J Biol Inorg Chem (2009) 14:727–739
DOI 10.1007/s00775-009-0486-8
ORIGINAL PAPER
DNA binding and oxidative DNA damage induced
by a quercetin copper(II) complex: potential mechanism
of its antitumor properties
Jun Tan Æ Bochu Wang Æ Liancai Zhu
Received: 1 October 2008 / Accepted: 30 January 2009 / Published online: 4 March 2009
Ó SBIC 2009
Abstract The interaction of a quercetin copper(II) com-
plex with DNA was investigated using UV–vis spectra,
fluorescence measurement, viscosity measurement, agarose
gel electrophoresis, and thiobarbituric acid reactive sub-
stances assay. The results indicate that the quercetin
copper(II) complex can promote the cleavage of plasmid
DNA, producing single and double DNA strand breaks, and
intercalate into the stacked base pairs of DNA. Moreover, the
complex can induce oxidative DNA damage involving
generation of reactive oxygen species such as H2O2 and
Cu(I)OOH. In addition, the cytotoxicity experiments carried
out with A549 cells confirmed its apoptosis-inducing activ-
ity. And we also demonstrate that the levels of survivin
protein expression in A549 cells decreased, and that relative
Electronic supplementary material The online version of this
article (doi:10.1007/s00775-009-0486-8) contains supplementary
material, which is available to authorized users.
J. Tan (&)
B. Wang (&)
L. Zhu
Bioengineering College,
Chongqing University,
No. 174 Shapingba Main Street,
400030 Chongqing, China
e-mail: tanjunmail@126.com
B. Wang
e-mail: wangbc2000@126.com
J. Tan
Department of Life Science and Chemistry,
Chongqing Education College,
400067 Chongqing, China
J. Tan
Mechanical Resource and Environment College,
Post-doctoral Research Center of Mechanics,
Chongqing University,
400030 Chongqing, China
activity of caspase-3 increased significantly after treatment
with the complex. So our results suggest that the antitumor
mechanism of the quercetin copper(II) complex involves not
only its oxidative DNA damage with generation of reactive
oxygen species but also its specific interaction with DNA.
Keywords Quercetin copper(II) complex

Oxidative DNA damage
DNA binding
Apoptosis

Antitumor activity
Introduction
A large part of weakly mutagenic and nonmutagenic car-
cinogens appear to induce carcinogenesis through metal-
mediated oxidative DNA damage. Particularly, endogenous
metal ions, such as copper and iron, react with superoxide
anion (O2
-) and hydrogen peroxide (H2O2) to produce
highly reactive species such as hydroxyl free radical (
OH) and
metal–oxygen complexes in Fenton-type reactions in bio-
logical systems, resulting in oxidative DNA damage [1–3].
Polyphenolic compounds, particularly bioflavonoids such
as quercetin (3,5,7,30 ,40 -pentahydroxyflavone), protect DNA
from damage induced by reactive oxygen species (ROS)
including
OH, H2O2, and O2
- [4]. The protective effect of
quercetin on DNA is attributed to its scavenging ROS either
as a free molecular species or at a site where quercetin binds
to DNA. On the other hand, quercetin, as a polyphenolic
compound, can prevent the production of ROS by com-
plexing cations such as copper and iron that participate in
hydroxyl radical formation [5, 6]. So polyphenolic com-
pounds are considered to possess anticancer and apoptosis-
inducing properties in cancer cells. Although generally
recognized as nature antioxidants, such compounds also
exhibit prooxidant properties under appropriate conditions
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[7, 8]. It is noted that the polyphenolic compounds cause both
single-strand and double-strand cleavage of DNA, and fur-
ther completely degrade DNA into smaller fragments in the
presence of copper(II). DNA single-strand or double-strand
breaks could contribute to the formation of chromosome
aberrations, which may lead to mutation or cancer [9]. Thus,
oxidative DNA damage seems to be relevant to the carcin-
ogenic process.
However, the prooxidant action might not always be
adverse for humans. It has been proposed that the prooxidant
action of plant-derived polyphenols may play a more crucial
role in their anticancer and apoptosis-inducing properties
than their antioxidant activity does, because ROS resulting
from the prooxidant action can mediate apoptotic DNA
fragmentation [10–12]. Evidence has demonstrated that
polyphenols such as the flavonoid quercetin and the stilbene
resveratrol can not only bind copper ions but also catalyze
their redox cycling. Quercetin could chelate copper(II) to
form a charge transfer complex which is also capable of
binding to DNA. It is proposed that the cleavage of DNA may
be related to the complexes involving in quercetin, DNA, and
copper(II) [13]. However, in these studies, the antioxidation
and prooxidation of only cooperative polyphenolic com-
pounds and copper cation were studied, and those of
synthetic polyphenol copper(II) complexes were considered.
Actually, quercetin can chelate metal ions to form metal
complexes that have better antioxidation and antitumor
activity than quercetin alone [14–16]. In a previous study, we
demonstrated that quercetin zinc(II) and manganese(II)
complexes could cleave DNA via a hydrolytic pathway, and
even intercalate into the DNA base pairs, inducing tumor cell
apoptosis [17–19]. However, it is not clear whether the DNA
cleavage induced by the quercetin copper(II) complex occurs
via a hydrolytic pathway or via an oxidative pathway. Fur-
thermore, little research has been devoted to determining
whether the DNA-binding properties and the DNA cleavage
mechanism of the quercetin copper(II) complex have an
intrinsic relationship with its antitumor activity.
In this study, the mode of DNA binding, oxidative DNA
cleavage activity, and apoptosis-inducing activity of the
quercetin copper(II) complex were investigated. We dem-
onstrate that the antitumor mechanism of the quercetin
copper(II) complex involves not only its oxidative DNA
damage with generation of ROS but also its specific
interaction with DNA.
