Ruthenium-porphyrin complexes induce apoptosis by inhibiting the generation of intracellular reactive oxygen species in the human hepatoma cell line (HepG2)

Article abstract of DOI:10.1002/ejic.201000968

Two ruthenium(II)-porphyrin complexes, [(3-Py)Ru(phen)₂(tmopp)] [1; phen = phenanthroline, tmopp = 5,10,15,20-tetrakis(4-methoxyphenyl) porphyrin] and [(4-Py)Ru(phen)₂(tmopp)] (2), have been synthesized and characterized for the first time. It was found that the two ruthenium(II)-porphyrin complexes show significant antitumor activity in HepG₂ cells. Flow cytometric analysis showed that complex 1 arrested the cell cycle in the G0/G1 phase and induced apoptosis in HepG₂ cells. Fluorescence microscopy and flow cytometric analyses demonstrated that the generation of intracellular reactive oxygen species (ROS) was significantly inhibited in cells treated with either complex. The total antioxidant capacity of the complexes was detected by a 2,2′-azinobis(3-ethylbenzthiazoline-6- sulfonic acid) (ABTS) assay; this showed that both complexes are good free-radical scavengers. Ruthenium(II)-porphyrin complex 1 also was found to scavenge hydroxy radicals, as measured by the Fenton system. These data demonstrate that ruthenium(II)-porphyrin complexes exhibit antioxidant properties, probably through the involvement of a direct scavenging effect on a hydroxy radical. Taken together, the findings show that ruthenium(II)-porphyrin complexes induce apoptosis in HepG₂ cells by inhibiting the generation of ROS and are potential anticancer therapeutic agents.

Full text of DOI:10.1002/ejic.201000968

FULL PAPER
DOI: 10.1002/ejic.201000968
Ruthenium–Porphyrin Complexes Induce Apoptosis by Inhibiting the
Generation of Intracellular Reactive Oxygen Species in the Human Hepatoma
Cell Line (HepG2)
Yanan Liu,[a,b] Xiaonian Zhang,[a] Rong Zhang,[a] Tianfeng Chen,[a] Yum-Shing Wong,[b]
Jie Liu,*[a] and Wen-Jie Zheng*[a]
Keywords: Ruthenium / Antitumor agents / Apoptosis / Antioxidants / Radical scavengers
Two ruthenium(II)–porphyrin complexes, [(3-Py)Ru(phen)2-
(tmopp)] [1; phen = phenanthroline, tmopp = 5,10,15,20-tet-
rakis(4-methoxyphenyl)porphyrin] and [(4-Py)Ru(phen)2-
(tmopp)] (2), have been synthesized and characterized for the
first time. It was found that the two ruthenium(II)–porphyrin
complexes show significant antitumor activity in HepG2 cells.
Flow cytometric analysis showed that complex 1 arrested the
cell cycle in the G0/G1 phase and induced apoptosis in
HepG2 cells. Fluorescence microscopy and flow cytometric
analyses demonstrated that the generation of intracellular re-
active oxygen species (ROS) was significantly inhibited in
cells treated with either complex. The total antioxidant ca-
pacity of the complexes was detected by a 2,2Ј-azinobis(3-
ethylbenzthiazoline-6-sulfonic acid) (ABTS) assay; this
showed that both complexes are good free-radical scaven-
gers. Ruthenium(II)–porphyrin complex 1 also was found to
scavenge hydroxy radicals, as measured by the Fenton sys-
tem. These data demonstrate that ruthenium(II)–porphyrin
complexes exhibit antioxidant properties, probably through
the involvement of a direct scavenging effect on a hydroxy
radical. Taken together, the findings show that rutheni-
um(II)–porphyrin complexes induce apoptosis in HepG2 cells
by inhibiting the generation of ROS and are potential anti-
cancer therapeutic agents.
tumor tissues. Porphyrins play a significant role in the diag-
nosis and treatment of cancer.[16] Over the last decade, ru-
thenium–porphyrin complexes have attracted much atten-
tion due to their potential application as anticancer
drugs.[17] However, the molecular mechanisms of the
apoptosis-inducing action of ruthenium porphyrin com-
plexes remain to be elucidated.
