CuII ions and the stilbene-chroman hybrid with a catechol moiety synergistically induced DNA damage, and cell cycle arrest and apoptosis of HepG2 cells: An interesting acid/base-promoted prooxidant reaction

Article abstract of DOI:10.1002/chem.201201545

Development of potential cancer treatment strategies by using an exogenous reactive oxygen species (ROS)-generating agent (prooxidant) or redox intervention, has attracted much interest. One effective ROS generation method is to construct a prooxidant system by polyphenolic compounds and Cu ^II ions. This work demonstrates that Cu^II and the stilbene-chroman hybrid with a catechol moiety could synergistically induce pBR322 plasmid DNA damage, as well as cell cycle arrest and apoptosis of HepG2 cells. Additionally, an interesting acid/base-promoted prooxidant reaction was found. The detailed chemical mechanisms for the reaction of the hybrid with Cu^II in acid, neutral and base solutions are proposed based on UV/Vis spectral changes and identification of the related oxidative intermediates and products. A view to a kill: This work demonstrates that Cu^II and the stilbene-chroman hybrid with a catechol moiety could synergistically induce DNA damage, and cell cycle arrest and apoptosis of HepG2 cells (see illustration). The acid/base-promoted prooxidant mechanism for the reaction of the hybrid with Cu^II is proposed based on UV/Vis spectral changes and identification of the related oxidative intermediates and products. Copyright

Full text of DOI:10.1002/chem.201201545

FULL PAPER
DOI: 10.1002/chem.201201545
CuII Ions and the Stilbene–Chroman Hybrid with a Catechol Moiety
Synergistically Induced DNA Damage, and Cell Cycle Arrest and Apoptosis
of HepG2 Cells: An Interesting Acid/Base-Promoted Prooxidant Reaction
Guo-Yun Liu, Jie Yang, Fang Dai,* Wen-Jing Yan, Qi Wang, Xiu-Zhuang Li,
De-Jun Ding, Xiao-Yan Cao, and Bo Zhou*[a]
Abstract: Development of potential
cancer treatment strategies by using an
exogenous reactive oxygen species
(ROS)-generating agent (prooxidant)
or redox intervention, has attracted
much interest. One effective ROS gen-
eration method is to construct a prooxi-
dant system by polyphenolic com-
pounds and CuII ions. This work dem-
onstrates that CuII and the stilbene–
chroman hybrid with a catechol moiety
could synergistically induce pBR322
plasmid DNA damage, as well as cell
cycle arrest and apoptosis of HepG2
cells. Additionally, an interesting acid/
base-promoted prooxidant reaction
was found. The detailed chemical
mechanisms for the reaction of the
hybrid with CuII in acid, neutral and
base solutions are proposed based on
UV/Vis spectral changes and identifica-
tion of the related oxidative intermedi-
ates and products.
Keywords: antitumor
agents
·
apoptosis · DNA damage · prooxi-
dants · reactive oxygen species
Introduction
deal with DNA damage. This difference provides an oppor-
tunity for the development of potential cancer treatment
strategies, namely, by using an exogenous ROS-generating
agent (prooxidant) or redox intervention further escalates
cellular ROS production to selectively kill cancer cells with-
out causing significant toxicity to normal counterparts.[4]
Chemical properties and biological activities of plant
polyphenols are the subject of intense research, as detailed
in the excellent review by Quideau et al.[5] It is well-known
that polyphenolic antioxidants can switch to prooxidants in
the presence of transition metal ions, such as copper, one of
the most redox-active ions present in cells;[6] this could trig-
ger a Fenton-type reaction leading to the generation of the
highly active HO·. Thus, one effective ROS generation
method is to construct a prooxidant system with polyphenol-
ic compounds and CuII ions. More importantly, elevated
levels of copper in cancer patients in comparison with
healthy subjects has been documented in a wide spectrum of
tumors, and this is closely related to cancer stage and/or pro-
gression.[4b] These discoveries have prompted interest in the
investigations of activity and biological mechanisms of DNA
damage[7] and cytotoxic action against cancer cells induced
by the polyphenol/CuII system.[8]