Materials and methods
Chemicals
All chemicals and reagents were purchased from com-
mercial sources and were used without further purification.
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J Biol Inorg Chem (2009) 14:727–739
The plasmid pBR322 DNA was purchased from TaKaRa
Biotechnology Co. (Japan). Quercetin, 3-(4,5-dimethylthi-
azol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), calf
thymus DNA (CT DNA), catalase, Hoechst33258, and
ethidium bromide were obtained from Sigma (Sigma
Chemical Co., St. Louis, MO, USA). Copper(II) chloride
(CuCl2
2H2O) was from Guangfu Fine Chemicals
(Tianjing, China). 2-Thiobarbituric acid (TBA) was from
Sinopharm Chemical Reagent Co. (Shanghai, China).
Agarose (molecular biology grade) was from Oxoid
(Basingstoke, UK). The tris(hydroxymethyl)aminomethane
(Tris–)HCl buffer solution was prepared with triple-dis-
tilled water. Anti-survivin (ZM-0468) were purchased from
Santa Cruz Biotechnology (Santa Cruz, CA, USA).
SP-9000 HistostainTM-Plus kits were from Zymed Labo-
ratories (South San Francisco, CA, USA). Caspase-3
colorimetric assay kits were from Keygen Biotech Co.
(Nanjing, China).
Preparation of quercetin copper(II) complexes
The quercetin copper(II) complex was prepared according
to the literature method [15]. The solid Que
2H2O (where
Que is quercetin; 2 mM) was dissolved in 60 mL of eth-
anol. Then the pH of the solution was adjusted to 6–7 with
NaOEt. After 5 min, CuCl2
2H2O (1 mM) was added to the
mixture. After it had been stirred and heated to reflux for
5 h at 60 °C, the reaction mixture was cooled to room
temperature and poured into H2O. The brown-yellow pre-
cipitate, which formed immediately, was set aside for 48 h,
filtered, and washed three times with 1:3 EtOH–H2O, The
solid product was dried under a vacuum for 48 h at room
temperature. Yield: 73%. Anal. found: C, 52.4%; H, 3.3%;
Cu, 9.1%. Calcd. for Cu(Que)2(H2O)2: C, 51.3%; H, 3.4%;
Cu, 9.0%. IR (KBr) (cm-1): vmax = 3,598–3,045 (b, m),
1,632 (s), 1,361 (s), 1,267 (s), 652 (w), 624 (w), 394 (m).
UV (EtOH) (nm): kmax = 427 (1.27 9 104), 265
(3.18 9 104). Attempts to grow single crystals suitable for
crystal structure determination were unsuccessful. These
data indicated that the stoichiometric ratio (copper to
quercetin) of Cu(Que)2(H2O)2 was 1:2 and quercetin could
chelate copper(II) via 3-OH and 4-oxo groups. A possible
structure model of the complexe is shown in Fig. 1.
Detection of plasmid pBR322 DNA cleavage
by agarose gel electrophoresis
DNA cleavage was measured by the conversion of super-
coiled pBR322 plasmid DNA to nicked circular and linear
DNA forms. Supercoiled pBR322 plasmid DNA (0.25 lg
per reaction) in Tris–HCl buffer (50 mM) with 50 mM
NaCl (pH 7.2) was treated with the indicated amount of the
quercetin copper(II) complex, followed by dilution with the
J Biol Inorg Chem (2009) 14:727–739
Fig. 1 A possible structure of the quercetin copper(II) complex
Tris–HCl buffer to a total volume of 10 lL. The samples
were incubated for 1 h at 37 °C. After the reaction had
been stopped by addition of 1/10 volume of the loading
buffer (0.25% bromophenol blue, 40% sucrose, 0.25%
xylene cyanole, and 200 mM EDTA), the samples were
loaded on 1% neutral agarose gel containing 40 mM Tris–
acetate and 1 mM EDTA (TAE buffer, pH 8.0), and were
subjected to electrophoresis in a horizontal slab gel appa-
ratus and 19 TAE buffer, which was performed at 75 V
for 1.5 h. The gel was stained with a solution of 0.5 lg/mL
ethidium bromide for 30 min, followed by destaining in
water. Agarose gel electrophoresis of plasmid DNA was
visualized by photographing the fluorescence of interca-
lated ethidium bromide under a UV illuminator. The
cleavage efficiency was measured by determining the
ability of the complexes to convert the supercoiled DNA to
the nicked circular form and the linear form. After elec-
trophoresis, the proportion of DNA in each fraction was
estimated quantitatively from the intensity of the bands
using Glyko BandScan software.
To study mechanism of the DNA cleavage reaction
performed by the Cu(Que)2(H2O)2 complex, different
scavengers or reactive oxygen intermediates such as
dimethyl sulfoxide (DMSO) (0.4 M), glycerol (0.4 M),
mannitol (0.2 M), catalase (15 U), oxidant H2O2 (50 lM),
and bathocuproine disulfonic acid (100 lM) were added to
reaction mixtures. Samples were treated as described
above.
Thiobarbituric acid reactive substances assay
Each sample containing 0.5 mM CT DNA, in 50 mM
phosphate buffer (pH 7.2), and Cu(Que)2(H2O)2 complex
729
(0, 25, 50, 100, 200, and 400 lM) was incubated at 37 °C
for 24 h in a total volume of 2 mL. After incubation,
samples were treated with 2 mL of a 1% (w/v) solution of
TBA in 50 mM NaOH and 2 mL of glacial acetic acid, and
were incubated at 100 °C for 1 h. After cooling, the
absorbance at 532 nm was measured. Blanks contained all
components except the complex.
