全 文 ：Chinese-German J Clin Oncol August 2013, Vol. 12, No. 8, P389–P392
DOI 10.1007/s10330-013-1204-0
Onychin (5-hydroxy-7-rhamnosylglucosyl flavone,
ONY) is one of dihydro-glycosides extracted from On-
ychium lucidum of Onychium Ching of Sinopteridaceae,
and it was reported that Onychin has the effects of anti-
tumor [1], oxidation resistance, and protecting endothelial
cells [2], but little reported was about the possible mecha-
nisms of Onychin. In this present study, experiments
were designed to investigate the effects of Onychin on
cells inhibition of proliferation, induction of apoptosis
and its related possible biological mechanism in HO-8910
cells.
Materials and methods
Materials
ONY was extracted, isolated, and purified in Tumor
Research Institute of University of South China. Human
ovarian cancer HO-8910 cells were purchased from the
China Center for Type Culture Collection. RPMI-1640
medium and fetal calf serum (FCS) were obtained from
Gibco Company (USA), and DMSO was purchased Com-
pany Amresco, USA. The 3-(4,5-dimethythiazol-2-yl)-
2,5-diphenylterazoliumbromide (MTT) was bought from
Sigma Company, USA. DTX was purchased from Hengrui
Medicine Co., Ltd, China. Mouse anti-bcl-2 monoclonal
antibody, rabbi anti-Bax monoclonal antibody, mouse
anti-cytochrome-c monoclonal antibody, mouse anti-
caspase-9 polyclonal antibody, and mouse anti-caspase-3
polyclonal antibody were obtained from Beyotime Com-
pany, China.
Cell culture
Human ovarian cancer HO-8910 cells were maintained
in RPMI-1640 medium supplemented with 10% fetal calf
serum at 37 ℃ in a humidified atmosphere of 5% CO2.
Exponentially growing cells were used in experiments [3].
Cell growth inhibition analysis
The effect of different concentrations of ONY (5, 10,
and 15 μg/mL) on the growth ability of HO-8910 cells
was determined by using MTT assay. The 2.5 × 104 cells/
mL exponentially growing cells were plated into 96-well
flat-bottom micro-plates, and treated with DTX 0.2 μg/
mL, ONY 5 μg/mL, ONY 10 μg/mL, ONY 15 μg/mL and
NS, respectively. The total incubational volume of each
well was 200 μL. After incubation for 24, 48, and 72 h,
Mechanism of induction apoptosis of Onychin in
ovarian cells in vitro
Yingxia Ning1, Jun Bai2

1 Department of Gynecology, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou 510120,
China
2 Department of Gynecology and Obstetrics, Hangzhou Red Cross Hospital, Hangzhou 310003, China
Received: 28 May 2013 / Revised: 19 June 2013 / Accepted: 15 July 2013
© Huazhong University of Science and Technology and Springer-Verlag Berlin Heidelberg 2013
Abstract Objective: The aim of the study was to investigate the possible mechanism of induction apoptosis of Onychin
(ONY) in ovarian cancer HO-8910 cells in vitro. Methods: Human ovarian cancer HO-8910 cells were cultured in vitro. Inhibi-
tory effect of ONY on the viability of HO-8910 cells was evaluated by the MTT assay. Apoptosis of HO-8910 cells treated with
different concentrations of ONY for 48 h was detected by FCM. Expression of proteins related to apoptosis was analyzed by
Western blot. Results: ONY significantly inhibited the viability of human ovarian cancer HO-8910 cells in a dose-dependent
and time-dependent manner, and the IC50 was 10.48 μg/mL for 48 h. The cells treated with ONY showed typical morphological
change of apoptosis and increased cells of sub-G1 population by FCM in a dose-dependent. Western blot showed that ex-
pression of Bax, cytochrome C, caspase-9 and caspase-3 proteins were upregulated and protein level of Bcl-2 was depressed
after treatment with ONY in a concentration dependent. Conclusion: Apoptosis of ovarian cancer HO-8910 cells was induced
by ONY through mitochondrial apoptosis pathway in vitro.
