRMS spectra (ESI, APCI, FAB)
were measured with an LTQ Orbitrap XL instrument (Thermo Fisher
Scientific, Waltham, MA, USA) or with a ZAB-EQ instrument (VG
Analytical, Manchester, UK).
d-scale and
rivatives have been prepared in vitro using chemical or enzymatic
procedures. Some derivatives have been designed mainly with the
aim of improving the physico-chemical and biological properties of
quercetin, which is, despite its beneficial (e.g. anti-inflammatory)
effects, unsuitable for medical applications due to low solubility
and low bioavailability [4]. Moreover, methods for the preparation
of quercetin metabolites have also been developed [5,6]. The
quercetin derivatives synthesized to date include, for instance, es-
ters of quercetin and of various aliphatic [7,8] and aromatic car-
boxylic acids [9e11], quercetin-amino acid conjugates with amino
acids attached to quercetin via a carbamate linkage [12], esters of
sulfuric acid (i.e. quercetin sulfates) [13,14], glycosides [15], glucu-
ronides [5], methyl ethers [16], etc.
One of the mechanisms mediating the beneficial action of
quercetin is the activation of the transcription factor NF-E2 p45-
related factor 2 (Nrf2; also called NFE2L2) [17,18]. Nrf2 regulates
the expression of genes encoding various cytoprotective enzymes
such as heme oxygenase-1 (HO-1), NAD(P)H:quinone oxidoreduc-
tase 1 (NQO1), and glutamate-cysteine ligase catalytic (GCLC) and
modifier subunits (GCLM) [19]. Being inspired by the fact that the
Nrf2 activation by catechins (flavanols) positively depends on the
presence of a galloyl moiety in the molecule [20], we have previ-
ously prepared galloyl esters of quercetin and of its natural deriv-
ative, the flavanonol taxifolin (2,3-dihydroquercetin), and
examined their effect on the Nrf2 pathway in murine macrophage
RAW264.7 cells. We have found that 3-O-galloylquercetin, in
contrast to quercetin, does not activate the Nrf2 pathway. On the
other hand, we have demonstrated the activation of Nrf2 in cells
treated with 7-O-galloyltaxifolin, while mere taxifolin was inactive.
The same study also found that 7-O-galloyltaxifolin was readily
oxidized to 7-O-galloylquercetin (Fig. 1) in RAW264.7 cells [11].
Since it was unclear which of these two molecules was responsible
for the biological response, we decided to prepare 7-O-galloyl-
quercetin and to investigate its effect on the Nrf2 pathway in vitro.
d
H
d
C
2
2.2. Synthesis of 7-O-galloylquercetin (3)
3,4,5-Tri-O-benzylgallic acid (1 g, 2.5 mmol) was suspended in
dry dichloromethane (15 mL) under an argon atmosphere. Oxalyl
chloride (4 mL, 46.6 mmol) was added to the solution, and dry
dimethylformamide (2 mL, 26.0 mmol) was added to the resulting
mixture dropwise under stirring. After another 4 h of stirring, the
reaction mixture was diluted with dry toluene (5 mL) and evapo-
rated to dryness in vacuo. The resulting white crude 3,4,5-tri-O-
benzylgalloyl chloride was dissolved in dry pyridine (15 mL).
Quercetin (0.5 g, 1.65 mmol), previously dried by coevaporation
ꢀ
with dry toluene (90 C, 3 ꢁ 10 mL) in vacuo, was dissolved in dry
ꢀ
pyridine (5 mL). This solution was added to the cooled (ꢂ60 C)
solution of 3,4,5-tri-O-benzylgalloyl chloride under an argon at-
mosphere and the reaction mixture was stirred overnight under
argon to slowly reach room temperature. Solvents were evaporated
and then coevaporated with toluene in vacuo to remove traces of
pyridine. The resulting solid was mixed with chloroform and water;
the organic phase was separated, washed with water, dried with
Na SO and evaporated to dryness. Flash chromatography on Sili-
2
4
cagel 60 (CHCl /toluene/acetone/HCO H, 85:5:5:1) yielded 7-O-
(3 ,4 ,5 -tri-O-benzylgalloyl)quercetin (1; 260 mg, 27%) and 3-O-
(3 ,4 ,5 -tri-O-benzylgalloyl)quercetin (2; 180 mg, 19%), which was
not used in further experiments.
