Download Anti-proliferative activity of Bupleurum scrozonerifolium in A549

Cancer Letters 222 (2005) 183–193
www.elsevier.com/locate/canlet
Anti-proliferative activity of Bupleurum scrozonerifolium in
A549 human lung cancer cells in vitro and in vivo
Yeung-Leung Chenga,1, Shih-Chun Leea,1, Shinn-Zong Linb, Wen-Liang Changc,**,
Yi-Lin Chenb, Nu-Man Tsaib, Yao-Chi Liud, Ching Tzaoa,
Dah-Shyong Yud, Horng-Jyh Harne,*
a
Division of Thoracic Surgery, Department of Surgery, Tri-Service General Hospital, National Defense Medical Center,
Taipei 114, Taiwan, ROC
b
Department of Neuro-Medical Scientific Center, Buddhist Tzu-Chi General Hospital, Tzu-Chi University, Hualien 970, Taiwan, ROC
c
School of Pharmacy, National Defense Medical Center, Taipei 114, Taiwan, ROC
d
Department of Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei 114, Taiwan, ROC
e
Division of Molecular Medicine, Department of Pathology, and Department of Emergency Medicine,
Buddhist Tzu-Chi General Hospital, Tzu-Chi University, Hualien 970, Taiwan, ROC
Received 20 August 2004; received in revised form 1 October 2004; accepted 7 October 2004
Abstract
Nan-Chai-Hu, the root of Bupleurum scorzonerifolium, is a traditional Chinese herb used in treatment of liver diseases such
as hepatitis and cirrhosis. We recently reported that the acetone extract of B. scorzonerifolium (BS-AE) could inhibit
proliferation and induce apoptosis in A549 human lung cancer cells. We further examined its anti-proliferative mechanisms and
in vivo anticancer effect. Our results showed that BS-AE had the ability to cause cell cycle arrest in G2/M phase, inducing
tubulin polymerization, and activating caspase-3 and -9 in A549 cells. BS-AE-induced apoptosis could be blocked by the broad
caspase inhibitor z-VAD-fmk in majority. The result of in vivo study showed that BS-AE could suppress growth in A549
subcutaneous xenograft tumors. These results indicate that BS-AE exerts antiproliferative effects on A549 cells in vitro and in
vivo, and prompted us to further evaluate and elucidate the chemical composition profile of BS-AE.
q 2005 Elsevier Ireland Ltd. All rights reserved.
Keywords: Bupleurum scorzonerifolium; Lung cancer; Apoptosis; Caspases; Cell cycle; Tubulin
1. Introduction
* Corresponding authors. Tel.: C886 3 856 1825x2223; fax:
C886 3 857 7161. ** Tel.: C886 2 8792 3100x18879; fax: C886
2 8792 3169.
E-mail addresses: [email protected] (W.-L. Chang),
[email protected] (H.-J. Harn).
1
The first two authors contributed equally to this work.
Cancer is still a serious clinical problem and has a
significant social and economic impact on the human
health care system. Despite modern advancements in
diagnosis, prevention and therapy, the disease still
0304-3835/$ - see front matter q 2005 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.canlet.2004.10.015
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affects millions of patients worldwide, reduces their
quality of life and one of the leading causes of death in
the world.
Natural products including plants, microorganisms
and marines provide rich resources for anticancer
drug discovery [1]. Based on ancient and modern
Chinese herbal medicine books such as Shen Nung
Pen Tsao Ching (200 AD) and Pharmacopoeia of
China, there are many other anticancer plants or
herbal formulations which should provide a guide,
along with clinical evidence, for the identification of
new anti-cancer compounds or a source of alternative
cancer therapy, and receive increasing scientific
attention recently [2–6].
As the different components in a herb may have
synergistic activities or buffering toxic effects,
mixtures or extracts of herbs might have more
therapeutic or preventive activity than alone [5,7].