In recent years, numerous studies showed that free radi-
cals, especially reactive oxygen species (ROS), are important
in the regulation of apoptosis.[18] Changes in the level of
ROS that precede morphological changes have been shown
to be an important way to induce apoptosis.[19] A great
number of studies have reported that, in the process of cell
apoptosis, ROS can also participate in cell-signal transduc-
tion, start the expression of some genes of apoptosis, and
have complicated correlations with a variety of biological
molecules, including those that belong to the B-cell lym-
phoma 2 (bcl-2) family, caspases, and mitochondrial perme-
ability transition (PT) pore complex.[20] Many ruthenium
complexes have been reported that exhibit both antioxidant
activity and antitumor activity.[21] Therefore, studies on the
relationship between the antitumor activity of drugs and
the level of ROS have vital significance.
Introduction
Since cisplatin was developed as an anticancer drug, the
synthesis and antitumor activity of inorganic metal com-
plexes have received widespread attention. The purpose is
to find drugs that have lower toxicity but more activity than
cisplatin.[1–3] Investigations into new anticancer drugs have
highlighted ruthenium as a potential metal center for new
therapies.[4–6] Ruthenium possesses several favorable proper-
ties: it exhibits cytotoxicity against cancer cells, has similar
ligand-exchange abilities as platinum complexes, has no
cross-resistance with cisplatin, is easily absorbed and
rapidly excreted by the body, and may have reduced toxicity
against healthy tissues due to transferrin transport.[7–11] Ru
complexes have previously been shown to be among the
most promising anticancer drugs.[12,13] A number of Ru
complexes have displayed promising anticancer ac-
tivity.[14,15] Porphyrins and their derivatives are widely used
in modern medicine as contrast agents for cancer diagnos-
tics and as sensitizers in photodynamic therapy due to their
spectral characteristics, phototoxicity, and high affinity for
[a] Department of Chemistry, Jinan University,
Guangzhou 510632, China
At present, studies on the mechanisms of action of ruthe-
nium complexes have been mainly focused on its targets,
such as DNA and protein, whereas its antioxidant activity
has been rarely reported.[22] Our current investigations in
this paper attempt to elucidate the molecular mechanism
Fax: +86-20-8522-1263
E-mail: tliuliu@jnu.edu.cn
[b] Department of Biology, The Chinese University of Hong Kong,
Hong Kong, China
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejic.201000968.
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Apoptosis Induced by Ruthenium–Porphyrin Complexes
that underlies such activity by ruthenium–porphyrin com-
plexes. We have examined the relationship between the anti-
tumor and antioxidant properties of ruthenium complexes
in the HepG2 tumor model. In addition, we assessed the
scavenging capability of free radicals of the two ruthenium
complexes by using in vitro molecular techniques. Ruthe-
nium–porphyrin complexes exhibit antioxidant activity and
might be responsible for complex-induced apoptosis.
Results and Discussion
Figure 1. Morphology of HepG2 cells upon incubation with 5, 10,
20, and 40 μm complexes 1 and 2.
Synthesis and Characterization
The ruthenium–porphyrin complexes were readily syn-
thesized in excellent purity by following the procedures re-
ported in the Exp. Section. Complexes 1 and 2 were charac-
terized by elemental analyses, UV/Vis spectroscopy, and
ESI-MS. The bonding arrangement was further confirmed
by 1H NMR spectra. The molecular structures of the ruthe-
nium–porphyrin complexes are shown in Scheme 1.
To further observe and study the antitumor activity of
these ruthenium–porphyrin complexes, cell viability was de-
termined by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-
razolium bromide (MTT) assay. As displayed in Figure 2,
increasing complex concentrations decrease the HepG2 cell
viability in a dose-dependent manner. Furthermore, com-
plex 1 appears to be more effective in inhibiting HepG2 cell
growth.
Figure 2. Cell viability after treatment with different concentrations
of the complexes for 48 h as examined by the MTT assay. The
significance of the differences between the mean values were deter-
mined by using a student’s t test. Results were considered signifi-
cant at p Ͻ 0.05, which are marked by *.
The IC50 values, calculated from the dose-survival curves
obtained after 48 h of drug treatment with the MTT assay,
are shown in Table 1. The IC50 values for complex 1 and 2
are (18.7Ϯ1.3) and (23.6Ϯ3.9) μgmL–1, respectively, thus
confirming that complex 1 is more active than complex 2
toward HepG2 cells.
Scheme 1. [(3-Py)Ru(phen)2(tmopp)] (1) and [(4-Py)Ru(phen)2-
(tmopp)] (2) [phen = phenanthroline; tmopp = 5,10,15,20-tetra-
kis(4-methoxyphenyl)porphyrin].