1 (260 mg) was dissolved in EtOAc (20 mL), and then Pd/C
(150 mg, 10% Pd) was added and stirred under H for 3 h (20 C).
3
2
0
0
0
0
0
0
ꢀ
2
Palladium catalyst was then removed by filtration through celite,
which was washed with acetone, and the solvent was evaporated.
The product was purified by gel filtration (Sephadex LH-20, MeOH)
2
. Experimental
2
.1. General
yielding 7-O-galloylquercetin (3; 180 mg, 89%). The purity (HPLC-
-
PDA) was 99.6%, HRMS (ESI) m/z [M ꢂ H] calcd for C22
13 11
H O
NMR analyses were performed using a Bruker Avance III
00 MHz spectrometer (Bruker Biospin, Rheinstetten, Germany)
453.04524, found 453.04553 (for NMR data see Table S1 and
Figs. S2eS6, for HPLC see Fig. S7, for MS and HRMS see Figs. S8 and
S9 in Supplementary Information).
6
1
13
(
600.23 MHz for H, 150.93 MHz for C) with samples dissolved in
DMSO-d (99.8% atom D; VWR International, Leuven, Belgium).
NMR spectra were referenced by the residual signal of the solvent
6
2.3. Reagents for biological testing
1
H C
(d 2.500 ppm, d 39.60 ppm). NMR experiments, including H
NMR, C NMR, COSY, H- C HSQC and H- C HMBC, were per-
formed using the software Topspin3 (Bruker Biospin). The 1D
13
1
13
1
13
Quercetin, gallic acid methyl ester, hemin, sulforaphane, N-
acetyl-L-cysteine, PD98059 (2-(2-amino-3-methoxyphenyl)-4H-1-

60
L. Roubalov aꢀ et al. / Chemico-Biological Interactions 260 (2016) 58e66
benzopyran-4-one),
methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole),
1,9-pyrazoloanthrone), dimethyl sulfoxide (DMSO) and solvents
for HPLC were purchased from Sigma-Aldrich.
SB203580
(4-(4-fluorophenyl)-2-(4-
TaqMan Gene Expression Assays, consisting of specific primers and
FAM dye-labeled TaqMan minor groove binder probes (Applied
SP600125
(
Biosystems). The assay ID was Mm00516005_m1 for Hmox1 and
ꢀ
Mm99999915_g1 for Gapdh. Amplification conditions were 50
C
ꢀ
ꢀ
for 2 min, 95 C for 10 min, followed by 40 cycles at 95 C for 15 s
ꢀ
2.4. Cell cultures and treatments
and 60 C for 1 min. Crossing point values, equivalent to C
T
, were
determined using second derivative maximum analysis. Relative
The stable human mammary AREc32 reporter cell line [21] was
cultured in Dulbecco's modified Eagle's medium (DMEM; No.
1966, Gibco, Grand Island, NY, USA) supplemented with 2 mM
changes in gene expression were calculated by the comparative C
T
ꢂ
DDC
T
method using the 2
Gapdh mRNA levels.
equation with results normalized to
4
glutamine and 10% fetal bovine serum (FBS). The murine macro-
phage RAW264.7 cell line (No. 91062702, ECACC, Salisbury, UK) was
cultured in DMEM (D5796, Sigma) supplemented with 100 U/mL
2.8. Western blot analysis
5
penicillin, 100
atoma Hepa1c1c7 cell line (No. 95090613, ECACC) was cultured in
Minimum essential medium (M0894, Sigma) supplemented with
mg/mL streptomycin and 10% FBS. The murine hep-
After the treatment of RAW264.7 cells (8 ꢁ 10 cells/well in a 6-
5
well plate) or Hepa1c1c7 cells (4 ꢁ 10 cells/well in a 6-well plate),
a
total cellular extracts were prepared as described previously [11].