Several studies have demonstrated that extracts from
several herbal medicines or mixtures had an anticancer potential in vitro or in vivo. Hu et al. [3]
demonstrated that an alcohol extract of Ganoderma
lucidum could induce apoptosis in MCF-7 human
breast cancer cells, and Lee et al. [8] demonstrated
that a water extract of Paeoniae lactiflora could
induce apoptosis in HepG2 and Hep3B hepatoma
cells. Kao et al. [4] reported that a water extract of
Bu-Zhong-Yi-Qi-Tang (mixture of 10 herbs) could
induce apoptosis in hepatoma cells. Yano et al. [6]
demonstrated that the water-soluble ingredients of
Sho-Saiko-To (mixture of seven herbs) inhibited the
proliferation of KIM-1 human hepatoma cells and
KMC-1 cholangiocarcinoma cells. PE-SPES (mixture
of eight herbs) had been developed for clinical
treatment of prostate cancer [7].
Nan-Chai-Hu, the root of Bupleurum scorzonerifolium Willd. (Umbelliferae), is a common and
important Chinese herb and frequently prescribed in
combination with other herbs to treat common cold,
febrile disorders, malaria, and chronic liver disorders
in China, Japan, and many other parts of Asia [9]. It is
also a major ingredient of several Chinese herbal
prescriptions such as Sho-Saiko-To (TJ9 in Japan) and
Bu-Zhong-Yi-Qi-Tang that were reported to have
anticancer activities [4,6]. Investigation of the possible anticancer activities of B. scorzonerifolium is one
of the major research programs in our laboratories.
Recently, we have reported that the acetone extract of
B. scorzonerifolium (BS-AE) can inhibit telomerase
activity and induce apoptosis in A549 human lung
cancer cells [10]. These results prompted us to further
evaluate the anticancer activity in vivo and clarify the
mechanism of BS-AE.
2. Material and methods
2.1. Chemicals and reagents
RPMI medium 1640, Eagle’s minimum essential
medium, FBS, penicillin/streptomycin, trypsin
/EDTA, and the NuPAGE Bis–Tris Electrophoresis
System (pre-cast polyacrylamide mini-gel) were
purchased from Life Technologies, Inc. (Grand
Island, NY, USA). Dimethyl sulfoxide (DMSO),
3-(4,5-dimethyl thizol-2-yl)-2,5-diphenyl tetrazolium
bromid (MTT), propidium iodine (PI), RNase A,
paclitaxel, vinblastine and anti-b-tubulin antibody
were purchased from Sigma Chemical Co. (St Louis,
MO, USA). Mycoplasma Removal Reagent was from
Dainippon Pharmaceutical Co. (Osaka, Japan).
Annexin V-FLOUS Staining Kit was from Roche
Molecular Biochemicals (Mannheim, Germany). A
goat antimouse IgG-FITC antibody was purchased
from Santa Cruz biotechnologies (Santa Cruz, CA,
USA). Western Lightninge Chemiluminescence
Reagent Plus was from Perkin–Elmer Life Science
(Boston, MA, USA). BSA Protein Assay Kit was from
Pierce (IL, USA). Caspase activity assay kits,
including the substrates of caspase-1 (YVAD-AFC),
caspase-3 (DEVD-AFC), caspase-8 (IETD-AFC),
caspase-9 (LEHD-AFC) and the broad-spectrum
caspase inhibitor, z-VAD-fmk, were purchased from
R&D Systems, Inc. (Minneapolis, MN, USA).
2.2. Cell lines and culture
Five common human solid tumor cell lines, A549
(lung adenocarcinoma), MCF-7 (breast cancer), HT29 (colon cancer), HepG2 (hepatocullular carcinoma)
and OVCAR-3 (ovarian cancer) were obtained from
American Type Culture Collection (Rockville, MD,
USA). A549, HT-29 and OVCAR-3 cancer cells were
maintained in RPMI medium supplemented with 10%
heat-inactivated FBS, 100 units/ml penicillin and
100 mg/ml streptomycin. MCF-7 and HepG2 cancer
Y.-L. Cheng et al. / Cancer Letters 222 (2005) 183–193
cells were maintained in Eagle’s minimum essential
medium with 10% heat-inactivated FBS, 100 units/ml
penicillin and 100 mg/ml streptomycin. All cultured
cells were incubated at 37 8C in a humidified
atmosphere containing 5% carbon dioxide. Cells
were fed with fresh cultured medium every 2–3
times per week and subcultured when 80% confluent.