Reactive oxygen species (ROS) are a family of oxygen-con-
taining molecules, including superoxide anion radical (O2·À),
hydrogen peroxide (H2O2), hydroxyl radical (HO·) and so
on, that are mainly produced in the mitochondrial electron-
transport chain and oxygen-metabolizing enzymatic reac-
tions as a consequence of aerobic life.[1] Although the over-
production and/or mismanagement of ROS, oxidative stress,
has been implicated as a causative role in cancer, aging and
neurodegenerative diseases,[2] emerging data show that ROS
can also function as signaling molecules regulating a wide
variety of essential physiological processes.[1,3] This dual
action (signaling molecule or toxin) of ROS might be strictly
controlled by their concentrations, pulse duration, subcellu-
lar localization, and cell types.[1b] Generally, low levels of
ROS can promote cell proliferation and survival, whereas
high levels of ROS reaching a toxic threshold could trigger
cell death.[1b,4] Compared to normal cells, cancer cells exhibit
increased levels of ROS and an altered redox status,[4] owing
to many factors, such as oncogenic stimulation, increased
metabolic activity and mitochondrial malfunction.[4f] As
such, cancer cells would be more vulnerable to further ROS
stress than normal cells,[4] despite their enhanced capacity to
However, the detailed prooxidation mechanisms of the
polyphenol/CuII system from a chemical point of view are
yet to be determined. Accordingly, we have previously re-
ported the prooxidation scenario in chemical detail for the
case of caffeic acid and resveratrol (3,5,4’-trihydroxy-trans-
stilbene, 1) derivatives in the presence of CuII ions, and pro-
vided a ground for further development of polyphenol-di-
rected and ROS-generating anticancer agents that interact
with this metal ion.[9] Prooxidative or ROS-generating effi-
[a] G.-Y. Liu, J. Yang, Dr. F. Dai, W.-J. Yan, Q. Wang, X.-Z. Li,
D.-J. Ding, X.-Y. Cao, Prof. Dr. B. Zhou
State Key Laboratory of Applied Organic Chemistry
Lanzhou University, 222 Tianshui Street S.
Lanzhou 730000 (P.R. China)
Fax : (+86)931-8915557
E-mail: bozhou@lzu.edu.cn
daifang@lzu.edu.cn
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Figure 2. Relaxation of supercoiled pBR322 DNA by CuII and/or com-
pounds 1–5. Cleavage was carried out at 378C for 1 h in PBS (pH 7.40)
then analyzed by agarose gel electrophoresis. A) Lane 1: DNA alone;
lane 2: 250 mm CuII; lanes 3–7: 100 mm 1–5, respectively. B) Lane 1: DNA
alone; lanes 2–6: 250 mm CuII and 100 mm 1–5, respectively. C) Effect of
CAT, GSH, and BCDS on 5 and CuII-mediated DNA strand breakage.
The experiment conditions were the same as in (A) and (B). Lane 1:
DNA alone; lane 2: 5 and CuII; lanes 3–6: 0.05, 0.1, 0.5, 1 mgmLÀ1 CAT,
respectively; lanes 7–10: 1, 2.5, 5, 10 mm GSH, respectively; lanes 11–15:
0.1, 0.2, 0.4, 0.8, 1 mm BCDS, respectively.
Figure 1. Molecular structures of compounds used in this study.
cacy and mechanisms of the polyphenol/CuII system are
strongly influenced by various factors including the molecu-
lar structure, acidity and electron-donating ability of poly-
phenols.[9a] We have recently synthesized the hydroxylated
stilbene–chroman hybrids (3–5, Figure 1) incorporating skel-
etons of two kinds of molecules (resveratrol and vitamin E),
and found that these compounds exhibit significantly in-
creased electron-donating ability compared with resveratrol
and 2,2,5,7,8-pentamethyl-6-hydroxychroman (2), PMC, a vit-
amin E analogue.[10] Thus, we were motivated to investigate
whether the hybrids in combination with CuII ions could
synergistically induce DNA damage, and cell cycle arrest
and apoptosis of cancer cells with an emphasis on chemical
mechanism study. In this study, we came upon an interesting
acid/base-promoted prooxidant reaction. Herein we provide
mechanistic interpretations and biological implications for
this reaction.
prooxidant system was also elucidated by the clear inhibi-
tion in DNA strand breakage with different concentrations
of ROS scavengers (glutathione (GSH) and catalase (CAT))
and CuI chelator (bathocuproinedisulfonic acid disodium
salt (BCDS)), respectively (Figure 2C).
