全 文 ：Chinese-German J Clin Oncol September 2014, Vol. 13, No. 9, P427–P431
DOI 10.1007/s10330-014-0015-2
Laggera alata, as a medical plant, posses potent anti-in-
flammatory, anti-bacterial, anti-febrile, anti-tumor, and
alexipharmic actions and so on, and it has been used in
folk and Chinese medicine for a few hundred years, and
the plant widely distribute in China [1]. Laggera alata fla-
vonen (LAF) is extract from laggera alata, nowadays, it
has been reported possessing anti-inflammatory, anti-vi-
rus, and so on, but its anti-tumor effect was not reported
[2]. In this present study, experiments were designed to
investigate the inhibiting effects of LAF on HO-8910 cells
proliferation, induction of apoptosis and its related pos-
sible biological mechanism in vitro.
Materials and methods
Materials
LAF was extracted, isolated, and purified in the De-
partment of Pharmacology of Guangzhou Medical Uni-
versity. Human ovarian cancer HO-8910 cells were
purchased from the China Center for Type Culture Col-
lection. RPMI-1640 medium and fetal calf serum, FCS)
was tained from Gibco Company (USA), and DMSO was
purchased Company Amresco (USA). The 3-(4,5-dimeth-
ythiazol-2-yl)-2,5-iphenylterazoliumromide (MTT) was
bought from Sigma Company (USA). DDP was purchased
from Hengrui Medicine Co., Ltd (China). AO/EB stain-
ing kit, mouse anti-Fas, anti-cyt-c, anti-truncated bind-
ing interface database (tBid) monoclonal antibody, rabbi
anti-caspase-8 monoclonal antibody were obtained from
Beyotime Company (China).
The inhibiting effects of Laggera alata flavone
on human ovarian cancer HO-8910 cells
proliferation and its mechanism in vitro
Min Tang1, Jun Bai2, Chunyan Chen1, Yingxia Ning3, Xiaochun Li4, Hanzhen He5
1 Department of Obstetrics and Gynecology, Hunan Provice Mawangdui Hospital, Changsha 410016, China
2 Department of Obstetrics and Gynecology, Hangzhou Red Cross Hospital, Hangzhou 310003, China
3 Department of Obstetrics and Gynecology, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou
510120, China
4 Department of Obstetrics and Gynecology, Longgang District Maternal and Child Healthcare Hospital of Shenzhen,
Shenzhen 518172, China
5 Department of Information, Longgang District Maternal and Child Healthcare Hospital of Shenzhen, Shenzhen
518172, China
Received: 30 June 2014 / Revised: 19 July 2014 / Accepted: 15 August 2014
© Huazhong University of Science and Technology 2014
Abstract Objective: The purpose of this study was to investigate the effect of Laggera alata flavonen (LAF) on the inhibit-
ing effect of human ovarian cancer HO-8910 cells proliferation and its possible mechanism in vitro. Methods: Human ovarian
cancer HO-8910 cells were cultured in vitro. Inhibitory effect of LAF on the viability of HO-8910 cells was evaluated by the
MTT assay. Apoptotic effect of different concentrations of LAF on HO-8910 cells was assessed by AO/EB staining and FCM
with propidium iodide (PI) staining. Expression of proteins related to apoptosis was analyzed by Western blot. Results: LAF
significantly inhibited the viability of HO-8910 cells proliferation in a dose-dependent and time-dependent manner, there were
statistical significance compared with NS group (P < 0.05), and the IC50 was 4.28 μg/mL for 48 h. The cells treated with LAF
showed typical morphological change and apoptotic rate increased by FCM in a dose-dependent, and there was notable dif-
ference compared with NS group (P < 0.05). Western blot showed that expression of Fas, caspase-8, tBid and Cyto-c proteins
were up-regulated after treatment with LAF for 48 h in a concentration dependent. Conclusion: LAF could inhibit HO-8910
cells proliferation and induce apoptosis, which may be through the pathway of death receptor in vitro.
Key words ovarian cancer; Laggera alata flavonen (LAF); apoptosis
Correspondence to: Yingxia Ning. Email: shushuanlao@163.com
428 http://zdlczl.chmed.net
Cell culture
Human ovarian cancer HO-8910 cells were maintained
in RPMI-1640 medium supplemented with 10% FCS at 37
℃ in a humidified atmosphere of 5% CO2. Exponentially
growing cells were used in experiments [3].
Cell proliferation inhibition analysis
The effect of different concentration of LAF (1, 3,
and 10 μg/mL) on the proliferation ability of HO-8910
cells was determined by using MTT assy. The 2.5 × 104
cells/mL exponentially growing cells were plated into 96-
well flat-bottom micro-plates, and treated with NS, 0.2%
DMSO, DDP 0.1 μg/mL, LAF (1, 3, and 10 μg/mL). The
total incubational volume of each well was 200 μL. After
incubation for 24, 48, and 72 h, 20 μL MTT was added
to each well and incubated for additional 6 h, and then
the supernatants were removed and 100 μL. DMSO was
added to dissolve the fromazan crystals. 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 LAF / Athe average value of
NS) × 100% [4]. The experiment was repeated three times.