All cultures were free of mycoplasma.
2.3. Preparation of BS-AE
The roots of B. scorzonerifolium were supplied
from Chung-Yuan Co., Taipei, and were identified by
Prof. Lin. A voucher herbarium specimen was
deposited at the School of Pharmacy, National
Defense Medical Center (NDMCP No. 900801). The
crude extract BS-AE was prepared as described
previously [10]. Briefly, the dried and powdered
roots of B. scorzonerifolium (2.0 kg) were extracted
with acetone (5 l, 3 times) at room temperature for
4 h. The extracts were concentrated and dried under
vacuum to yield an acetone extract (BS-AE). The
extract was stored at 4 8C before each experiment.
2.4. Cytotoxicity and cell viability analysis
Viability of control and treated cells were evaluated
using the MTT assay in triplicate [10]. Briefly, cancer
cells (5!103) were incubated in 96-well plates
containing 200 ml of the growth medium. Cells were
permitted to adhere for 16–18 h, washed with PBS, and
then treated with agents dissolved in medium. After
treatment, the drug-containing medium was replaced
by fresh medium. Then cells in each well were
incubated at 37 8C in 50 ml of MTT (5 mg/ml) for
4 h. After the medium and MTT were removed, 200 ml
of DMSO and 25 ml of glycine buffer (0.1 M glycine,
0.1 M NaCl, pH 10.5) were added to each well.
Absorbance at 570 nm of the mixture was detected
using a microplate ELISA reader (MRX II, DYNEX
Technologies, Chantilly, VA, USA). The absorbance
of untreated cells was considered as 100%. The results
were determined by three independent experiments.
2.5. Cell cycle analysis
Analysis of the cell cycle of control and treated
cancer cells was determined. Using standard methods
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[11], the DNA of cells were stained with PI, and the
proportion of non-apoptotic cells in different phases
of the cell cycle was recorded. Briefly, 5!105 cancer
cells were treated with BS-AE, and washed twice with
PBS. The cells were fixed overnight with cold 70%
ethanol, and then stained with PI solution consisting
of 45 mg/ml PI, 10 mg/ml RNase A, and 0.1% Triton
X-100. After a 1-h incubation at room temperature in
the dark, fluorescence-activated cells were sorted in a
FACScan flow cytometer (equipped with a 488-nm
argon laser) using CellQuest 3.0.1 software (Becton
Dickinson, Franklin Lakes, NJ), and the data were
analyzed using ModFitLT V2.0 software. In the nonapoptotic population (as 100% after excluded sub-G1
population), the percentage of cells in each phase of
the cell cycle was determined as least triplicate and
expressed as meanGSD.
2.6. Detection of apoptosis
A modified method used for detection of PS
(phosphatidylserine) in adherent cell lines was
described by van Engeland et al. [12]. Externalization
of PS and membrane integrity was quantified using an
Annexin V-FLOUS Staining Kit. In brief, 106 cells
were grown in 35 mm diameter plates before
treatment. Treated and control cancer cells were
labeled with Annexin V-FLOUS (10 mg/ml) and PI
(20 mg/ml) prior to harvesting. After labeling, all
plates were washed with binding buffer and harvested
by scraping. Cells were resuspended in binding buffer
at a concentration of 2!105 cells/ml before analysis
by flow cytometry (FACScan). The data were
analyzed using WinMDI V2.8 software. The percentage of cells undergoing apoptosis was determined by
three independent experiments.