CuII and compound 5 induced synergistically cell cycle
arrest and apoptosis of HepG2 cells: We next employed
flow cytometry to assess the effect of the active compound 5
or/and CuII on cell cycle distribution of human hepatoma
HepG2 cells by staining with propidium iodide.[12] A trend
similar to DNA strand breakage was obtained. Compound 5
(80 mm) alone had no clear effect on the cell cycle distribu-
tion but resulted in a significant accumulation of cells in S
and G2/M phase in the presence of CuII (50 mm), a phenom-
enon that fits with the DNA damage (Figure 3). When the
Results and Discussion
CuII and compounds 1–5 induced synergistically DNA
damage: The DNA-cleaving ability of the three hybrids (3–
5) was firstly investigated together with that of the parent
molecules (1 and 2) by using agarose gel electrophoresis
analysis to measure the relaxation of supercoiled pBR322
plasmid DNA into the open circular (single-strand break-
age) and linear (double-strand breakage) forms.[11] No visi-
ble DNA strand breakage was found when either 250 mm
CuII or 100 mm compounds 1–5 alone was used (Figure 2A).
However, their cooperation induced synergistically DNA
strand breakage with an activity sequence of 5>4>3>2>
1 (Figure 2B), suggesting that this hybrid mode is an effi-
cient strategy in improving prooxidant activity of the parent
molecules. It is not surprising that compound 5 with a cate-
chol moiety is the most active, resulting in a complete con-
version from supercoiled DNA to the open circular and
linear forms, because it has the lowest oxidation potential
(the strongest electron-donating ability)[10] among the com-
pounds examined. Additionally, this catechol moiety is in
favor of a complex formation with the CuII ion as a bidentate
ligand, hence facilitating the intramolecular electron trans-
fer.[9] The role of ROS and CuII/CuI redox cycle in the 5/CuII
Figure 3. Synergistic induction of cell cycle arrest by 5 and CuII at the in-
dicated concentrations in HepG2 cells. After the cultured cells were
treated with CuII and/or 5 for 6 h, the cells were incubated in fresh
medium for 24 h, then harvested, and analyzed by flow cytometry. Each
experiment was performed in triplicate.
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An Interesting Acid/Base-Promoted Prooxidant Reaction
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apoptosis-inducing activity of this prooxidant system in
HepG2 cells was further characterized by assessing changes
in nuclear morphology by fluorescence microscopy with
Hoechst 33258 staining,[13] the synergistic action was again
observed (Figure 4) and CuII acted in concert with 5 to
induce a significant reduction in the number of living cells,
and caused fragmentation and condensation of chromatin,
a hallmark of apoptosis. Moreover, addition of N-acetylcys-
teine (NAC, 10 mm) almost completely reversed the apopto-
sis, clearly indicating the involvement of ROS (Figure 4).
(bottom-right inset in Figure 5A). The product was previ-
ously obtained by oxidizing 5 with tris(2,4,6-trichloro-3,5-di-
nitrophenyl) methyl radical.[10]
Figure 4. Synergistic induction of apoptosis by 5 and CuII, and the inhibi-
tion of NAC, at the indicated concentrations in HepG2 cells as assessed
by fluorescence microscopic analysis of cells stained with Hoechst 33258
after treatment with these compounds.