Determination of oxidative DNA damage
DNA damage was determined by using the ethidium bro-
mide binding assay [20, 21]. A total of 3 mL of 10 mM
phosphate-buffered saline (PBS; pH 7.4) containing CT
DNA (60 lg/mL) with the quercetin copper(II) complex
(0, 25, 50, 100, 200, and 400 lM) was incubated at 37 °C
for 1 h under aerobic conditions. After incubation, ethi-
dium bromide (50 lL, 0.75 mg/mL) was added and the
fluorescence was measured using a PerkinElmer LS-50B
fluorescence spectrophotometer with excitation at 500 nm
and emission at 587 nm. A solution containing all reagents
and DNA but without the complex was used as the control
for 100% fluorescence, and zero fluorescence was assessed
in a solution identical to the control except it did not
contain DNA. The loss of the fluorescence was used as a
measure of DNA damage.
UV–vis spectral measurements
UV–vis spectra were measured using a Lambda 900 UV–
vis–near-IR spectrometer (PerkinElmer) in 0.01 M Tris
buffer. Spectroscopic titrations were carried out at room
temperature to determine the binding affinity between
DNA and the quercetin metal complex. Initially, the
solutions (2,000 lL) of the blank buffer and the
Cu(Que)2(H2O)2 complex sample (10 lM) were placed in
the reference and sample cuvettes (1-cm path length),
respectively, and then first spectrum was recorded in the
range 300–500 nm. During the titration, an aliquot (20 lL)
of buffered DNA solution (concentration of 1 mM in base
pairs) was added to each cuvette to eliminate the absorption
from DNA itself, and the solutions were mixed by repeated
inversion. After the solutions had been mixed for 10 min,
the absorption spectra were recorded. The titration pro-
cesses were repeated until there was no change in the
spectra for four titrations at least, indicating that binding
saturation had been achieved. The changes in the metal
complex concentration were negligible owing to dilution at
the end of each titration.
Viscosity measurements
Viscosity measurements were carried out using an
Ubbelodhe viscometer maintained at a constant temperature
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at 30.0 ± 0.1 °C in a thermostatic bath. Flow time was
measured with a digital stopwatch and each sample was
measured three times and an average flow time was calcu-
lated. Data are presented as (g/g0)1/3 versus binding ratio
[22], where g is the viscosity of DNA in the presence of the
complex and g0 is the viscosity of DNA in the absence of the
complex. Viscosity values were calculated from the
observed flow time of DNA-containing solutions
(t [ 100 s) corrected for the flow time of buffer alone (t0):
g = t - t0.
Fluorescence measurements
Fluorescence measurements were made using a PerkinEl-
mer LS-50B fluorescence spectrophotometer with a slit
width of 5 nm for the excitation and emission beams.
Fluorescence measurements were carried out by adding CT
DNA (40 lM bas pairs) directly to the cell containing the
solution of the Cu(Que)2(H2O)2 complex (c = 30 lM,
0.01 M Tris buffer, pH 7.2). To determine the selective
binding of the complex to DNA with different sequences,
another two kinds of DNA [poly(dA)
poly(dT) and poly
(dG)
poly(dC)] were introduced to displace CT DNA.
Emission spectra were recorded in the region 330–370 nm
using an excitation wavelength of 312 nm. All measure-
ments were performed at 25 °C.
Cytotoxicity evaluation
The cytotoxicity/survival of cells in the presence or
absence of the experimental agent was determined using a
system based on MTT as described previously [23]. Human
lung adenocarcinoma cell lines (A549) were obtained from
the American Type Culture Collection (Rockville, MD,
USA). The tumor cells (A549) were plated into 96-well
microtiter plates at a density of 1 9 104 cells per well.
After 24 h, culture medium was replaced by 200 lL RPMI
1640 medium (Roswell Park Memorial Insititue, Hyclone)
supplemented with 10% fetal bovine serum in the presence
of various concentrations (0, 6.25, 12.5, 25, 50, and
100 lM) of the Cu(Que)2(H2O)2 complex or (Que)2 (its
concentration was calculated as two molecules), and the
cells were incubated for 24, 48, and 72 h. The final con-
centration of solvent was less than 0.1% in the cell culture
medium. Culture solutions were removed and replaced by
90 lL culture medium. Ten microliters of sterile filtered
MTT solution (5 mg/mL) in PBS (pH 7.4) was added to
each well, reaching a final concentration of 0.5 mg MTT/
mL. Then the cells were incubated at 37 °C for 4 h. After
the medium and unreacted dye had been removed, DMSO
(200 lL) was added to each well. The absorbance at
490 nm of the dissolved solution was measured with a Bio-
Rad 680 microplate reader (Bio-Rad, USA). The relative
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J Biol Inorg Chem (2009) 14:727–739
cell viability (percent) related to control wells containing
cell culture medium without the compounds tested was
calculated by dividing the absorbance of the treated cells
by that of the control in each experiment. The IC50 value
was calculated using SPSS statistical software as the con-
centration of the compound tested which inhibits growth of
50% of cells relative to nontreated control cells.
Apoptosis assessment by Hoechst33258 staining
Chromatin condensation was detected by nuclear staining
with Hoechst33258. The cells were cultured on cover slips,
which were kept in a 60 Petri dish for 24 h before treat-
ment. After treatment with 0, 20, 40, or 80 lM
Cu(Que)2(H2O)2 complex for 24 h, A549 cells were fixed
with ice-cold methanol and acetic acid (3:1) at room
temperature for 5 min and exposed to Hoechest33258
staining solution (5 lg/mL) at room temperature in the
dark for 15 min. Samples were observed under a fluores-
cence microscope (Olympus BX-51, Japan).