2.7. Caspase activity assay
Activity of caspase-1, -3, -8 or -9 was detected by
using a fluorometric assay kits according to the
manufacturer’s protocol. In brief, 2!106 control or
treated cells were lysed in 50 ml of cold lysis buffer
and incubated in ice for 10 min. Fifty microliters cell
lysates added to 50 ml of reaction buffer and 5 ml
of fluorogenic report substrates specific for caspase-1,
-3, -8, or -9 in a 96-well microplate. After incubation
at 37 8C for 1 h, the fluorescence from the cleaved
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AFC side chain was detected by a fluorescence
microplate reader (Fluoroskan Ascent; Labsystems)
with excitation at 400 nm and emission at 505 nm.
2.8. Immunofluorescence and confocal microscopy
The immonostaining method was described by
Yoon et al. [13]. Briefly, cells were cultured on glass
slides in six-well plates. Control and treated cells
growing on the slides were fixed with cold 3.7%
formaldehyde in PBS for 30 min. The fixed cells were
washed twice in PBS, and then incubated in cold
permeabilization solution (0.1% Triton X-100C0.1%
sodium citrate) for 10 min. The cells were washed
with PBS three times and incubated with anti-btubulin antibody diluted with PBS (1:200) at 37 8C for
1 h. After washing with PBS, the cells were blocked
with 5% serum. The cells were washed with PBS
twice and then treated with antimouse IgG linked to
FITC for 30 min at 37 8C. After then, the cells were
washed with PBS three times, and the images of
tubulin conjugated with FITC staining were taken
with an LSM-510 confocal laser scanning microscope
system (Carl Zeiss, Oberkochen, Germany).
2.9. Tubulin polymerization by immunoblot assay
A simple assay for quantitation of tubulin polymeraization as described by Giannakakou et al. was
used [14]. Briefly, 2!106 cells were cultured in sixwell plates and treated in the following day. After
treatment, cells were washed twice with PBS and
lysed with 100 ml of hypotonic buffer (1 mM MgCl2,
2 mM EGTA, 1% NP40, 2 mM phenylmethysulfonyl
fluoride, 1 mg/ml aprotinin, 2 mg/ml pepstatin, and
20 mM Tris–HCl, pH 6.8) at 37 8C for 5 min. The
cytosolic and cytoskeletal fractions were separated by
high-speed centrifugation for 10 min. The supernatant
contained the soluble cytosolic form and was
collected. The pellet contained polymerized form
and was dissolved in 100 ml of hypotonic buffer. After
addition of 20 ml sample buffer (45% glycerol, 20%
b-mercaptoethanol, 9.2% SDS, 0.04% bromphenol
blue, and 0.3 M Tris–HCL, pH 6.8) to each 100 ml
sample, the samples were vortexed and boiled for
5 min. Electrophoresis was performed using a
NuPAGE Bis–Tris Electrophoresis System (4–12%
NuPAGE Bis–Tris gels) using 20 ml of each sample.
Immunoblotting was performed using monoclonal
anti-b-tubulin antibody conjugated to antimouse IgGHRP antibody, and detected by the Western Lightninge Chemiluminescence Reagent Plus. The
expressions of b-tubulin were quantified using a
densitometer. The experiment was repeated three
times with similar results.
2.10. Antitumor activity in vivo
Female congenital athymic BALB/c nude (nu/nu)
mice were purchased from Charles River Laboratories
(Boston, MA, USA). They were maintained under
specific pathogen-free conditions and provided with
sterile food and water. The Committee on Animal
Research approved all procedures. All experiments
were carried out using 6–8-week-old mice weighting
18–22 g. Murine tumor models were used as previously described [15,16]. In vitro cultured A549
cancer cells (1!107) were injected s.c. into the back
of mice. When the tumor reached 80–120 mm3 in
volume, animals were divided randomly into test
groups consisting of 6–8 mice per group (day 0).
Daily i.p. administration of BS-AE, dissolved by 10%
Tween 80 in normal saline (v/v), from day 0 to day 4
was performed. The mice were weighed three times a
week up to days 21–28 to monitor toxic effects.
Tumor response was determined by measurement of
the length (L) and width (W) of the tumor mass three
times a week up to days 21–28. The tumor volume at
day n (TVn) was calculated as TV (mm3)Z(L!W2)/2.