UV/Vis spectral changes of 5 in the presence of CuII in acid,
neutral and base solutions: Finally, we turned to a detailed
chemical mechanism study on this prooxidant system by as-
sessing the UV/Vis spectral changes of 5 in the presence of
CuII and the influence of ethylenediaminetetraacetic acid
(EDTA) and pH value on their interaction, as well as identi-
fying the related oxidative products. Addition of CuCl2 to 5
in phosphate buffered saline (PBS)/methanol (1:1, v/v,
pH 7.2) under aerobic conditions, induced the rapid disap-
pearance of the maximal absorption at 321 nm, whereas
a new band with a characteristic absorption at 324 nm and
a shoulder in the l range of 334–338 nm developed over
time (Figure 5A). They remained unchanged upon addition
of EDTA (data not shown), a well-known chelating agent
for metal ions; this suggests that this spectral change can be
assigned to the rapid formation of oxidation product, in-
stead of a chelate complex. This was also confirmed further
by identifying the only oxidation product as a benzofuran
derivative, 6 (Figure 1), with a completely identical absorp-
tion spectrum to that produced in the 5/CuII system
Figure 5. Absorption spectral changes of 5 (50 mm) in the presence (solid
lines) of CuII (100 mm) in PBS/MeOH buffer (1:1, v/v) at 258C in air. The
dotted lines show the absorption spectrum of 5 in the absence of CuII:
A) pH 7.20, 2 min intervals. Top-right inset: enlargement of absorbance
in the range l=240–360 nm, 30 s intervals; bottom-right inset: absorption
spectrum of the oxidation product,
6 in PBS/MeOH (1:1, v/v);
B) pH 5.89, 2 min intervals. Inset: enlargement of absorbance in the
range l=240–360 nm, 30 s intervals; C) pH 8.75, 8 min intervals;
D) pH 9.97, 8 min intervals. Inset: effect of EDTA (300 mm) on the ab-
sorption spectrum of the 5/CuII system at different times (from top to
bottom, 0, 1, 2, 4 and 6 h, respectively).
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When this reaction was conducted in acid medium
(pH 5.89), the same spectral changes with that in pH 7.2
were observed (Figure 5B). However, formation of 6 is
faster in acid medium than in neutral medium (insets of Fig-
ure 5A and B), that is, acid facilitates the reaction of 5 with
CuII. Noticeably, the acid-promoted result is completely op-
posite to that obtained previously in the case of caffeic acid
for which acid conditions remarkably inhibited its reaction
with CuII.[9a] In contrast, significantly different spectral evo-
lutions occurred in base medium (Figure 5C and D, pH 8.75
and 9.97, respectively). Specifically, at pH 8.75 (Figure 5C),
addition of CuII led to the prompt disappearance of the ab-
sorption band at 321 nm along with the appearance of a red-
shifted peak at 340 nm, ascribable to the formation of a com-
plex of 6 phenolate anion–CuII (see below). This redshifted
peak then decayed with time, whereas the absorption band
at about 411 nm increased due to the production of 6 o-qui-
none (see below). The presence of an isosbestic point at
371 nm supports a direct transition from one species (6
phenoxide–CuII chelate) to another (6 o-quinone). Compa-
ratively, decay of the redshifted peak was relatively fast at
pH 9.97 (Figure 5D). Upon addition of EDTA at different
time points, the redshifted peak at 340 nm returned to
324 nm (a characteristic absorption of 6) with a decrease in
absorbance (Figure 5D, inset). These outcomes indicate un-
ambiguously that the redshifted peak should be attributed
to formation of a complex of 6 phenolate anion–CuII, and
base promotes a further reaction of 6, a stable oxidative
product in acid and neutral medium, with CuII to form other
oxidative products including o-quinone. Furthermore, the
reaction of 5 with CuII to afford 6 was conducted in con-
trolled form, that is, in the absence CuII ions. As expected,
the spectrum of 5 alone did not change during the 2 h reac-
tion time under either acid (pH 5.89) or basic (pH 9.03; data
not shown) conditions, unambiguously confirming the pivo-
tal role of CuII ions.
Figure 6. Elucidation of the intermediate o-quinone formed from the re-
action of 5 or 6 with CuII in PBS/MeOH (1:1, v/v, pH 9.0) at 258C in air
based on the trapping of phenylmethanethiol or MBTH. A) Absorption
spectral comparison. 1: 5 (50 mm); 2: phenylmethanethiol (50 mm); 3:
products obtained from the overnight reaction of 5 (50 mm) with CuII
(100 mm); 4: products and phenylmethanethiol (50 mm). B) MBTH color
test. The concentrations of 6, MBTH and CuII were fixed at 1, 2 and
2 mm, respectively; 1: CuII +MBTH; 2: 6+MBTH; 3: 6+CuII; 4: 6+
CuII +MBTH. C) Absorption spectra of 6 (50 mm) and MBTH (100 mm)
before and after reaction with CuII (100 mm). 1: 6; 2: CuII +MBTH 2 h; 3:
6+MBTH 2 h; 4: 6+CuII 9 h; 5: 6+CuII 9 h+MBTH 1 h; 6: 6+CuII
9 h+MBTH 2 h.