Aliquots containing an equal amount of protein were subjected to
electrophoresis through 4e12% sodium dodecyl sulfate-
polyacrylamide gel, proteins were transferred to polyvinylidene
difluoride membrane by electroblotting, and the membranes were
probed with appropriate primary antibodies. Rabbit polyclonal
heme oxygenase-1 (sc-10789), rabbit polyclonal Nrf2 (sc-722), goat
polyclonal Keap1 (sc-15246) and goat polyclonal actin (sc-1616)
antibodies were obtained from Santa Cruz Biotechnology (Santa
Cruz, CA, USA). Antibodies against NQO1, GCLC and GCLM were
kindly provided by Dr. John D. Hayes (University of Dundee, Dun-
dee, UK). Primary antibodies were visualized with rabbit anti-goat
or goat anti-rabbit horseradish peroxidase-conjugated secondary
antibodies using a chemiluminescent reaction.
2
.2 g/L NaHCO
3
and 10% heat- and charcoal-treated FBS. Cells were
ꢀ
maintained at 37 C in a humidified atmosphere containing 5% CO
2
.
For experiments, cells were seeded into multiwell plates in serum-
containing medium. Experiments on AREc32 and Hepa1c1c7 cells
were performed after 24 h of stabilization in fresh serum-
containing medium. For RAW264.7 cells, the culture medium was
replaced with serum-free medium after 8 h of stabilization, and
following overnight incubation the experiments were performed in
fresh serum-free conditions. Cells were treated with the tested
compounds (in 0.1% (v/v) DMSO) and negative controls were
treated with 0.1% DMSO alone.
2.5. Cell viability assay
5
RAW264.7 cells (1.7 ꢁ 10 cells/well in a 24-well plate) and
2.9. HPLC/MS analysis of biotransformation products
4
Hepa1c1c7 cells (1 ꢁ 10 cells/well in a 96-well plate) were treated
for 6 or 48 h, respectively, with 0.1% DMSO (control), 1.5% (v/v)
Triton X-100 (positive control) or with the tested compounds. After
treatment, the cell viability was determined using the MTT reduc-
tion assay. Cells were washed with phosphate-buffered saline (PBS)
5
RAW264.7 cells (8 ꢁ 10 cells/well in a 6-well plate) were
incubated for 0e6 h with 15 mM 7-O-galloylquercetin. After incu-
bation, cells were scraped from the plates, collected by gentle
centrifugation, washed twice with PBS, resuspended in 0.4 mL of
methanol containing 5% (v/v) acetic acid, and sonicated 10 times at
50% amplitude with a cycle set at 0.5 s using an Ultrasonic Processor
UP200s equipped with a Sonotrode Microtip S2 sonicator probe
(Hielscher, Teltow, Germany). Afterwards, the cell lysates were
centrifuged for 2 min at 14 000ꢁ g at room temperature and the
supernatants were analyzed by HPLC/MS. Aliquots of culture me-
dium were diluted (1:1, v/v) in methanol containing 5% (v/v) acetic
acid, centrifuged for 2 min at 14 000ꢁ g and the supernatants were
analyzed by HPLC/MS. The chromatographic separation was per-
formed in an Agilent Zorbax Eclipse XDB-phenyl column
ꢀ
and incubated for 2 h at 37 C in fresh serum-free medium con-
taining
0.5
mg/mL
3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT). After this, the medium was
removed and formazan produced by active mitochondria was dis-
solved in DMSO. The absorbance at 540 nm was measured on a
spectrophotometric plate reader and used to calculate relative cell
viability, where cells treated with DMSO alone represented 100%
viability.
2
.6. Gene reporter assay
(150 mm ꢁ 2.1 mm i.d., 5
mm; Agilent Technologies, CA, USA) using
4
AREc32 cells were seeded into a 96-well plate at 1 ꢁ 10 cells/
an Acquity UPLC system (Waters, Milford, MA, USA) equipped with
a binary solvent manager, sample manager, column manager and
photodiode array detector. A Waters QqTof Premier Mass Spec-
trometer (Waters, Manchester, UK) was connected to the UPLC
system via an electrospray ionization (ESI) interface. Acquiring data
enabled the collection of intact precursor ions as well as fragment
ion information in an unbiased manner. Post-acquisition processing
of the data was performed using Metabolynx V4.1 software (Wa-
ters, Milford, MA, USA). For more details, see Ref. [22].
well. On the next day, cells were exposed for 24 h to the tested
ꢀ
compounds. Afterwards, the plate was frozen at ꢂ20 C for 24 h and
then the luciferase activity was measured on
a GloMax-
Multiþ microplate luminometer (Promega, Madison, WI, USA) us-
ing the Bright-Glo Luciferase Assay System (Promega). The lumi-
nescence values were normalized to the protein content of the cells
and used for calculation of fold changes versus the control.