The relative tumor volume at day n (RTVn) was
expressed according to the following formula:
RTVnZTVn/TV0. Tumor regression (T/C%) in treated
versus control mice was expressed as follows:
T/C%Z(mean RTV of treated group)/(mean RTV of
control group)!100. Xenograft tumors of treated and
control mice were harvested and fixed in 4% formalin,
embedded in paraffin, and cut in 4-mm sections for
histologic study.
2.11. Statistical analysis
The data are shown as meanGSD. Statistical
significance of differences were analyzed using the
Student’s t-test for normally distributed values and
by the non-parametric Mann–Whitney U-test for
Y.-L. Cheng et al. / Cancer Letters 222 (2005) 183–193
Table 1
Cytotoxicities of BS-AE in human cancer cells
Cell lines
Origin
IC50 (mg/ml)
A549
MCF7
HT-29
HepG2
OVCAR-3
Lung adenocarcinima
Breast carcinoma
Colon carcinoma
Hepatocellular carcinoma
Ovarian carcinoma
60G5
74G6
55G6
52G5
58G5
The values of IC50 are result after 24-h treatment (meanGSD from
three independent experiments).
valuables that were not normally distributed. Values
of P!0.05 were considered significant.
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BS-AE (0–200 mg/ml) for 24 h. Cells incubated with
0.2% DMSO was used as a control. The IC50 values of
BS-AE tested in these cancer cells were ranged from
50 to 80 mg/ml after 24-h treatment (Table 1). HepG2
cells showed the highest susceptibility (IC50Z
46–58 mg/ml), and MCF-7 cells displayed the lowest
susceptibility to BS-AE (IC50Z79–82 mg/ml). After
exposure of BS-AE (60 mg/ml) on these cancer cell
lines for 12, 24 or 48 h, the cell viability was
significantly suppressed after 24-h treatment
(Fig. 1). The results indicated that BS-AE-induced
growth inhibition presented with a time-dependent
manner on these cancer cells.
3.2. Cell cycle analysis
3. Results
3.1. Antiproliferative effect of BS-AE on human
solid tumor cells
The antiproliferative effect of BS-AE was on
various human cancer cell lines was determined.
Cells were treated at different concentrations of
The cell cycle were analyzed in A549 cells treated
with BS-AE (60 mg/ml) for 24 or 48 h. The percentage
of cells in each phase of cell cycle in control (exposed
to 0.2% DMSO) and treated cells were determined by
flow cytometry and the results were showed in Fig. 2.
The sub-G1 population indicated apoptotic-associated
chromatin degradation [17]. As compared to
Fig. 1. Anti-proliferative activity of BS-AE on A549, MCF-7, HT-29, HepG2 or OVCAR-3 cells detected by MTT assay after 12, 24 or 48 h
treatment. The control is cells treated with solvent (0.2% DMSO). Values are MeanGSD (means of three experiments). BS-AE shows a timedependent antiproliferative activity in these cancer cells. *P!0.05 versus the control.
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Fig. 2. Cell cycle analysis of A549 cells cultured with either 0.2% DMSO (A, control), 60 mg/ml of BS-AE for 24 h (B), or for 48 h (C). The
percentage of non-apoptotic cells within each cell cycle was determined by flow cytometry. Compared to the control (A), BS-AE could induce
apoptosis (increasing sub-G1 population) and G2/M arrest in A549 cells.
control, the sub-G1 group significantly increased after
cells cultured with BS-AE (24 h: 12.6G3.2%; 48 h:
21.8G4%). In non-apoptotic population, we observed
that A549 cells accumulated in G2/M phase after
administration of BS-AE for 24 and 48 h (42.9G3 and
75.2G4%, respectively). On the other hand, BS-AE
caused S phase attenuation after 48 h treatment
(control: 31.3G3%; 24 h: 24.5G3%; 48 h: 11.5G
2%). These results suggested that BS-AE can induce
cell cycle arrest in G2/M phase and apoptosis in A549
cells with a time-dependent manner.