Immunocytochemistry analysis
The cells were cultured on cover slips, which were kept in
a 60 Petri dish for 24 h before treatment. After treatment
with 0, 20, or 60 lM Cu(Que)2(H2O)2 complex for 24 h,
the cells were washed three times with isotonic PBS (pH
7.4) and then fixed in 4% paraformaldehyde solution in
PBS for 15 min at 37 °C. Then the cells were washed three
times with PBS (pH 7.4) and incubated with 0.3% Triton
X-100 in PBS for 20 min at 37 °C. The slides were
immersed in 3% H2O2 in methanol at 20 °C for 15 min to
block endogenous peroxidase activity. Then the cover slips
were washed three times with PBS, and nonspecific bind-
ing sites were blocked in PBS containing 10% normal goat
serum for 30 min. The cells were incubated with rabbit
anti-human survivin antibody (1:100) in PBS containing
10% normal goat serum overnight at 4 °C and washed
three times with PBS. Then the cells were incubated with
biotin-conjugated goat anti-rabbit/anti-mouse IgG in PBS
containing 10% normal goat serum for 40 min at 37 °C and
washed three times with PBS. The cells were incubated
with horseradish peroxidase labeled streptavidin in PBS for
60 min at 37 °C. After the slides had been washed three
times, horseradish peroxidase was visualized by freshly
prepared 3,30 -diaminobenzidine tetrahydrochloride/H2O2
in the dark for 10 min. The coloring reaction was stopped
by washing the slides with water and then the nuclei were
counterstained with Mayer’s hematoxylin (Zhongsha Bio,
Beijing) for 1 min. Finally, the slides were dehydrated with
ethanol, cover-slipped with DPX [1,10 -(1,4-phenylene-
bis(methylene))bis-pyridiniudibromide], and observed and
photographed with an Olympus optical microscope.
J Biol Inorg Chem (2009) 14:727–739
Negative controls in which the primary antibody was
omitted and positive controls for each antibody were
included in each batch of immunocytochemistry.
Determination of caspase-3 activity by absorption
spectroscopy
The measurement of caspase-3 activity was performed
using caspase-3 colorimetric protease assay kits (Keygen
Biotech. Co., China). The A549 cells were plated at a
density of 1 9 106 cells per well in complete RPMI 1640
medium for 24 h. After treatment with 0, 20, or 60 lM
Cu(Que)2(H2O)2 complex for 24 h, cells were harvested
and washed three times with cold PBS by centrifuga-
tion (1,000g, 3 min) to stop the incubation. Cells were
resuspended in 50 lL of chilled cell lysis buffer contain-
ing N-(2-hydroxyethyl)piperazine-N0 -ethanesulfonic acid
(50 mM, pH 7.4), 3-[(3-cholamidopropyl)dimethylammo-
nio]propanesulfonate (5 mM), and dithiothreitol (5 mM).
After they had been incubated on ice for 10 min and cen-
trifuged for 1 min in a microcentrifuge (10,000g), the
supernatants (cytosolic extract) were transferred to a fresh
tube and put on ice to assay the protein concentration. The
protein concentration was adjusted with the cell lysis buf-
fer, then the sample (50 lL) was added to twofold
concentrated reaction buffer [50 lL; 40 mM N-(2-
hydroxyethyl)piperazine-N0 -ethanesulfonic acid, pH 7.4,
3 mM 3-[(3-cholamidopropyl)dimethylammonio]propane-
sulfonate,10 mM dithiothreitol, and 4 mM EDTA] and
DEVD-pNA (caspase-3; 5 lL, 4 mM), then incubated at
37 °C for 24 h. After incubation, every sample was read at
405 nm in a Bio-Rad 680 microplate reader (Bio-Rad,
USA).
Statistical evaluation
The cell assay results shown represent the mean ± the
standard deviation from triplicate experiments performed
in a parallel manner unless otherwise indicated. Statistical
analyses were performed using an unpaired, two-tailed
Student’s t test. All comparisons are made relative to
untreated controls and significance of difference is indi-
cated as *P \ 0.05 and **P \ 0.01.
Results
Strand breakage of plasmid pBR322 DNA induced
by the quercetin copper(II) complex
The double-stranded plasmid pBR322 exists in a compact
supercoiled conformation. Upon formation of strand
breaks, the supercoiled form of DNA is disrupted into the
731
nicked circular form and the linear form. If one strand is
cleaved, the supercoiled form will relax to produce a
nicked circular form. If both strands are cleaved, a linear
form will be produced. Relatively fast migration is
observed for the supercoiled form when the plasmid DNA
is subjected to electrophoresis. The nicked circular form
migrates slowly and the linear form migrates between the
supercoiled form and the nicked circular form [24]. Hence,
DNA strand breaks were quantified by measuring the
transformation of the supercoiled form into nicked circular
and linear forms.
The ability of the Cu(Que)2(H2O)2 complex to induce
DNA cleavage was studied by gel electrophoresis using
supercoiled pBR322 DNA in 50 mM Tris–HCl/50 mM
NaCl buffer (pH 7.2). Both quercetin (data not shown) and
Cu2? (Fig. 2, lane 2) alone induced little DNA cleavage.
Figure 2 shows the results of the gel electrophoresis
experiment carried out with supercoiled DNA cleavage
induced by various concentrations of the complex. The
complex scarcely catalyzed the cleavage of plasmid DNA
at 10 lM. When the concentration of the complex was up
to 25 lM, the cleavage of plasmid DNA was observed.
More than 50 lM complex could promote the conversion
of the substrate DNA almost completely into the nicked
circular and linear forms, and the increase in the amounts
of linear DNA was observed to be associated with the
increase of the concentration of the complex.