AO/EB staining assay
The effect of LAF on morphological changes of HO-
8910 cells apoptosis was determined by using AO/EB
staining assay. 900 μL (5 × 103 cells/mL ) exponentially
growing cells were plated into 24-well flat-bottom micro-
plates, after incubation for 24 h, 100 μL of different ex-
perimental factors was added to each well and incubated
for additional 48 h and the final concentration were NS,
0.2% DMSO, DDP 0.1 μg/mL and LAF 1, 3, and 10 μg/
mL). And then removed the culture medium and stained
in AO/EB. At last we used 340 nm wavelength ultravio-
let excitation florescene observed under microscope, and
camera [5].
Cell apoptosis analysis
The effect of LAF on apoptotic rate of HO-8910 cells
was determined by using FCM assay. Exponentially
growing cells were simultaneously dealt in RPMI-1640
medium supplemented with 1% FCS for 24 h, and then
the cells were exposed in different experimental drug
groups and continued to incubate in RPMI-1640 medium
supplemented with 10% FCS for 48 h. Then the cells were
harvested and washed by cold PBS twice and fixed in eth-
anol 70% (4 ℃), then stained in propidium iodide (PI) in
darkness. Distribution of cell cycle was analyzed by flow
cytometry [6]. The experiment was repeated three times.
Western blot analysis
HO-8910 cells were exposed to NS, 0.2% DMSO, DDP
0.1 μg/mL and LAF (1, 3, and 10 μg/mL) for 48 h, 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 primary
antibodies overnight at 4 ℃, and then membranes were
incubated with HRP-conjugated second antibody. Pro-
tein-antibodies complexes were detected by enhanced
chemiluminescene according to the manufacturer’s rec-
ommendations. Band densities in western blot measured
using Imaging J for windows software [7]. The experiment
was repeated three times.
Statistical analysis
All the experimental data were expressed in χ ± s, and
statistic analyses were performed using SPSS 15.0 soft-
ware. Differences between groups were examined with
One-Way ANOVA, and a probability level of 0.05 was
chosen for statistical significance.
Results
Effect of LAF on the inhibiting proliferation
of HO-8910 cells
MTT assay showed that LAF significantly inhibited
the viability of human ovarian cancer HO-8910 cells in a
dose-dependent and time-dependent manner. There were
statistical significance when cells exposed to the different
concentration of LAF (1, 3, and 10 μg/mL) for 24, 48, and
72 h compared with NS group (P < 0.05), and there were
significantly difference between any two different con-
centration of LAF experimental groups (P < 0.05), and the
IC50 was 4.28 μg/mL for 48 h. While the inhibiting effect
of DDP 0.1 μg/mL group was approximately equal to LAF
3 μg/mL (P > 0.05; Fig. 1).
Effect of LAF on the apoptotic morphological
changes of HO-8910 cells
AO/EB staining assay showed there were not apoptotic
morphological changes when HO-8910 cells were ex-
posed to NS group, 0.2% DMSO group for 48 h. While
cells were treated with LAF (1, 3, and 10 μg/mL) for 48
h, there were typical apoptotic morphological changes in
dose-dependant, such as nuclear condensation, nuclear
fragmentation and so on, the effect of LAF 3 μg/mL was
similar to DDP 0.1 μg/mL group (Fig. 2).
Effect of LAF on the apoptotic rate of HO-8910
cells
FCM with PI staining showed apoptosis of HO-8910
cells increased in dose-dependant when they exposed in
different concentrations of LAF (1, 3, and 10 μg/mL) for
429Chinese-German J Clin Oncol, September 2014, Vol. 13, No. 9
48 h, the LAF groups superior to the NS groups in the
apoptotic rate (P < 0.05). There was no statistical differ-
ence between NS groups and 0.2% DMSO group. There
were significantly difference among different groups of
LAF in the apoptotic rate (P < 0.05). DDP group supe-
rior to NS group in the apoptotic rate (P < 0.05), and the
apoptotic rate of DDP 0.1 μg/mL was similar to the rate of
LAF 3 μg/mL (P > 0.05; Fig. 3).
Effect of LAF on the expression of proteins
HO-8910 cells related to the pathway of
death receptor
Western blot assay showed the expressions of Fas, Cas-
pase-8, tBid and Cyto-c proteins were up-regulated in
dose-dependant manner when HO-8910 cells exposed to
different concentration of LAF (1, 3, and 10 μg/mL) for
48 h compared with NS groups (P < 0.05), which showed
LAF inhibited HO-8910 cells proliferation and induced
apoptosis might be through the pathway of death recep-
tor in vitro (Fig. 4).