Elucidation of the intermediate o-quinone: Note that all at-
tempts to isolate the o-quinone proved unsuccessful, due to
its instability and easy conversion to the pristine 6 under the
present experimental conditions. In view of o-quinone being
a Michael acceptor and vulnerable to attack by nucleophilic
reagents, we used phenylmethanethiol and 3-methyl-2-ben-
zothiazolinone hydrazone (MBTH)[14] to demonstrate its for-
mation based on UV/Vis spectral analysis and color test.
When the reaction of 5 (50 mm) with CuII (100 mm) was car-
ried out in PBS/MeOH (1:1, v/v, pH 9.0) solution, the initial-
ly colorless solution gradually became pale brown, which
then rapidly returned to colorless by addition of phenyl-
methanethiol (50 mm); this smoothened the absorption band
at about 411 nm (Figure 6A), supporting the formation of
phenylmethanethiol-o-quinone adducts. Similarly, introduc-
tion of MBTH also gave rise to the color change from black-
ish green to dark brown (Figure 6B) and the spectral
smooth at about 411 nm (Figure 6C) in the case of 6, due to
the formation of o-quinone derived from 6. Moreover, this
outcome is in accordance with the previous observation of
the absorbances of caffeic acid[15a,b] and 1,2-dehydro-N-ace-
tyldopamine[15c] o-quinones at 400 and 390 nm, respectively.
Mechanisms and biological implications: On the basis of the
above information, detailed prooxidant mechanisms for the
5/CuII system are proposed (Scheme 1). At neutral pH, the
formation of 6 can be easily rationalized by sequential
proton loss from the 4’-OH and electron transfer in the 5
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Scheme 1. Probable mechanisms for the reaction of 5 with CuII at neutral (A), base (A) and acid (B) pH. Base pH facilitates the deprotonation from 5
and 6 to promote the SPLET reaction and the formation of o-quinone as indicated by the dotted arrow, which does not occur in neutral medium. Under
acidic conditions the formation of the quinone methide radical cation leads to a relatively high efficiency in the following 5-exo-trig electrophilical cycli-
zation and production of 6 in comparison with the quinone methide radical produced in neutral pH.
phenolate anion–CuII complexes (sequential proton loss
electron transfer, SPLET),[16] to give a neutral radical, which
could be delocalized over the whole molecule by resonance
to obtain the quinone methide radical with a Michael ac-
ceptor moiety, followed by intramolecular nucleophilic
attack and the second CuII-mediated oxidation to afford 6
under the driving force of aromaticity restoration
(Scheme 1A). Alternatively, at acid pH, an intramolecular
electron transfer reaction could take place in the parent
molecule–CuII complexes due to the low oxidation potential
hand, basic pH facilitates the deprotonation from 5 and 6 to
promote the SPLET reaction and the further formation of
o-quinone (Scheme 1A), which is involved in DNA damage
and apoptosis induction.[17] Therefore, both acidic and basic
pH make the overall prooxidant reaction very efficient. Ad-
ditionally, CuI generated in the reaction of 5 with CuII, can
be oxidized back to CuII by oxygen, resulting in the forma-
tion of O2·À, and the subsequent production of H2O2 and
HO·. The CuII/CuI redox cycle and formation of ROS play
a pivotal role in inducing DNA damage, cell cycle arrest
and apoptosis of cancer cells.
of 5, giving rise to
a highly acidic radical cation
(Scheme 1B). This radical cation is in equilibrium with its
quinone methide radical cation, which is more favorable for
the electrophilic 5-exo-trig cyclization than the quinone
methide radical produced in neutral pH because of protona-
tion of the carbonyl group (Scheme 1B). Thus, acid pH ac-
celerates the production of 6 (Scheme 1B). On the other
Conclusion
In summary, CuII and 5, the stilbene–chroman hybrid with
a
catechol moiety, could synergistically induce DNA
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F. Dai, B. Zhou et al.
lution as
a
blank. In the case of ethylenediaminetetraacetic acid
damage, and cell cycle arrest and apoptosis of HepG2 cells.