Effects of ionic strength and time on DNA cleavage
induced by the quercetin copper(II) complex
The effects of ionic strength on the double-strand DNA
cleavage by adding NaCl were studied. Figure 3 shows that
the process of cleavage was sensitive to the change of ionic
strength. When the concentration of NaCl was not more
than 100 mM, the ionic strength could promote DNA
cleavage. However, the extent of DNA cleavage became
Fig. 2 Agarose gel (1%) of pBR322 (0.25 lg) incubated for 1.5 h at
37 °C and pH 7.2 [50 mM tris(hydroxymethyl)aminomethane (Tris)–
HCl] with increasing concentrations of the Cu(Que)2(H2O) 2 complex
(where Que is quercetin). Lane 1 DNA control, lane 2 Cu2? 100 lM,
lanes 3–7 Cu(Que)2(H2O)2 complex, 10, 25, 50, 100, 200, and
400 lM, respectively. NC nicked circular, L linear, SC supercoiled
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Fig. 3 Agarose gel (1%) of pBR322 DNA incubated for 1.5 h at
37 °C and pH 7.2 with 100 lM Cu(Que)2(H2O)2 complex for
increasing NaCl concentrations. Lane 1 DNA control, lanes 2–8
NaCl concentration was 10, 25, 50, 100, 200, 400, and 800 mM,
respectively
Fig. 4 Agarose gel (1%) of pBR322 (0.25 lg) at 37 °C and pH 7.2
(50 mM Tris–HCl) with 100 lM Cu(Que)2(H2O)2 complex for
increasing reaction time. Lane 1 DNA control, lanes 2–9 15, 30,
60, 90, 120, 150, 180, and 240 min, respectively

obviously weakened and the amount of the linear form

decreased when the ionic strength changed from 200 to
800 mM. Generally speaking, a high concentration of NaCl
could make the conformation of DNA tighter, going
against the DNA binding of the complex, which inhibits the
DNA cleavage induced by the complex. The results suggest
that electrostatic interaction may contribution to the DNA
cleavage induced by the Cu(Que)2(H2O)2 complex.
The time dependence of the reaction was determined in
the presence and the absence of the complex, and pBR322
DNA was incubated with 100 lM Cu(Que)2(H2O)2 com-
plex in 50 mM Tris–HCl buffer at 37 °C for 15–240 min.
The increase in the amounts of linear DNA was observed
to be associated with the increase of reaction time Fig. 5 Disappearance of the supercoiled form and formation of the
(Fig. 4). The amounts of linear DNA were 16.8 and nicked circular form of pBR322 DNA in the presence of the
Cu(Que)2(H2O)2 complex (100 lM) with incubation time (pH 7.2,
26.6% when the reaction time was 3 and 4 h, respectively
37 °C). Inset: ln(% supercoiled form) versus time for a complex
(Fig. 4). concentration of 100 lM
Figure 5 shows the extent of DNA cleavage by the
Cu(Que)2(H2O)2 complex with reaction time. The decrease
in the amount of the supercoiled form and the formation of
nicled circular DNA with time shows the expected expo-
nential nature of the curves. The plot of ln(% supercoiled
DNA) versus time is linear, which confirms the process is
pseudo-first-order. The rate constant k1 (4.82 9 10-4 s-1),
the slope of the linear plot, was obtained using a complex
concentration of 100 lM (Fig. 5, inset).

Mechanism of the DNA cleavage

To clarify the roles of oxygen-derived active species in
the DNA strand breakage induced by the Cu(Que)2(H2O)2
complex, hydroxyl radical scavengers (0.4 M DMSO and
glycerol), catalase (15 U), reducing agent (1 mM ascorbic
acid), oxidant (50 lM H2O2), and copper(I) chelator
(100 lM bathocuproine disulfonic acid) were introduced
to the system (Fig. 6). Hydroxyl radical scavengers
(DMSO and glycerol) and bathocuproine disulfonic acid
showed little inhibitory effect on the DNA cleavage.
Ascorbic acid resulted in a slight increase in the amount
of linear DNA. However, H2O2 could markedly promote
Fig. 6 Agarose gel (1%) of pBR322 (0.25 lg) incubated for 1.5 h at
37 °C and pH 7.2 (50 mM Tris–HCl) with 100 lM Cu(Que)2(H2O)2
complex and different scavengers or H2O2: lane 1 DNA control, lane
2 complex control, lane 3 ascorbic acid (1 mM), lane 4 dimethyl
sulfoxide (0.4 M), lane 5 H2O2 (50 lM), lane 6 glycerol (0.4 M),
lane 7 catalase (15 U), lane 8 bathocuproine disulfonic acid (100 lM)
the strand breakage of plasmid pBR322 DNA induced by
the complex, and DNA was completely degraded as
indicated by the disappearance of the main bands of
DNA in the gel and by the presence of a smear in the
corresponding electrophoretic lane. On the other hand,

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catalase showed an obvious inhibitory effect on the DNA
cleavage and the linear form disappeared. The results
indicated the participation of both ROS, especially H2O2,
is important in DNA cleavage. So DNA cleavage pro-
moted by the complex might occur via an oxidative
pathway.
DNA damage induced by the quercetin copper(II)
complex
Copper-dependent oxidative DNA damage can also be
quantitatively measured by the ethidium bromide binding
assay [25, 26]. Ethidium bromide generally shows no
apparent emission intensity in buffer solution because of
solvent quenching. However, ethidium bromide emits
intense fluorescent light in the presence of DNA owing to
its strong intercalation between the adjacent DNA base
pairs. It has been reported that several forms of DNA
lesions, including strand scission, base oxidation, and base
liberation, could contribute to the loss of fluorescence
[21, 27]. The CT DNA was remarkably damaged by the
complex in a concentration-dependent manner (Fig. 7).
To determine whether the deoxyribose ring in the DNA
skeleton was broken owing to the formation of ROS, we
analyzed the influence of the complex on the oxidative
damage of CT DNA by measuring the formation of TBA
reacting species (TBARS). The amounts of TBARS obvi-
ously increased with the concentration of the complex
(Fig. 8), so the complex could induce the oxidative deg-
radation of CT DNA. These data suggest that the
deoxyribose ring in the DNA skeleton was cut by ROS and
oxidative cleavage of DNA occurred, which agrees well
with the results reported above.