Table 1. Growth inhibition of ruthenium–porphyrin complexes on
HepG2 cells.[a]
Blocking the Growth of HepG2 Cells
Complex
1
2
IC50 [μgmL–1]
18.7Ϯ1.3
23.6Ϯ3.9
Cell death is characterized by obvious morphological
characteristics. HepG2 cells treated by ruthenium complexes
with different concentrations showed clear morphological
change and quantity decrease, as shown in Figure 1. The
morphological change of the cells is positively correlated
with dosage, and significant apoptotic bodies were observed
with higher complex concentration. These phenomena sug-
gest that ruthenium–porphyrin complexes block the growth
of HepG2 cells.
[a] Cells were treated with various concentrations of tested com-
plexes for 48 h.
G0/G1-Phase Arrest and Apoptosis of HepG2 Cells
In general, inhibition of cancer-cell proliferation by cyto-
toxic drugs could be the result of induction of apoptosis or
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of cell cycle arrest, or a combination of these two cein diacetate (DCFH-DA), which is subsequently hy-
modes.[20,23] According to the results of the MTT assay drolyzed by intracellular esterase to form dichlorofluores-
(Table 1), complex 1 is more active than complex 2 in in- cein (DCFH). The nonfluorescent substrate is oxidized by
hibiting HepG2 cell growth. Thus, this complex was used intracellular free radicals to produce a fluorescent product
for further investigation of the underlying mechanisms. DCF. The level of ROS production is linearly proportional
Flow cytometric analysis was carried out to determine the to the fluorescence intensity of DCF.[24] As shown in Fig-
possible mechanisms of cell-growth inhibition. Figure 3 ure 4, ROS generation in HepG2 cells treated with complex
shows the representative DNA distribution histograms of 1 decreased with increasing complex concentration as indi-
HepG2 cells incubated in the absence or presence of 10 and cated by decreasing DCF intensity. Therefore, ruthenium–
20 μgmL–1 complex 1 for 72 h. Exposure of HepG2 cells to porphyrin complexes reduced the level of intracellular ROS.
complex 1 led to a marked dose-dependent increase in the
We obtained similar results with a DCFH-DA flow cyto-
proportion of apoptotic cells, as reflected by the subdiploid metric assay (Figure 5). When cells were exposed to dif-
peaks. Moreover, treatment with complex 1 caused a dose- ferent concentrations of complex 1 or 2 for 15, 30, and
dependent increase in the percentage of cells at the G0/G1 60 min, we observed a marked dose-dependent decrease in
phase, accompanied by a corresponding reduction in the the level of intracellular ROS. The results showed that both
percentage of cells at G2/M phase and S phase, thereby in- complexes inhibit the level of ROS generation to different
dicating the induction of G0/G1-phase arrest by complex 1. extents, thereby suggesting that ruthenium complexes may
have antioxidant activity in vivo.
Figure 3. Effects of complex 1 on apoptosis and cell-cycle distribu-
tion in HepG2 cells. The cells were treated for 72 h, collected, and
stained with propidium iodide after fixation as described in the
Exp. Section. These values were from a representative result of
three independent experiments.
Intracellular ROS Levels
ROS, as signal molecules and effector molecules in
apoptosis, have been shown to play an important role in
apoptosis induced by a variety of factors. We next investi-
gated whether the cell death induced by ruthenium–por-
phyrin complexes is dependent on ROS levels.
We treated HepG2 cells with complex 1 and measured
their level of intracellular ROS by using 2Ј,7Ј-dichlorofluo-
Figure 5. Intracellular ROS generation in HepG2 cells treated with
rescin (DCF) fluorescence. This assay is based on the cellu-
complexes 1 and 2. Cells were treated with different concentrations
lar uptake of a nonfluorescent probe, 2Ј,7Ј-dichlorofluores-
of the complexes for 15, 30, and 60 min. The significance of the
differences between the mean values were determined by using a
student’s t test. Results were considered significant at p Ͻ 0.05,
which are marked by *.