The acid/base-promoted prooxidant mechanisms for the
system are proposed based on UV/Vis spectral changes and
identification of the related oxidative intermediates and
products. This work gives us useful information for design-
ing a prooxidant system constructed by polyphenolic com-
pounds and CuII ions, and for understanding the chemical
processes leading to ROS formation, and development of
cancer treatment with the ROS-generating strategy.
(EDTA), spectra were run against blanks containing the PBS/MeOH (v/
v, 1:1) solution and CuII.
Oxidative product analysis of compound 5 in the presence of CuII: Com-
pound 5 (1 equiv) and CuII (2 equiv) were mixed in the PBS/MeOH (1:1,
v/v, pH 7.4) solution, which was stirred at 258C for 4 h until completion
of the reaction as monitored by thin layer chromatography (TLC).
EDTA (6 equiv) was then added to the solution and MeOH was removed
in vacuo. The residue was dissolved in ethyl acetate and washed with dis-
tilled water. Then the organic layer was collected and dried overnight,
with anhydrous Na2SO4, followed by evaporation in vacuo. Purification
by column chromatography (petroleum ether/ethyl acetate) gave the oxi-
dation product 6 with the yield of 95.9%. The product was previously ob-
tained by oxidizing 5 with tris(2,4,6-trichloro-3,5-dinitrophenyl) methyl
radical,[10] and for its characterization (1H and 13C NMR and HRMS
data), please see the Supporting Information in our previous work.[10]
Experimental Section
Materials: Ethidium bromide, catalase (CAT, 3000 U per mg protein), re-
duced glutathione (GSH), pBR322 DNA, N-acetylcysteine (NAC), propi-
dium iodide (PI), ribonuclease A (RNaseA), Hoechst 33258, phenylmeth-
anethiol and 3-methyl-2-benzothiazolinone hydrazone (MBTH) were
purchased from Sigma–Aldrich and were used as received. Antifade
mounting medium and bathocuproinedisulfonic acid disodium salt
(BCDS) were from Beyotime Institute Biotechnology and Fluka, respec-
tively. CuCl2·2H2O was of analytical grade. All other chemicals were of
the highest quality available. Compounds 1–5 were prepared in our previ-
ous study,[10] and their purity was >97% by HPLC.
Elucidation of the intermediate o-quinone by trapping experiments
Phenylmethanethiol assay: A solution of PBS/MeOH (1:1, v/v, pH 9.0)
containing 5 (50 mm) and CuII (100 mm) was incubated, overnight, at 258C
in air, and then the spectrum tracing was started by addition of phenyl-
methanethiol (50 mm).
MBTH assays
1) Color test: The concentrations of 6, MBTH and CuII used in the ex-
periment were 1, 2 and 2 mm, respectively. The color change from black-
ish green to dark brown appeared immediately based on their mixing in
PBS/MeOH (1:1, v/v, pH 9.0) solution. MBTH solution was freshly pre-
pared before use.
Assay for oxidative DNA strand breakage: DNA cleavage was deter-
mined following the procedure described previously.[9a] Samples of
pBR322 plasmid DNA (100 ng) were prepared in a final volume of 25 mL
in microcentrifuge tubes (0.5 mL) including DNA (5 mL), compound
(5 mL), CuII (5 mL), and PBS (10 mL). Inhibition reactions were carried
out by prior incubation of CAT, GSH or BCDS before addition of CuII.
2) Spectral analysis: Compound
6
(50 mm) was incubated with CuII
(100 mm) in PBS/MeOH (1:1, v/v, pH 9.00) at 258C for 9 h, the corre-
sponding spectra were then recorded upon addition of MBTH (100 mm).