Fig. 7 Extent of calf thymus DNA damage induced by the
Cu(Que)2(H2O)2 complex. DNA (0.5 mM) was incubated at 37 °C
for 60 min with different concentrations of the complex in phosphate-
buffered saline (PBS; pH 7.2). Each value represents the mean ± the
standard deviation (SD) of three experiments
733
Fig. 8 The formation of 2-thiobarbituric acid reacting species
(TBARS) induced by the Cu(Que)2(H2O)2 complex. DNA
(0.5 mM) was incubated at 37 °C for 24 h with different concentra-
tions of the complex in PBS (pH 7.2). TBARS formation was
determined by measuring the absorbance at 532 nm. Each value
represents the mean ± SD of three experiments. **P \ 0.01 com-
pared with control group
Interaction of the quercetin copper(II) complex
with DNA
Electronic absorption spectra studies
The binding of the complex to DNA was also characterized
classically through absorption titration. A complex bound
to DNA through intercalation is characterized by the
change in absorbance (hypochromism) and redshift in
wavelength, due to the intercalative mode involving a
stacking interaction between the aromatic chromophore
and the DNA base pairs. But inconspicuous hypochromism
could imply electrostatic interactions or groove binding of
the complex with DNA. The electronic absorption spectra
of the Cu(Que)2(H2O)2 complex exhibited broad absorp-
tion bands in the region 260–440 nm, typical for transitions
between the p-electron energy levels of the quercetin
skeleton. The electronic spectra of the complex in the
presence and absence of DNA were monitored over the
wavelength range 260–440 nm, as shown in Fig. 9. With
increasing concentration of CT DNA (0–50 lM), the
absorptivity at about 270 nm decreased moderately, and
then the absorptivity increased slightly when the concen-
tration of CT DNA was higher than 30 lM, which is
consistent with the findings of another study [28]. This
could result from two causes: (1) the modes of binding of
the complex with DNA were both intercalation and elec-
trostatic interaction; (2) the complex could induce the
breakage of DNA, which was determined in the electro-
phoresis measurements described earlier, resulting in a
decrease in the amounts of the complex intercalated into
DNA.
123
734
Fig. 9 Absorption spectra of
the Cu(Que)2(H2O)2 complex
(10 lM) in 0.01 M Tris–HCl
buffer at pH 7.2, in the absence
and presence of increasing
amounts of calf thymus DNA
(CT DNA): a 0 lM base pairs,
b 10 lM base pairs,
c 20 lM base pairs,
d 30 lM base pairs,
e 40 lM base pairs,
f 50 lM base pairs
Viscometric studies
To investigate further the DNA-binding mode of the
Cu(Que)2(H2O)2 complex, viscosity measurements on
solutions of CT DNA incubated with the complex were
performed. It is well known that intercalative DNA binding
would cause elongation of the DNA polymer by effecting
separation of the DNA base pairs, resulting in an increase
in viscosity. In contrast, a partial and/or nonclassical
intercalation of the ligand could bend or kink the DNA,
resulting in a decrease in its effective length with a con-
comitant decrease in its viscosity [29, 30].
The relative specific viscosity (g/g0) of DNA generally
reflects the increase in contour length associated with
separation of DNA base pairs caused by intercalation.
Figure 10 shows an obvious increase in the relative specific
viscosity of DNA when the Cu(Que)2(H2O)2 complex is
added to CT DNA solution, but there is a slight decrease in
the relative specific viscosity of DNA when the concen-
tration ratio of the complex to DNA, r, is more than 0.15.
So it was demonstrated that the Cu(Que)2(H2O)2 complex
could intercalate into DNA, resulting in an increase in the
relative specific viscosity of DNA, and that the chain of the
DNA helix became short owing to the breakage of DNA,
resulting in a decrease in g/g0. The result is consistent with
the absorption spectra results described earlier and with the
findings of another study [30].
Fluorescence measurements
The additional evidence for intercalation into the DNA was
obtained from fluorescence spectra studies. The fluores-
cence spectra of the Cu(Que)2(H2O)2 complex, exhibiting a
broad emission band in the range 340–360 nm, were
monitored at a fixed concentration of 30 lM. Enhanced
123
J Biol Inorg Chem (2009) 14:727–739
fluorescence without a wavelength shift was observed
(Fig. 11) when CT DNA was added to the complex solu-
tion. The result suggests that the stronger enhancement of
fluorescence intensity for the complex may be largely due
to the interaction between adjacent base pairs of CT DNA
and the complex. Furthermore, the binding of the complex
to the DNA helix could decrease the collision frequency of
solvent molecules with the complex, which usually leads to
emission enhancement of the complex. So the complex
may intercalate into the adjacent base pairs of CT DNA. In
addition, Fig. 11 shows the fluorescence intensity of the
complex when the different kinds of DNA were added
to the complex solution. The result illustrates that there
is the largest increase in fluorescence intensity when
poly(dG)
poly(dC) is added to the complex solution. So
the binding affinity between the complex and poly(dG)

poly(dC) may be larger than that between the complex and
CT DNA [or poly(dA)
poly(dT)].
Cytotoxicity study
The potential antiproliferative effects of the Cu(Que)2
(H2O)2 complex on the viability of tumor cells lines (A549)
were detected by the MTT assay. The concentrations which
produced 50% (IC50) inhibition of the cell viability were
analyzed by SPSS software and the results are listed in
Table 1. From Table 1, the Cu(Que)2(H2O)2 complex
significantly inhibits growth and proliferation of A549 cells
in a dose- and time-dependent manner. The viability of
A549 cells was 33.1% after treatment with 100 lM
Cu(Que)2(H2O)2 complex for 48 h. As shown in Table 1, the
estimated IC50 values of the Cu(Que)2(H2O)2 complex were
78.1 ± 1.0, 46.7 ± 0.9, and 21.5 ± 0.5 lM for 24, 48, and
72 h of treatment of A549 cells, respectively, but those of
(Que)2, which is equivalent to 2 mol of quercetin, were
J Biol Inorg Chem (2009) 14:727–739
Fig. 10 Effect of increasing amounts of the Cu(Que)2(H2O)2 com-
plex on the relative viscosity of CT DNA in 0.01 M Tris–HCl buffer
(pH 7.2) at 30 ± 0.1 °C. cDNA = 50 lM, r = ccomplex/cDNA
Fig. 11 The change of emission fluorescence spectra of the
Cu(Que)2(H2O)2 complex (30 lM) in 0.01 M Tris–HCl buffer at
pH 7.2, in the absence (a) and in the presence of 30 lM base pairs of
CT DNA (b), poly(dA)
poly(dT) (c), and poly(dG)
poly(dC) (d).