Key words ovarian cancer; Onychin (ONY); apoptosis
Correspondence to: Jun Bai. Email: shushuanlao@163.com
390 www.springerlink.com/content/1613-9089
20 μL MTT was added to each well and incubated for ad-
ditional 6 h, and then the supernatants were removed and
100 μL. DMSO was added to dissolve the formazan crys-
tals. The viable cell number was directly proportional to
the production of formazan. The plate was then read in a
micro-plate reader (ELX-800) at 570 nm. We counted the
relative viable cells inhibit rate (IR), IR = (1 – Athe average value
of ONY 15 μg/mL／Athe average valule of NS) × 100% [3]. The experiment
was repeated three times.
Cell apoptosis analysis
Exponentially growing cells were simultaneously dealt
in RPMI-1640 medium supplemented with 1% fetal calf
serum for 24 h, and then the cells were exposed in differ-
ent experimental drug groups and continued to incubate
in RPMI-1640 medium supplemented with 10% fetal calf
serum for 48 h. Then the cells were harvested and washed
by cold PBS twice and fixed in ethanol 70% (4 ℃), then
stained in PI in darkness. Distribution of cell cycle was
analyzed by flow cytometry [3]. The experiment was re-
peated three times.
Western blot
HO-8910 cells were exposed to NS, 5 μg/mL ONY, 10
μg/mL ONY, 15 μg/mL ONY for 48 h, respectively, and
then washed 3 times in cold PBS, then harvested and
treated with cell lysate. The protein amount was detect-
ed with BCA kit, samples containing 30 μg of proteins
were separated by 10% SDS-PAGE gel electrophoresis,
and then electro-transferred to the PVDF membranes.
Membranes were blocked with TBST containing 5%
non-flat dry milk and incubated with the indicated pri-
mary antibodies overnight at 4 ℃, then membranes were
incubated with HRP-conjugated second antibody. Pro-
tein-antibodies complexes were detected by enhanced
chemiluminescene (ELC) according to the manufacturer’s
recommendations. Band densities in Western blot mea-
sured using Imaging J for windows software (NIH) [3]. The
experiment was repeated three times.
Statistical analysis
All the experimental data were expressed in χ ± s, and
statistic analyses were performed using SPSS 13.0 soft-
ware. Differences between groups were examined with
One-Way ANOVA, and a probability level of 0.05 was
chosen for statistical significance.
Results
ONY inhibited the growth of human ovarian
HO-8910 cells
MTT assay showed that the growth of HO-8910 cells
was significantly inhibited when cells exposed to the
same concentration of ONY for 24, 48, and 72 h (P < 0.05)
compared with NS group, at the same time, the growth of
HO-8910 cells was significantly inhibited when cells ex-
posed to different concentrations of ONY at 24/48/72 h (P
< 0.05) compared with NS group, and ONY significantly
inhibited the viability of human ovarian cancer HO-8910
cells in a dose-dependent and time-dependent manner,
and the IC50 was 10.48 μg/mL for 48 h. The inhibition
rate of DTX group lied in between 10 μg/mL ONY and 15
μg/mL ONY (Fig. 1).
Effect of ONY on the apoptotic rate of human
ovarian HO-8910 cells
The HO-8910 cells showed typical morphological
change of apoptosis and increased cells of sub-G1 popu-
lation when they exposed in different concentrations of
ONY for 48 h, the ONY groups superior to the NS group
in the apoptotic rate (P < 0.05). There were significantly
difference among different groups of ONY in the apop-
Fig. 1 Effect of ONY with different concentrations for different times
on proliferation of HO-8910 cells. a: P < 0.05 vs NS group; b: P < 0.01
vs NS group
Fig. 2 Apoptosis on HO-8910 cells treated with different concentrations
of ONY for 48 h. Note: 1: NS group; 2: DTX 0.2 μg/mL group; 3: ONY 5
μg/mL group; 4: ONY 10 μg/mL group; 5: ONY 15 μg/mL group. a: P <
0.05 vs NS group; b: P < 0.01 vs NS group
391Chinese-German J Clin Oncol, August 2013, Vol. 12, No. 8
totic rate (P < 0.05). DTX group superior to NS group in
the apoptotic rate (P < 0.05), and the apoptotic rate of
DTX was similar to the rate of 10 μg/mL ONY (P > 0.05;
Fig. 2).
ONY regulated the expression of proteins
related to mitochondrial apoptosis pathway
in HO-8910 cells
Expressions of bax, cytochrome-c, caspase-9 and cas-
pase-3 proteins were up-regulated when HO-8910 cells
exposed to different ONY concentrations compared to
NS group (P < 0.05). The expression of bcl-2 protein was
showed to be repressed when HO-8910 cells exposed to
different ONY concentrations compared to NS group (P
< 0.05; Fig. 3).