2.7. Reverse transcription and quantitative real-time PCR
2
.10. NQO1 activity assay
5
After the treatment of RAW264.7 cells (8 ꢁ 10 cells/well in a 6-
4
well plate), total RNA was extracted using TRI Reagent Solution
Applied Biosystems, Foster City, CA, USA). RNA samples (2
reverse transcribed using a High-Capacity cDNA Reverse Tran-
scription Kit (Applied Biosystems) and real-time PCR was per-
a LightCycler 480 II system (Roche Diagnostics,
Mannheim, Germany) using TaqMan Universal PCR Master Mix and
After the treatment of Hepa1c1c7 cells (1 ꢁ 10 cells/well in a
(
mg) were
96-well plate), the activity of NQO1 was determined spectropho-
tometrically as described previously [23]. Cells were washed four
times with PBS and lysed with 75
digitonin, 2 mM EDTA, pH 7.8) by shaking on an orbital shaker for
20 min at room temperature. One part of the cell lysate (20 L) was
mL of digitonin solution (0.8 g/L
formed in
m

L. Roubalov aꢀ et al. / Chemico-Biological Interactions 260 (2016) 58e66
61
Scheme 1. Synthesis of 7-O-galloylquercetin (3).
Reagents and conditions: (a) 3,4,5-tri-O-benzylgalloyl chloride (1.5 equiv), pyridine, e60 C, 1 h, 27%; (b) H
ꢀ
2
ePd/C, EtOAc, room temp, 3 h, 89%.
used to determine the protein content. The remaining lysate (55
m
L)
reduction assay, the viability of RAW264.7 cells treated for 6 h with
15 M 3 or quercetin was 80% and 86%, respectively. The effect of the
was mixed with 200 L of 0.5 M Tris-Cl buffer containing 10%
m
m
bovine serum albumin, 1.5% Tween-20, 7.5 mM FAD, 150 mM
tested compounds on the viability of RAW264.7 and Hepa1c1c7
cells after 6 and 48 h of incubation, respectively, is shown in Fig. 2.
glucose-6-phosphate, 2 U/mL glucose-6-phosphate dehydrogenase
þ
(
Roche), 50 mM NADP , 25 mM menadione and 0.7 mM MTT. The
mixture was incubated for 5 min at room temperature and the
reaction was stopped with 50 L of dicumarol solution (0.3 mM
3.3. Activation of Nrf2 by 7-O-galloylquercetin (3) in AREc32
reporter cells
m
dicumarol, 5 mM potassium phosphate, 0.5% DMSO). The absor-
bance of the reduced MTT corresponding to the activity of NQO1
was measured at 610 nm on a spectrophotometric plate reader. The
absorbance values were normalized to the protein content of the
cells and used for the calculation of fold changes versus the control.
To evaluate the effect on the Nrf2 pathway, we first examined
whether compound 3 and its structural components could induce
the transcriptional activity of Nrf2 in the stably transfected human
AREc32 reporter cell line. AREc32 cells contain a luciferase reporter
gene controlled by eight copies of the antioxidant response element
(ARE), through which Nrf2 activates the expression of its target
2.11. Statistical analysis
genes [21]. The treatment of AREc32 cells for 24 h with 5 mM sulfo-
Results were expressed as means ± standard deviation (SD). The
raphane, used as a positive control [21], resulted in an 8.2-fold in-
crease in luciferase activity compared to the control (Fig. 3). A
significant increase in luciferase activity was also induced by 3 and
quercetin, with the latter compound being more effective. In
contrast, a mild or negligible elevation in luciferase activity was
detected after cell exposure to methyl gallate. After 24 h of incuba-
tion, the activity of luciferase reached 4.4-fold, 1.8-fold and 1.3-fold
differences in mean values were analyzed by one-way ANOVA with
Tukey's post hoc test. A p value of less than 0.05 was considered to
be statistically significant.