Discussion
Human ovarian cancer is one of common malignant
tumors in human, its occurrence and development are
not ease to perceive in clinic, and nowadays the occur-
rence of ovarian cancer tends to younger staff, which se-
riously threaten human’s life quality [8]. Chemotherapy is
one of effective treatment to ovarian cancer in clinic, and
the new generation of chemotherapy medicine and the
application of neoadjuvant chemotherapy improve ovar-
ian cancer survival rate and life quality, so it is prime task
to develop new chemotherapy medicine to cure ovarian
cancer [9].
Apoptosis is a manifestation of individual cell of pro-
grammed death in vivo, it determined by factors outside
the body which trigger cell death program stored within
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 degeneration, atrophy and aging as well as
autoimmune diseases and tumor development and so on
[10]. LAF is extract from Laggera alata that is used tradi-
tional herbal plant curing inflammatory, febrile, alexi-
pharmic drug, tumors and so on, and it has been reported
possessing anti-inflammatory, anti-virus, and so on, but
its anti-tumor effect was not reported [11]. In this pres-
ent study, we found that LAF could significantly inhibit
human ovarian cancer HO-8910 cells proliferation and
induce them apoptosis, which suggested LAF is a proper
Fig. 1 The inhibiting effect of LAF with different concentrations for dif-
ferent time on HO-8910 cells proliferation. * P < 0.05 vs NS group
Fig. 2 The effect of LAF on the
apoptotic morphological changes of
HO-8910 cells. (a) NS group; (b) 0.2%
DMSO group; (c) DDP 0.1 μg/mL group;
(d) LAF 1 μg/mL group; (e) LAF 3 μg/mL
group; (f) LAF 10 μg/mL group
430 http://zdlczl.chmed.net
candidate to therapy human ovarian tumors.
The apoptotic pathway of death receptor mediation is
one of typical dead ways of cells, in this dead way, dead
ligant, such as TNF-α, Fas L, can combine with Fas recep-
tor which lies in cell membrane [12], and the activated Fas
owning the domain that possess the special function of
collecting adpter proteins with function combining pro-
caspase-8 to construct a new complex called Fas L-Fas-
FADD-procaspase-8, and in this complex, the high dose
of procaspase-8 can be self-activated, and then caspase-8
were released into cell cytoplasm, and trigger the caspase
cascade reaction and induce apoptosis [13–14]. Simultane-
ously caspase-8 can activate the protein of Bid which can
form tBid that possess capacity to damage mitochodria and
cause Cyto-c release and trigger apoptosis [15]. Our study
showed the proliferation of HO-8910 cells were inhibited
and apoptosis were induced when cells exposed to dif-
Fig. 4 The expression of proteins of HO-8910 cells treated with differ-
ent concentrations of LAF for 48 h. (1) NS group; (2) LAF 1 μg/mL group;
(3) LAF 3 μg/mL group; (4) LAF 10 μg/mL group. a P < 0.05 vs NS group;
b P < 0.01 vs NS group
Fig. 3 The apoptotic induction of LAF
with different concentrations on HO-8910
cells for 48 h. 1: NS group; 2: 0.2% DMSO
group; 3: DDP 0.1 μg/mL group; 4: LAF
1 μg/mL group; 5: LAF 3 μg/mL group; 6:
LAF 10 μg/mL group. a P < 0.05 vs NS
group; b P < 0.01 vs NS group
431Chinese-German J Clin Oncol, September 2014, Vol. 13, No. 9
ferent concentration of LAF (1, 3, and 10 μg/mL). Mean-
while, the expression of Fas, Caspase-8, tBid, and Cyto-c
proteins were up-regulated in a dose-dependent of LAF,
which suggest the inhibiting effect of proliferation and
the inducing action of apoptosis of ovarian cancer HO-
8910 cells were induced by LAF through the apoptotic
pathway of death receptor mediation in vitro.
In summary, LAF could inhibit the proliferation of
human ovarian cancer HO-8910 cells and induce apop-
tosis through the death receptor pathway, in this study
we observed the inhibiting effect of proliferation and the
inducing action of apoptosis HO-8910 cells that LAF in-
duced and its possible mechanism, which show LAF has
great value to develop as a new kind of chemotherapy
medicine. However, the current study is also limited in
vitro, and the in vivo study of related to the antitumor
action of LAF needs further to perform in vivo.
Conflicts of interest
The authors indicated no potential conflicts of interest.
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DOI 10.1007/s10330-014-0015-2
Cite this article as: Tang M, Bai J, Chen CY, et al. The inhibiting effects
of Laggera alata flavone on human ovarian cancer HO-8910 cells prolif-
eration and its mechanism in vitro. Chinese-German J Clin Oncol, 2014,
13: 427–431.