kex = 312 nm
103.9 ± 1.3, 72.1 ± 1.0, and 34.9 ± 1.0 lM in the same
conditions. The results illustrate that the cytotoxicity of the
Cu(Que)2(H2O)2 complex is higher than that of quercetin
alone.
A549 cells display apoptotic characteristics after
treatment with the quercetin copper(II) complex
To further address the death pattern, A549 cells were
stained with Hoechst 33258 after treatment with the
Cu(Que)2(H2O)2 complex. The Hoechst 33258 staining is
sensitive to DNA and was used to assess changes in nuclear
morphology. The results of Hoechst33258 staining assay
showed that cells demonstrated apoptotic features such as
nuclear shrinkage, chromatin condensation, or fragmenta-
tion after treatment with the Cu(Que)2(H2O)2 complex for
24 h. The ratio of cells with a profile of chromatin
735
Table 1 Effect of quercetin and quercetin copper(II) complex on the
viability of A549 cells
Time (h) IC50 (lmol/L)
(Quercetin)2 Cu(quercetin)2(H2O)2
24 103.9 ± 1.3 78.1 ± 1.0
48 72.1 ± 1.0 46.7 ± 0.9
72 34.9 ± 1.0 21.5 ± 0.5
Cell viabilities were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide assays. Cells were incubated for 0–72 h
without (control) or with 0–100 lM tested compound. The IC50
values are the concentrations which produced 50% inhibition of the
cell viability. The results are expressed as the mean ± the standard
deviation (n = 3)
condensation and fragmented fluorescent nuclei increased
in a dose-dependent manner (Fig. 12a).
The apoptotic rate occurred in a dose-dependent manner
as determined from Hoechst33258 staining assay, as shown
in Fig. 12b. After treatment with 80 lM Cu(Que)2(H2O)2
complex for 24 h, the apoptotic rate of A549 cells was
more than 55%. This suggest that A549 cells undergo the
typical morphologic changes of apoptosis after exposure to
the Cu(Que)2(H2O)2 complex.
The expression of survivin protein
by immunocytochemistry
We further examined whether the Cu(Que)2(H2O)2 com-
plex induces cell apoptosis by modulating the expression of
survivin protein by immunocytochemistry. After treatment
with the Cu(Que)2(H2O)2 complex for 24 h, the levels of
survivin protein decreased significantly (Fig. 13). The
levels of survivin protein expression occurred in a dose-
dependent manner and were determined from observation
with an optical microscope. After treatment with 60 lM
Cu(Que)2(H2O)2 complex for 24 h, the levels of survivin
protein expression in A549 cells decreased by 43.9%.
Activity of caspase-3 by absorption spectroscopy
To demonstrate the molecular mechanism of apoptosis of
A549 cells induced by the Cu(Que)2(H2O)2 complex, we
further measured the caspase-3 activity from the absorption
spectra. After treatment with 20 or 60 lM quercetin for
24 h, the relative activity of caspase-3 increased by 181
and 246%, respectively (Fig. 14).
Discussion
In this study, the oxidative DNA damage, DNA binding,
and antitumor activity of the quercetin copper(II) complex
123
736 J Biol Inorg Chem (2009) 14:727–739

Fig. 12 a The morphological

changes of Hoechst33258-
stained A549 cells after
treatment with the
Cu(Que)2(H2O)2 complex as
observed under a fluorescence
microscope (A0–A3). Cells were
treated without the complex
(A0) or with the complex at
20 lM (A1), 40 lM (A2), and
80 lM (A3) for 24 h. b
Apoptotic cells were analyzed.
The results are expressed
as the mean ± SD (n = 3).
**P \ 0.01 compared
with the control group

were investigated. Our results demonstrate that the quer-
cetin copper complex can effectively promote the cleavage
of plasmid DNA at physiological pH and temperature via
an oxidative path. As for quercetin metal complexes, the
rate constant of the oxidative DNA cleavage found in this
study is much higher than that of hydrolytic DNA cleavage
found in our previous studies [18, 19]. In vitro studies
showed that DNA damage was induced at thymine and
guanine by generating ROS in the presence of copper(II)
and H2O2 [31, 32]. Additionally copper can induce oxi-
dative DNA damage by activating H2O2 to form a reactive
copper(II)–hydroperoxo complex [1].
Furthermore, it is suggested that the complex can
intercalate into DNA owing to favorable planarity of the
ligand quercetin, and that copper cation may coordinate
with the negatively charged oxygen in the phosphodiester
backbone of DNA, displacing a water molecule, which
enhances the binding affinity between the complex and
DNA. To clarify the cleavage mechanism of pBR322 DNA
induced by the Cu(Que)2(H2O)2 complex, the effects of
several additional agents on DNA cleavage were examined.