3.3. Apoptosis assay
In our previous study, BS-AE induced chromatin
change in A549 cells [10]. Shrinkage of cells and
disorganization of chromatin might be related to
apoptosis [18]. Simultaneous staining with Annexin
V-FLOUS and PI distinguished between intact cells,
early apoptosis, late apoptosis or cell death [19]. In
A549 control culture (exposed to 0.2% DMSO for
48 h), 92G3% of the cells were viable, 5G2% were
in early apoptosis and 3G1% were in late apoptosis or
cell death (Fig. 3A). In A549 cells treated with BS-AE
(60 mg/ml) for 48 h, 29G3% cells were in early
apoptosis, and 5G2% were in late apoptosis or
cell death, respectively (Fig. 3B). Moreover, after
A549 cells exposed to different dosages of BS-AE
(10, 50, 100 or 200 mg/ml) for 48 h, the apoptotic cells
were significantly increased with a dose-dependent
(data not shown). These results suggested that BS-AE
can induce apoptosis in A549 cells.
3.4. Caspase activity assay
For evaluation the molecular effector pathway of
apoptosis induced by BS-AE, activity of caspase-1,
-3, -8, and -9 were measured using specific fluorogenic peptide substrates after addition of 60 mg/ml
BS-AE to A549 cells for 6, 12, 24 or 48 h. As shown
in Fig. 4A, activation of caspase-3 and caspase-9 were
significant in BS-AE-treated cells since 12 h of
treatment and continued to rise until 48 h. But BSAE did not activate caspase-1 or -8. For further
definition, the role of caspases in BS-AE-induced
apoptosis, A549 cells were pretreated with the broadspectrum caspase inhibitor, z-VAD-fmk. A549 cells
were treated with 100 mM of z-VAD-fmk 2 h prior
BS-AE treatment. The apoptosis was detected 24 or
48 h after treatment. As shown in Fig. 4B, z-VADfmk attenuated BS-AE-induced apoptosis at both 24
and 48 h. But z-VAD-fmk did not inhibit BS-AEinduced apoptosis completely. Otherwise, these
results indicated that activation of caspases involved
BS-AE-mediated apoptosis.
Y.-L. Cheng et al. / Cancer Letters 222 (2005) 183–193
189
Tubulin in the cytosolic and cytoskeletal fractions
from A549 cells exposed to BS-AE (60 mg/ml),
paclitaxel (100 nM) or vinblastine (50 nM) were
isolated and analyzed by immunoblotting for btubulin (Fig. 5C). These results showed that BS-AE
caused significant shifts of soluble tubulin to the
polymerized form, thus stabilizing microtubules in
A549 cells. This shift contributes to G2/M arrest
caused by BS-AE.
3.7. Anticancer activity in vivo
To evaluate the antitumor activity of BS-AE in
vivo, we created human lung cancer xenografts by s.c.
Fig. 3. Fluorescence-activated sorting analysis of annexin VFLOUS and PI for quantification of BS-AS-induced apoptosis in
A549 cells. The percentage of apoptosis was determined by flow
cytometry. (A) Control A549 cells, (B) cells after 48 h of treatment
with BS-AE (60 mg/ml). The data shown are representative of three
independent experiments.
3.5. In situ fluorescent staining of b-tubulin
For b-tubulin immnunofluorescent staining, A549
cells were treated with BS-AE (60 mg/ml) for 24 h.
Using confocal microscopy, compared to control cells
(Fig. 5A), we found that BS-AE can influence
microtubule dynamic stability by increasing tubulin
polymers in A549 cells (Fig. 5B).
3.6. Tubulin polymerization
The distribution of tubulin in soluble or polymerized forms was analyzed by immunoblotting.
After separation, the soluble tubulin (S) was
contained in the cytosolic fraction, and the polymerized tubulin (P) was in the cytoskeletal fraction.