3
. Results and discussion
3.1. Synthesis of 7-O-galloylquercetin (3)
in cells treated with 15 mM quercetin, 3 and methyl gallate, respec-
tively (Fig. 3). These results demonstrated that 3 activates the Nrf2-
The synthesis of 3 was performed by direct galloylation of
dependenttranscription, albeit with a lower potency thanquercetin.
quercetin with 3,4,5-tri-O-benzylgalloyl chloride in pyridine
Scheme 1). 3,4,5-Tri-O-benzylgalloyl chloride in pyridine was
prepared by treating tribenzylgallic acid with oxalyl chloride so-
lution in CH Cl and a catalytic amount of dimethylformamide
(
3.4. Induction of HO-1 by 7-O-galloylquercetin (3) in
RAW264.7 cells
2
2
immediately before use. The reaction yielded two regioisomers 1
and 2 that were separated by flash chromatography. Benzyl-
protected intermediate 1 was then deprotected by catalytic
hydrogenolysis catalyzed with Pd/C, giving the final galloyl ester 3.
The study further compared the effect of 3 and its structural
components on the expression of HO-1 in RAW264.7 cells. HO-1,
3.2. Effect of 7-O-galloylquercetin (3) on cell viability
The aim of the biological part of the study was to investigate
whether 3 activates the Nrf2 pathway. For that purpose, we used
three cell models including murine macrophage RAW264.7 cells,
murine hepatoma Hepa1c1c7 cells, and stably transfected AREc32
reporter cells derived from human breast cancer MCF7 cells [21]. To
compare the effect of 3 with those of its structural components, we
also included quercetin and gallic acid methyl ester (methyl gallate)
in our experiments (Fig.1). Methyl gallate was chosen instead of free
gallic acid, since the acid was previously shown to have only a weak
stimulating effect on the Nrf2 pathway [11]. In this study, we tested
both 3 and its structural components at concentrations of up to
Fig. 2. Effect of tested compounds on viability of RAW264.7 and Hepa1c1c7 cells.
RAW264.7 and Hepa1c1c7 cells were treated for 6 or 48 h (as indicated) with 0.1%
DMSO (control), 15 mM quercetin (QUE) or with 3.75e15 mM 7-O-galloylquercetin (3) or
methyl gallate (MG). The cell viability was determined by the MTT reduction assay. Data
15 mM. At these concentrations, the tested compounds had a weak
or negligible effect on the viability of cells used. We found that 3
only decreased cell viability below 90% in RAW264.7 cells, and this
effect was comparable to that of quercetin. As shown by the MTT
are means ± SD of three experiments. *p < 0.05, significantly decreased versus control.

62
L. Roubalov aꢀ et al. / Chemico-Biological Interactions 260 (2016) 58e66
mRNA detected at concentrations from 7.5 mM. The levels of Hmox1
mRNA were also elevated in cells treated with quercetin and methyl
gallate, but their effect was weaker than that of 3. At the concen-
tration of 15 mM, Hmox1 mRNA levels induced by 3, quercetin and
methyl gallate reached 7.9-fold, 3.0-fold and 2.0-fold, respectively,
compared to the control (Fig. 4A). Western blot analysis showed that
the induction of Hmox1 gene expression by 3 was also associated
with the upregulation of the HO-1 protein. After 6 h of cell exposure
to 3, the highest level of HO-1 was found at the concentration of
7.5 mM. We also found that quercetin (15 mM) upregulated HO-1 to a
level comparable to that induced by 3, whereas methyl gallate
produced lower levels of HO-1 (Fig. 4B). It should be mentioned that
the induction of HO-1 in RAW264.7 cells by quercetin was reported
previously [11,25] and the ability of methyl gallate to upregulate HO-
1
was demonstrated in mouse mesangial cells [26].
The expression of the Hmox1 gene is regulated by Nrf2. Under
Fig. 3. 7-O-Galloylquercetin (3) induces ARE-driven gene expression. AREc32 reporter
cells were treated for 24 h with 0.1% DMSO (control), 5 M sulforaphane (SFN; positive
control) or with 3.75e15 M 3, quercetin (QUE) or methyl gallate (MG). After treat-
ment, luciferase reporter activity was determined luminometrically and normalized to
protein content. Data are means ± SD of three experiments. *p < 0.05; **p < 0.01,
significantly increased versus control.