Although hydroxyl radical scavengers (DMSO and glyc-
erol) and bathocuproine disulfonic acid showed little
inhibitory effect on the DNA cleavage, it is not suggested
that there is no hydroxyl radical and copper(I) in the sys-
tem. It is possible that the chelation of bathocuproine
disulfonic acid and copper(I) was sterically inhibited by the
ligand quercetin. But the strand breakage of plasmid
pBR322 DNA induced by the complex was markedly
promoted by H2O2 and inhibited by catalase. It is suggested
that the participation of both ROS, especially H2O2, is
important in DNA cleavage and that DNA cleavage

123
J Biol Inorg Chem (2009) 14:727–739
Fig. 13 a The expression of
survivin protein in A549 cells
after treatment for 24 h with
various concentrations of the
Cu(Que)2(H2O)2 complex:
0 lM, control (A0); 20 lM
(A1); 60 lM (A2). b The
percentage of the positive
expression was analyzed. The
results are expressed as the
mean ± SD (n = 3).
*P \ 0.05, **P \ 0.01
compared with the control
group
Fig. 14 The activity of caspase-3 in A549 cells after treatment with
the Cu(Que)2(H2O)2 complex for 24 h. The optical density at 405 nm
values was measured and the activity of caspase-3 was expressed
relative to the untreated control, designated as 100. The results are
expressed as the mean ± SD (n = 3). *P \ 0.05, **P \ 0.01
compared with the control group
promoted by the complex might occur via an oxidative
pathway. These results can be explained by assuming the
possible mechanism of DNA damage induced by the
Cu(Que)2(H2O)2 complex as shown in Fig. 15. The cop-
per(II), linked to a 3-hydroxo-4-keto moiety, can be in
equilibrium with the copper(I) complex formed when
the copper(II) complex takes one electron from hydroxo
oxygen and then is converted into a radical (Que
). The
copper(I) complex intercalates into the stacked base
pairs of DNA owing to the planarity of quercetin. There
are electrostatic interactions between copper(I) and the
737
phosphate diester backbone and hydrogen bonding inter-
actions between coordinated –NH– and two carbonyl
oxygen atoms with the functional groups positioned at the
edge of the DNA bases, which promotes strong DNA-
binding affinities of the Cu(Que)2(H2O)2 complex to form
the DNA–(Que–Cu(I)–Que
) complex. Then copper(I)
reacts with O2 to generate superoxide anion (O2
-) and
subsequently H2O2. The copper(I) formed and bound to
DNA reacts with H2O2 to produce a peroxide complex such
as DNA–Cu(I)OOH [5]. In the proximity of DNA, fur-
thermore, the reduction of the peroxide complex (DNA–
Cu(I)OOH) produces the ROS in abundance, i.e., hydroxyl
radical
OH, which would immediately attack the adjacent
deoxyribose ring in the DNA skeleton and produce more
chain breaks on DNA than in the case of ROS produced in
the bulk before it can be scavenged by
OH scavengers
such as DMSO.
In addition, we found that the Cu(Que)2(H2O)2 complex
could induce cell growth inhibition and apoptosis in A549
cells. The Cu(Que)2(H2O)2 complex has more significant
inhibition of growth and proliferation of A549 cells in a
dose- and time-dependent manner than quercetin alone.
Copper-complex-induced A549 cells stained with Hoe-
chst33258 were characterized by nuclear shrinkage,
chromatin condensation, or fragmentation (Fig. 12). This
suggested that A549 cells underwent the typical morpho-
logic changes of apoptosis after exposure to the complex.
Moreover, the levels of survivin proteins decreased, while
123
738
Fig. 15 A possible mechanism
of oxidative DNA damage
induced by the Cu(Que)2(H2O)2
complex
relative activity of caspase-3 increased in the A549 cells
after treatment with the complex. It is suggested that the
Cu(Que)2(H2O)2 complex could downregulate survivin
expression and promote the activation of caspase-3,
resulting in the apoptosis of tumor cells.
Evidence has been presented that the antioxidant prop-
erties of polyphenols such as quercetin may not entirely
account for their chemopreventive effects [7, 33]. It was
suggested that the antioxidant effects of those polyphenols
123
J Biol Inorg Chem (2009) 14:727–739
may be essential but not sufficient for their activity against
tumor promotion. In addition, the latest studies have indi-
cated that quercetin as an inhibitor of tyrosine kinase was
not an effective cancer therapeutic agent [34]. However,
the cytotoxicity of the Cu(Que)2(H2O)2 complex against
cancer cells is higher than that of quercetin alone, which
may be attributed to the intercalation of the complex into
DNA and the effective oxidative DNA damage induced by
it. Furthermore, the Cu(Que)2(H2O)2 complex could not
J Biol Inorg Chem (2009) 14:727–739
only induce apoptosis of A549 cells but could also down-
regulate the expression of survivin protein. Additionally,
fluorescence measurements have shown the larger binding
affinity between the complex and poly(G)
poly(C). On the
basis of these results, we could audaciously speculate that
the Cu(Que)2(H2O)2 complex might preferentially inter-
calate into the GC-rich core promoter region of survivin
like hedamycin [35], resulting in downregulation of sur-
viving gene expression. Besides, hydroxyl radical produced
by the peroxide complex attacks the adjacent deoxyri-
bose ring in the DNA skeleton, which shows a moderate
selectivity in oxidative DNA damage. In other words, the
selective oxidative DNA damage induced by the
Cu(Que)2(H2O)2 complex depends on the preference of its
intercalation into DNA. However, the real apoptosis
mechanism needs to be confirmed through further detailed
research.
In conclusion, we have demonstrated that the quercetin
copper(II) complex could induce oxidative DNA damage
related to its intercalation into DNA, which may be an
important mechanism of its anticancer and apoptosis-
inducing activities. Actually, the oxidative DNA damage is
associated with the transient reduction of copper(II) to
copper(I) and the generation of ROS such as H2O2 and
Cu(I)OOH. The selective DNA binding mechanism of the
complex and the mechanism of cell apoptosis induced by
the complex are under investigated.
Acknowledgments This research was financially supported by the
Science and Technology Project of Education Commission of
Chongqing (grant no. KJ051503) and a China Postdoctoral Science
Foundation funded project.
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