Cell culture: Human hepatoma (HepG2) cells obtained from the Shang-
hai Institute of Biochemistry and Cell Biology, Chinese Academy of Sci-
ences, were maintained in RPMI 1640 supplemented with heat-inactivat-
ed fetal bovine serum (10%, v/v), penicillin (100 kULÀ1), streptomycin
(100 kULÀ1), and glutamine (2 mm), and incubated at 378C in a humidi-
fied atmosphere of 95% air and 5% CO2. Exponentially growing cells
were used throughout these experiments. Compound 5 was dissolved in
DMSO before the experiments and the volume of DMSO solution added
to the cell suspension was less than 0.1% (v/v) of the culture medium.
Acknowledgements
Special thanks to the three anonymous referees for their very helpful and
constructive comments and suggestions on improving the manuscript.
This work was supported by the National Natural Science Foundation of
China (Grant No.: 20972063), the 111 Project, Program for New Century
Excellent Talents in University (NCET-06–0906) and the Fundamental
Research Funds for the Central Universities.
Cell cycle analysis by using flow cytometry: HepG2 cells were plated at
a density of 2ꢁ105 cells per well in six-well plates and incubated for 24 h.
After treatment with compound 5 (80 mm) and/or CuII (50 mm) for 6 h, the
cells were incubated in fresh medium for 24 h. Then the cells were har-
vested, washed with PBS twice, and fixed with ice-cold ethanol (75%) at
48C, overnight. The fixed cells were resuspended in PBS containing
DNase-free RNaseA (200 mgmLÀ1) and PI (50 mgmLÀ1) and incubated at
378C for 30 min in the dark. Fluorescence-activated cell sorting was car-
ried out by using a FACSCanto flow cytometer (Becton–Dickinson, San
Jose, CA, USA) and the FACSDiva software program. Cell cycle distri-
butions were analyzed by using Modfit LT 3.0 software. Ten thousand
events were collected per sample.
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Analysis of cell apoptosis: Fragmentation and condensation of chromatin
are the key features of apoptosis and can be detected by staining with
Hoechst 33258. HepG2 cells at a density of 2ꢁ105 cells per well were cul-
tured on coverslips, which were incubated in six-well plates for 24 h.
After 6 h treatment with compound 5 (80 mm) and/or CuII (50 mm), the
cells were incubated in fresh medium for 24 h, and then Hoechst 33258
staining was performed according to the manufacturerꢂs instructions (Be-
yotime Institute Biotechnology, China). In the case of inhibition, NAC (5
or 10 mm) was incubated for 1 h before addition of compound 5 and CuII.
UV/Vis spectral measurements: After mixing PBS (10 mm, pH 7.4) solu-
tion with an equal volume of methanol, concentrated HCl or NaOH was
used to adjust the pH of the system to the appropriate values. The oxida-
tion of 5 (50 mm) by CuII (100 mm) in PBS/MeOH (v/v, 1:1) at 258C was
followed with a Cary 300 spectrophotometer. Spectra were taken every
appointed time after addition of CuII by using a PBS/MeOH (v/v, 1:1) so-
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An Interesting Acid/Base-Promoted Prooxidant Reaction
FULL PAPER
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Received: May 3, 2012
Published online: && &&, 0000
Chem. Eur. J. 2012, 00, 0 – 0
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These are not the final page numbers!

F. Dai, B. Zhou et al.
A view to a kill: This work demon-
strates that CuII and the stilbene–chro-
man hybrid with a catechol moiety
could synergistically induce DNA
damage, and cell cycle arrest and
apoptosis of HepG2 cells (see illustra-
tion). The acid/base-promoted prooxi-
dant mechanism for the reaction of the
hybrid with CuII is proposed based on
UV/Vis spectral changes and identifi-
cation of the related oxidative inter-
mediates and products.
Antitumor Agents
G.-Y. Liu, J. Yang, F. Dai,* W.-J. Yan,
Q. Wang, X.-Z. Li, D.-J. Ding,
X.-Y. Cao, B. Zhou* ......... &&&& — &&&&
CuII Ions and the Stilbene–Chroman
Hybrid with a Catechol Moiety
Synergistically Induced DNA Damage,
and Cell Cycle Arrest and Apoptosis
of HepG2 Cells: An Interesting Acid/
Base-Promoted Prooxidant Reaction
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www.chemeurj.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 0000, 00, 0 – 0
ÝÝ
These are not the final page numbers!