m
m
unstressed conditions, Nrf2 is targeted for proteasomal degradation
through its interaction with the repressor protein Keap1 (Kelch-like
ECH-associated protein 1). Conversely, stress conditions may impair
the interaction between Nrf2 and Keap1 via the phosphorylation of
encoded by the Hmox1 gene, is an inducible form of heme oxygen-
2
þ
ase, an enzyme that degrades heme to Fe , the antioxidant bili-
verdin and anti-inflammatory agent carbon monoxide [24]. As
shown by quantitative real-time PCR, the treatment of
RAW264.7 cells for 6 h with 5 mM hemin, a positive control, signif-
icantly increased the level of Hmox1 mRNA to 53-fold when
normalized to Gapdh mRNA (Fig. 4A). After 6 h of incubation, 3 was
found to induce the expression of the Hmox1 gene in RAW264.7 cells
in a dose-dependent manner, with a significant increase in Hmox1
Fig. 5. Effect of MAPK inhibitors and N-acetyl-L-cysteine (NAC) on 7-O-galloylquercetin
(compound 3)-induced Hmox1 gene expression in RAW264.7 cells. (A) Cells were
Fig. 4. 7-O-Galloylquercetin (3) induces HO-1 expression and Nrf2 accumulation in
RAW264.7 cells. Cells were treated for 6 h with 0.1% DMSO (control), 5
positive control), 15 M quercetin (QUE) or with 3.75e15 M 3 or methyl gallate (MG).
A) After treatment, relative changes in Hmox1 mRNA levels were determined by
quantitative real-time PCR with results normalized to Gapdh mRNA. Data are
means ± SD of five experiments. *p < 0.05; **p < 0.01, significantly increased versus
control. (B) After treatment, protein levels of HO-1, Nrf2, Keap1 and actin in the whole
m
M hemin (HM;
pretreated for 30 min with 0.1% DMSO (control), 15
30 M SP600125 and then incubated in the absence or presence of 15
additional 6 h. (B) Cells were pretreated for 30 min with 2.5 or 5 mM NAC and then
incubated in the absence or presence of 15 M 3 for an additional 6 h. The levels of
Hmox1 mRNA were determined by quantitative real-time PCR and normalized to
Gapdh mRNA. Results are expressed as the percentage of compound 3-induced Hmox1
mRNA expression. Data are means ± SD of three experiments. **p < 0.01, significantly
decreased versus 3 in the absence of MAPK inhibitors or NAC.
m
M PD98059, 15
m
M SB203580 or
m
m
m
m
M 3 for an
(
m
cell lysates (20
mg/lane) were analyzed by Western blotting. Representative Western
blots are shown.

L. Roubalov aꢀ et al. / Chemico-Biological Interactions 260 (2016) 58e66
63
Nrf2 or through the oxidation or covalent modification of Keap1,
which in turn undergoes proteasomal degradation. The disruption
of the Keap1eNrf2 interaction results in the accumulation of Nrf2
and its translocation to the nucleus, where it binds to the DNA at the
ARE and triggers the expression of Nrf2 target genes [27,28].
Accordingly, the Western blot analysis showed that 5 mM hemin
increased the level of Nrf2 and concurrently decreased the level of
Keap1 in RAW264.7 cells after 6 h of incubation (Fig. 4B). We found
significantly suppressed by N-acetyl-
ROS scavenger. For instance, 30 min of the cell pretreatment with
2.5 mM NAC decreased the induction of Hmox1 mRNA by 6 h of
L
-cysteine (NAC), a nonspecific
exposure to 15 mM 3 by 69% (Fig. 5B). These results suggest that the
activation of Nrf2 by 3 is mediated through the enhanced produc-
tion of ROS and activation of p38 MAPKs. As shown in our previous
study [11], both of these events were also involved in HO-1 induc-
tion in RAW264.7 cells exposed to 7-O-galloyltaxifolin, which only
differs from 3 in the absence of a 2,3-double bond. Taking into ac-
count the oxidation of 7-O-galloyltaxifolin to 3 [11] and essentially
the same mechanism of HO-1 induction found for both compounds,
it may be supposed that compound 3 was responsible for the bio-
logical effect of 7-O-galloyltaxifolin in RAW264.7 cells [11].
that 3 at a concentration of 15 mM also upregulated Nrf2, but the
level of Keap1 remained unchanged after 6 h of exposure. Similarly,
quercetin and methyl gallate also increased Nrf2 without affecting
Keap1 levels (Fig. 4B). The accumulation of Nrf2 together with the
induction of HO-1 indicates that 3, like quercetin, may activate the
Nrf2 pathway in RAW264.7 cells.