Clean Up of the Oxygen Free Radicals in In Vitro Tests
The 2,2Ј-azinobis(3-ethylbenzthiazoline-6-sulfonic acid)
(ABTS) radical cation decolorization assay, established by
Miller in 1996,[25] has been widely used to determine the
total antioxidant capacity of biological samples. We used
this assay to study the radical scavenging activity of the
ruthenium–porphyrin complexes. This method is based on
the ability of antioxidant molecules to quench the stable
radical cation ABTS·+, a blue-green chromophor with char-
Figure 4. DCF fluorescence images of HepG2 cells treated with
complex 1. Cells were treated with various concentrations of tested
complex for 30 min.
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Apoptosis Induced by Ruthenium–Porphyrin Complexes
acteristic absorption at 734 nm. In this system, greater tor for different antioxidants.[25] To understand the charac-
bleaching of the solution indicates stronger total antioxi- teristics of the ABTS system reaction kinetics used in this
dant capacity of the detected material.
experiment, different concentrations of ruthenium–por-
phyrin complexes were added to the ABTS radical solution
and the absorbance was detected with equal time intervals.
The results are shown in Figure 7. We observed that, after
adding the two complexes (100–1600 μm) to the ABTS radi-
cal solution, both complexes produced a significant de-
crease of characteristic absorption peak at 734 nm. How-
ever, the characteristic absorption peak at 734 nm of com-
plex 1 was reduced to a greater extent than that of complex
2 under the same concentration and time conditions. There-
fore, complex 1 shows stronger free-radical scavenging abil-
ity and antioxidant activity.
Determining the Wavelength of the ABTS Reaction System
In this study, the UV/Vis absorption spectra of ABTS
and ruthenium–porphyrin complexes were measured,
respectively. The results show that the radical cation
(ABTS·+) has a characteristic absorption spectrum with
maxima at 415 and 734 nm, and the ruthenium–porphyrin
complex has a strong absorption peak in the 400–500 nm
range but no absorption peak at 734 nm. Therefore, to pre-
vent interference caused by the absorption peaks of the ru-
thenium–porphyrin complex, we used 734 nm as the detec-
tion wavelength for the ABTS free-radical scavenging assay.
UV/Vis Absorption Spectrum Titration Experiments
The UV/Vis absorption spectrum was determined upon
addition of complex 1 and 2 to the ABTS radical solution.
We observed dose-dependent decreases in the absorption
peak at 734 nm (Figure 6), thereby suggesting that both ru-
thenium–porphyrin complexes can clear ABTS free radi-
cals. This can explain why ruthenium–porphyrin complexes
have antioxidant activity. Again, there is evidence that com-
plex 1 has a stronger free-radical scavenging ability, and
hence stronger antioxidant activity because the ABTS ab-
sorption peak of complex 1 was reduced more significantly
when used at the same concentration as for complex 2.
Figure 7. Changes in absorbance of ABTS after adding different
concentrations of complexes 1 and 2 for different times.
Effect of Ruthenium Complex 1 on Hydroxy Radicals
Hydroxy radicals (OH·) were generated in a Fenton-type
system. The ability of ruthenium complex 1 to scavenge this
free radical was tested. Figure 8 depicts the inhibitory effect
Figure 6. Changes in absorbance spectra of ABTS with addition of
complexes 1 and 2.
The Reaction Kinetics of the ABTS System
In the ABTS free-radical scavenging experiment, the re-
sponse time of the reaction system is a key influencing fac- periments were performed in triplicate.
Figure 8. Scavenging effect of complex 1 on hydroxy radicals. Ex-
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J. Liu, W.-J. Zheng et al.
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of ruthenium complex 1 on OH·; the average suppression
ratios for OH· increase with the increasing concentration of
ruthenium complex 1 in the range of 1–16 μm. The suppres-
sion ratio against OH· ranged from 20.69 to 95.40%. Due
to the lower IC50 (3.04 μm) value, ruthenium complex 1 may
be a potential free-radical scavenger.
uct dissolved in chloroform was purified by filtration through silica
gel (60–100 mesh). The second purple band was collected. Then,
by evaporation of the solution, purple powder was obtained, and
it was dried in vacuo; yield 0.722 g (5.3%).