Discussion
Ovarian cancer is one of common malignant tumors
in women, its occurrence and development are not easy
to perceive in clinic and its morality is higher among dis-
eases in women, which are the typical features of ovarian
cancer. Chemotherapy is one of effective treatments to
ovarian cancer in clinic, the new generation of chemo-
therapy medicine and the application of neoadjuvant che-
motherapy improve ovarian cancer survival rate and life
quality, so it is prime task to develop new chemotherapy
medicine to cure ovarian cancer [4]. Apoptosis is a mani-
festation of individual cell of programmed death in vivo,
it determined by factors outside the body which trigger
cell death program stored within in cells and lead cells to
death, apoptosis play an important role in the occurrence
and development of embryo, cell transition from the old
to mature, hormone-dependent physiological degenera-
tion, atrophy and aging as well as autoimmune diseases
and tumor development and so on [5].
ONY is one of dihydro-glycosides extracted from On-
ychium lucidum of Onychium Ching of Sinopteridaceae,
it were reported that Onychin have effects of anti-tumor,
oxidation resistance, and protecting endothelial cells. One
research of Tang et al showed ONY could induce ovarian
cancer COC1 cells and COC1/DDP COC1 cells apoptosis
[1], while there was little inhibiting actions to the Chi-
nese hamster ovary cell line CHO-K1 cells, but the re-
lated mechanism of induction apoptosis was still not re-
ported. Our study showed the growth of human ovarian
cancer HO-8910 cells were inhibited and apoptosis were
induced when cells exposed to ONY, which is similar to
the results of Tang et al.
Bcl-2 family members regulate intrinsic mitochondrial
pathway through regulating mitochondrial signal trans-
duction system, it can inhibit the permeability of mito-
chondrial outer membrane and block the release of cyto-
chrome-c as a kind of inhibiting protein of cell apoptosis
[6]. Bax lying in mitochondrial outer membrane is a kind
of protein promoting apoptosis, and form channels in mi-
tochondrial outer membrane to promote cytochrome-c
release. The change of the permeability of mitochondria
is the key role of cytochrome-c release [7]. Bax homodi-
mer may form complete channels in mitochondrial outer
membrane to allow cytochrome-c release, and the forma-
tion of bax homodimer is one of essential factors of in-
trinsic mitochondrial apoptotic pathway [6, 7]. Bcl-2 play
its anti-apoptotic effect through forming heterodimer
with Bax, therefore the relative expression level between
Bcl-2 and Bax protein is key to regulate apoptosis [8]. Cy-
tochrome-c combined with Apaf-1 and ATP/dATP into
apoptotic complex when it come into cytoplasm [9], apop-
tosis complex can activate caspase-9, subsequently cas-
pase-9 can activate caspase-3 [10], while caspase-3 which
is key enzymes of apoptosis and core protease inducing
apoptosis protease cascade is one of executors of apopto-
sis, and directly destroy the functional protein and struc-
tural protein, and induces cell apoptosis, and make the
cytoplasm, nudeus, structural protein inactivation [11].
Our study showed the growth of human ovarian can-
cer HO-8910 cells were inhibited and apoptosis were in-
duced when cells exposed to ONY, the expression of Bax,
cytochrome C, caspase-9 and caspase-3 proteins were
upregulated and protein level of Bcl-2 was depressed af-
ter treatment with ONY in a concentration dependent,
which suggest apoptosis of ovarian cancer HO-8910 cells
was induced by ONY through mitochondrial apoptosis
pathway in vitro.
In summary, ONY could inhibit the growth of ovarian
cancer HO-8910 and induce apoptosis through intrinsic
mitochondrial pathway, in this study, we observed the ef-
fect that ONY induced apoptosis of HO-8910 cells and its
possible mechanism, which show ONY has great value to
develop as a new kind of chemotherapy medicine. How-
ever, the current study is also limited to in-vitro study,
and the in-vivo study of related to the antitumor action
Fig. 3 The expression of proteins of HO-8910 cells treated with differ-
ent concentrations of ONY for 48 h. A: NS group; B: ONY 5 μg/mL group;
C: ONY 10 μg/mL group; D: ONY 15 μg/mL group
392 www.springerlink.com/content/1613-9089
of ONY needs further to do.
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