3.6. Uptake and biotransformation of 7-O-galloylquercetin (3) by
3.5. Induction of HO-1 by 7-O-galloylquercetin (3) involves ROS
RAW264.7 cells
and p38 MAPK activity
The study also investigated the metabolic fate of
RAW264.7 cells. The cells were incubated for up to 6 h with 15
and the cells and culture medium were separately analyzed using the
previously developed high-performance liquid chromatography-
mass spectrometry (HPLC/MS) method in Ref. [22]. As recorded with
an ESI-MS detector operating in negative mode, compound 3 gave a
3
in
As described above, the accumulation of Nrf2 in RAW264.7 cells
by compound 3 was not associated with a downregulation of Keap1.
Since the activation of Nrf2 may also be mediated through its
phosphorylation [28], we investigated, using pharmacological in-
hibitors, whether mitogen-activated protein kinases (MAPKs)
including p38 MAPKs, extracellular signal-regulated kinases
mM 3,
R
well-resolved chromatographic peak at a t of 14.6 min (Fig. 6A). We
(
ERKs), and c-Jun N-terminal kinases (JNKs) could play a role in the
found that during the incubation period, the concentration of 3 in the
culture medium evidently decreased. In contrast, the concentration of
3 in cell extracts increased at the beginning of the incubation, reached
a maximum at 2 h and then a decline in the concentration occurred
cell response to 3. We found that the increase in Hmox1 mRNA
induced by 3 was reduced significantly by the p38 MAPK inhibitor
SB203580, and nonsignificantly by the ERK inhibitor PD98059
(
Fig. 5A). Pretreatment of RAW264.7 cells for 30 min with 15
SB203580 or 15 M PD98059 decreased the induction of Hmox1
mRNA by 6 h of exposure to 15 M 3 by 71% and 15%, respectively.
On the other hand, cell pretreatment with 30 M SP600125, a JNK
inhibitor, potentiated the effect of 3 on Hmox1 mRNA expression by
81% under the same experimental conditions (Fig. 5A).
m
M
(Fig. 6B). These findings indicated that 3 was absorbed by
m
RAW264.7 cells and that the galloyl ester underwent some kind of
metabolic transformation and/or degradation. Analyses showed that
compound 3 was stable in terms of possible hydrolytic cleavage, since
no significant amounts of quercetin and gallic acid were detected in
either the cells or medium (Fig. 6B). Thus, we could rule out the
possibility that the biological effects of 3 were mediated by its struc-
tural components. Furthermore, we found that 3 was not converted to
7-O-galloyltaxifolin. On the other hand, two types of phase II
m
m
1
The MAPK pathways may be activated by various stimuli
including reactive oxygen species (ROS) [29]. As shown in Fig. 5B,
the elevation in Hmox1 mRNA induced in RAW264.7 cells by 3 was
Fig. 6. Biotransformation and uptake of 7-O-galloylquercetin (3) by RAW264.7 cells. Cells were treated for 0e6 h with 15 mM 3 or 0.1% DMSO (control), and cell extracts and culture
medium were analyzed by HPLC with negative ESI-MS detection. (A) HPLC/MS chromatograms of 3 (inset) and of its metabolites found in cells after 2 h of treatment. (B) Time course
of distribution of 3, quercetin (QUE) and gallic acid (GA) in cells and medium. Data are means ± SD of three experiments.

64
L. Roubalov aꢀ et al. / Chemico-Biological Interactions 260 (2016) 58e66
Fig. 7. Full and fragmentation (insets) MS spectra of 7-O-galloylquercetin (3) and of its metabolites. The MS spectra were obtained from the chromatographic peaks shown in Fig. 6.
A) Parent compound 3, m/z 453, (B) glucuronide of 3, t 13.1 min, m/z 629 (C) methylated 3, m/z 467, (D) methylated and glucuronidated 3, m/z 643.