(4-Py)tmopp: This ligand was synthesized in an identical manner
to that described for (3-Py)TMOPP with 4-pyridinecarbaldehyde
in place of 3-pyridinecarbaldehyde; yield 0.871 g (6.4%).
cis-[Ru(phen)2Cl2]·2H2O: [Ru(phen)2Cl2]·2H2O was prepared fol-
lowing the literature and was determined to be the identical com-
pound.[27] A mixture of RuCl3·nH2O (6 mmol), phenanthroline
(12 mmol), and lithium chloride (28 mmol) in DMF (10 mL) was
heated to reflux under argon for 8 h. The cooled, mixed solution
was diluted with acetone (50 mL) and frozen for 24 h. The mixture
was filtered. The precipitates were washed with ice water and ace-
tone several times and dried in vacuo. A purple-black microcrystal-
line was obtained; yield 1.66 g (49%) (calculated from phen-
anthroline).
Conclusion
The complexes [(3-Py)Ru(phen)2(tmopp)] (1) and
[(4-Py)Ru(phen)2(tmopp)] (2) were synthesized and charac-
terized. In vitro experiments show that both complexes have
growth-inhibitory effects on HepG2 cells, with complex 1
being more highly effective at inducing G0/G1 cell-cycle ar-
rest and apoptotic cell death in HepG2 cells. The level of
intracellular ROS decreased significantly after the treatment
with both complexes. An ABTS assay revealed that both
complexes are able to scavenge free oxygen radicals. Com-
plex 1 was also found to be able to scavenge hydroxy radi-
cals. From these data we conclude that ruthenium–por-
phyrin complexes may have antioxidant activity. One pos-
sible and important mechanism is the reduction of the level
of intracellular ROS by scavenging hydroxy radicals, and
thus inducing HepG2 cell apoptosis.
[(3-Py)Ru(phen)2(tmopp)] (1):
A
mixture of (3-Py)tmopp
(0.36 mmol) and cis-[Ru(phen)2Cl2]·2H2O (0.3 mmol) in acetic acid
(20 mL) was heated to reflux under argon for 3 h. Then, by evapo-
ration of the solution, the crude product was obtained. The crude
product was dissolved in chloroform and purified by filtration
through silica gel (60–100 mesh) with chloroform and methanol
(2:1 v/v) as eluant. The band that was predominantly purple was
collected. The solvent was removed under reduced pressure and a
purple-black powder was obtained; yield 0.086 g (20.9%). Elemen-
tal analyses were performed with a Perkin–Elmer LS55 Elemental
Analyzer. 1H NMR: δ = 8.83 (d, 2 H), 8.73 (d, 2 H), 8.61 (d, 4 H),
8.32 (s, 4 H), 8.15 (d, 2 H), 7.88–7.94 (m, 2 H), 7.52 (d, 2 H), 7.21–
7.34 (m, 12 H), 7.12 (d, 2 H), 4.02 (d, 8 H), 3.31 (s, 9 H) ppm.
[C70H51N9O3ClRu]Cl (1202.28): calcd. C 69.90, H 4.27, N 10.48;
found C 70.03, H 4.42, N 10.73. UV/Vis (CH3CN): λ (10–4 ε,
Experimental Section
General Conditions: RuCl3·nH2O (AR) was obtained from the
Kunming Institute of Precious Metals. Tris(hydroxymethyl)amino-
methane (Tris) and 2,2Ј-azinobis-3-ethylbenzothiazolin-6-sulfonic
acid (ABTS) were purchased from the Sigma Company. Tris/HCl
buffer (50 mm, pH = 7.4) was prepared by physiological saline.
Stock solutions (0.4 mm) of the ruthenium–porphyrin complexes
were prepared in the appropriate culture medium with 10% fetal
bovine serum and diluted by culture medium to various working
concentrations, which were obtained by serial dilutions. All other
chemicals and solvents were reagent grade, purchased commercially
and used without further purification unless stated otherwise.
m
–1 cm–1) = 654.0 (0.2), 594.0 (0.5), 560.5 (0.9), 523.0 (1.4), 410.5
(12.4) and 267.5 (2.6) nm. ESI-MS: m/z: 1202 [M]+.
[(4-Py)Ru(phen)2(tmopp)] (2): This complex was synthesized in an
identical manner to that described for 1 with (4-Py)tmopp in place
of (3-Py)tmopp; yield 0.065 g (14.2%). 1H NMR: δ = 8.88–8.73 (d,
6 H), 8.58 (d, 2 H), 8.31 (s, 4 H), 8.19 (d, 2 H), 8.05 (d, 6 H), 7.71–
7.83 (m, 2 H), 7.52–7.75 (m, 10 H), 7.48 (d, 2 H), 7.24 (d, 2 H),
7.15 (d, 2 H), 3.35 (s, 9 H) ppm. [C70H51N9O3ClRu]Cl (1202.28):
calcd. C 69.90, H 4.27, N 10.48; found C 70.11, H 4.59, N 11.00.