(
R
conjugation reactions, i.e. glucuronidation and methylation, appeared
to be involved in the transformation of 3 in RAW264.7 cells. Analyses
revealed three different glucuronides of the parent compound eluted
this cell line [31]. Moreover, the expression of UDP-
glucuronosyltransferases was reported in macrophages [32]. It
might be mentioned that we previously showed the formation of
glucuronides and methyl derivatives of 3-O-galloylquercetin in hu-
man hepatocytes [22]. Hence, it may be supposed that the biotrans-
formationpatternof3 foundinRAW264.7cells canalsobe expectedin
other cell types expressing the phase II conjugating enzymes.
at t
5.8 min, and also a methylated glucuronide that appeared as a minor
metabolite at a t of 13.6 min (Fig. 6A). The full and fragmentation MS
R R
of 12.7, 13.1 and 14.4 min, a methyl derivative eluted at a t of
1
R
spectra of 3 and of its metabolites are shown inFig. 7. Wewere not able
to identify which hydroxyl groups in the molecule of 3 were conju-
gated. Nonetheless, some of the conjugations could occur at the same
positions which are conjugated in quercetin and gallic acid metabo-
3.7. Induction of NQO1 activity by 7-O-galloylquercetin (3) in
Hepa1c1c7 cells
0
lites such as 3 -O-methylquercetin-3-O-glucuronide [3] and 4-O-
methylgallic acid [30]. The metabolic activityof RAW264.7 cellswas in
accordance with the study showing the methylation of quercetin in
To evaluate whether 3 could stimulate the Nrf2 pathway in cells
other than macrophages, we tested the effect of the compound on
Fig. 8. 7-O-Galloylquercetin (3) induces NQO1 activity and Nrf2 accumulation in Hepa1c1c7 cells. (A) Cells were treated for 48 h with 0.1% DMSO (control), 5
positive control) or with 3.75e15
three experiments. *p < 0.05; **p < 0.01, significantly increased versus control. (B) Cells were treated for 3 or 24 h (as indicated) with 0.1% DMSO (control), 5
positive control) or with 15
30 g/lane) were analyzed in duplicate by Western blotting. Representative Western blots are shown.
m
M sulforaphane (SFN;
m
M 3, quercetin (QUE) or methyl gallate (MG). After treatment, the activity of NQO1 was determined using the NQO1 assay. Data are means ± SD of
mM sulforaphane (SFN;
m
M 3, quercetin (QUE) or methyl gallate (MG). After treatment, the protein levels of Nrf2, NQO1, HO-1, GCLC, GCLM, and actin in the whole cell lysates
(
m

L. Roubalov aꢀ et al. / Chemico-Biological Interactions 260 (2016) 58e66
65
murine hepatoma Hepa1c1c7 cells, a well-established model for
the identification of possible NQO1 inducers [23]. The NQO1 assay
showed that the treatment of Hepa1c1c7 cells for 48 h with 5
sulforaphane, a positive control [23], elevated the activity of NQO1
.7-fold compared to the control (Fig. 8A). The induction of NQO1
activity by sulforaphane was accompanied by an obvious increase
in the levels of Nrf2 and Nrf2-regulated proteins, namely, NQO1,
HO-1, GCLC and GCLM, as demonstrated by Western blot analysis
after 3 and/or 24 h of exposure (Fig. 8B). We found that the activity
of NQO1 in Hepa1c1c7 cells was also elevated by 3 and quercetin,
but not by methyl gallate. However, 3 significantly increased NQO1
activity only at the highest concentration tested, thus it was a less
effective NQO1 inducer than quercetin. In cells treated for 48 h with
Research UK (C20953/A18644).
mM
Appendix A. Supplementary data
3
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.cbi.2016.10.015.
Supplementary data include H and 13C NMR data in extenso, the
COSY spectrum, HSQC spectrum, HMBC spectrum, HPLC chro-
matogram and MS data of compound 3.
1
Transparency document
Transparency document related to this article can be found
online at http://dx.doi.org/10.1016/j.cbi.2016.10.015.
15
m
M quercetin, 3 and methyl gallate, the activity of NQO1 reached
.1-fold, 1.8-fold and 0.9-fold, respectively, compared to the control
Fig. 8A). In Hepa1c1c7 cells, 3 (15 M) was also found to induce a
mild increase in the protein levels of Nrf2, HO-1 and GCLM after
4 h of incubation, while the levels of NQO1 and GCLC were un-
affected. Quercetin (15 M) clearly elevated Nrf2 as early as after 3 h
2
(
m
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