UV/Vis (CH3CN): λ (10–4 ε, m–1 cm–1)=656.0 (0.3), 598.0 (0.6),
567.0 (1.0), 523.5 (1.3), 432.5 (14.6) and 268.5 (3.5) nm. ESI-MS:
m/z: 1202 [M]+.
Cell Culture: The HepG2 human hepatoma cell line was obtained
from the American Type Culture Collection (ATCC, Manassas,
VA) and was cultured in RPMI 1640 medium that contained 10%
fetal bovine serum, 100 unitsmL–1 penicillin, and 50 unitsmL–1
streptomycin at 37 °C in a humidified incubator under 5% CO2
atmosphere. Cells from a confluent monolayer were removed from
flasks by 0.25% trypsin/ethylenediaminetetraacetic acid (EDTA)
solution. Cell viability was determined by the trypan blue dye ex-
clusion test.
Cell Viability Assay: The cytotoxicity of ruthenium–porphyrin
complexes was determined by the MTT assay, a colorimetric assay
based on the ability of the viable cells to reduce a soluble yellow
tetrazolium salt to blue formazan crystals.[28,29] Cells were removed
from flasks by trypsin solution and their viability determined by
the trypan blue exclusion test. They were seeded into 96-well plates
at a density of 2ϫ104 cells per mL and left to grow overnight. The
cells were treated by adding a solution (100 μl) that contained the
test complexes, with each reaction in triplicate. In each instance,
cells were left to grow for 48 h before treatment with MTT (20 μl,
Synthesis and Characterization
(3-Py)tmopp: (3-Py)tmopp was synthesized according to the litera-
ture[26] Briefly: A mixed solution that contained propionic acid
(30 mL), 3-pyridinecarbaldehyde (0.019 mol), and pyrrole 5 mgmL–1) reagent for 5 h (Sigma, St. Louis, MO). After this time,
(0.075 mol) was added dropwise into propionic acid solution
(120 mL) that contained p-methoxybenzaldehyde (0.056 mol) with
stirring and was heated to reflux at 120 °C for 40 min. The cooled,
mixed solution was diluted with ethanol (100 mL) and frozen for
24 h. The mixture was filtered and the purple precipitates were
washed with ethanol several times and then dried. The crude prod-
the medium was aspirated and replaced with DMSO (150 μl) per
well to dissolve the formazan salt. The color intensity of the
formazan solution, which reflects the cell-growth conditions, was
measured at 570 nm with an ELX800 type Microplate Reader (Bio-
Tek, USA). The percentages of surviving cells relative to untreated
controls were determined. The IC50 values, defined as the drug con-
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Apoptosis Induced by Ruthenium–Porphyrin Complexes
centration that inhibits cell growth by 50%, were estimated from
semilogarithmic dose–response plots.[30]
Acknowledgments
Cell-Cycle Analysis and Apoptosis Assays: Flow cytometric analysis
was carried out according to our previous method. Briefly, one
million cells, as determined by trypan blue exclusion test, were
washed in cold phosphate buffer solution (PBS) and fixed in 70%
ethanol at –20 °C overnight. The fixed cells were washed twice with
PBS, re-suspended in PBS (1 mL) that contained RNase A
(0.25 mg), EDTA (2 mm), and propidium iodide (0.1 mg). This was
incubated at 37 °C for 30 min, and cells were analyzed. The fluores-
cence of 10000 cells was measured with a Beckman Coulter Epics
XL-MCL flow cytometer (Miami, FL). The cell-cycle distribution
was analyzed by MultiCycle software (Phoenix Flow Systems, San
Diego, CA). The proportions of cells in the G0/G1, S, and G2/M
phases were represented as DNA histograms. Apoptotic cells were
measured by quantifying the sub-G1 peak.
This work was supported by the National Natural Science Founda-
tion of China (NSFC) (grant number 20871056), the Natural Sci-
ence Foundation of Guangdong Province (grant number
8251063201000008), the Planned Item of Science and Technology
of Guangdong Province (C1011220800060), the 211 Project Grant
of Jinan University, and the Fundamental Research Funds for the
Central Universities.
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ABTS Assay: The scavenging capability of free radicals of the two
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Received: September 10, 2010
Published Online: March 16, 2011
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