Fig. 4. (A) Effects of BS-AE on activity of caspase-1, -3, -8 and -9 in
A549 cells using fluorogenic peptide substrates. A549 cells treated
with BS-AE (60 mg/ml) for indicated times. Data represents for
three experiments (meanGSD). (B) Effects of caspase inhibitor,
z-VAD-fmk, on BS-AE-induced apoptosis detected by annexin
V-FLUOS staining. Apoptotic cells were determined 24 or 48 h
after treatment with BS-AE (60 mg/ml) in the presence or absence of
z-VAD-fmk. *P!0.05; **P!0.01 versus the control.
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Fig. 5. Tubulin assays. (A) and (B) Confocal scanning of immunofluorescent staining of b-tubulin in A549 cells treated with BS-AE (60 mg/ml)
for 24 h. Compared to untreated cells (A), BS-AE-treated cells (B) showed multiple intracellular spindle-like fibers (magnification !400). (C)
Immunoblotting assay of b-tubulin after electrophoresis of soluble (S) and polymerized (P) fractions of untreated A549 cells and A549 cells
exposed to BS-AE (60 mg/ml) for 24 or 48 h, paclitaxel (Taxol, 100 nM), or vinblastine (Vinbl, 50 nM) for 24 h. The ratio of S/P was
determined by a densitometer. Similar to paclitaxel, BS-AE cause shift of microtubule to polymerized form. Otherwise, vinblastine shifts
microtubule to a soluble (depolymerized) form. Scale bars, 8 mm.
injection of A549 cells into nude mice. After daily i.p.
administration of 100, 300 or 500 mg/kg of BS-AE on
day 0 to day 4, a significant and dose-dependent
suppression of growth was found on BS-AE-treated
A549 xenografts (P!0.001), but not in that of control
group (administration of 100 ml 10% Tween 80)
(Fig. 6A). Furthermore, BS-AE could also induce
regression of A549 tumors at dosages 300 and
500 mg/kg. Regarding to general toxicity, body
weight of the tumor-bearing mice had significantly
decreased at dosage of BS-AE 500 mg/kg before day
6, and recovered progressively after then. Otherwise,
there was not significant change in body weight at
dosages less than 300 mg/kg (Fig. 6B). Compared to
the control tumor-bearing mice, the histology showed
that a massive necrosis and infiltration of inflammatory cells in the BS-AE-treated A549 xenograft tumor
was found (300 mg/kg, D9; Fig. 6C and D). These
results demonstrated that BS-AE had antitumor
activity in nude mice bearing A549 lung cancer
xenografts.
4. Discussion
In the present study, we found that BS-AE had
antiproliferative effect to human cancer cells. BS-AE
can cause cell cycle arrest in G2/M phase, induce
apoptosis, shift tubulin to a polymerized form and
activate caspases in A549 cells. Furthermore, BS-AE
can inhibit tumor growth and induce tumor regression
in an A549 xenograft model, and a dose-dependent
manner was also found. In vivo toxicity was also
roughly examined by body weight change and
histologic study of vital organs (data not shown),
and there were no significant toxic effects below a
dose of 500 mg/kg of BS-AE
Apoptosis is an important homeostatic mechanism
that balances cell division and cell death and
maintaining the appropriate cell number in the body.
Disturbances of apoptosis in cancer cells have been
studied in detail, and induction of apoptosis in cancer
cells was one of the strategies for anticancer drug
development [20,21]. There are several crucial
Y.-L. Cheng et al. / Cancer Letters 222 (2005) 183–193
191
Fig. 6. Effect of BS-AE on subcutaneous A549 tumor-bearing mice. BS-AE was administered i.p. on day 0 to day 4 at doses of 100, 300 or
500 mg/kg. The figure shows the relative tumor volume (A), and body weight change (B) of untreated and therapeutic groups. (C) shows the
untreated A549 xenograft tumor, and (D) shows massive necrosis with infiltration of inflammatory cells in an A549 xenograft tumor (right side)
after administration of 300 mg/kg BS-AE at day 9. Scar bars, 100 mm.
cellular and molecular biological features involving
apoptosis, including cell shrinkage, disorganization of
chromatin, externalization of PS, and activation of
caspase [22,24]. Activation of the family of caspases
was known as a crucial mechanism for induction of
death signals to apoptosis. Caspases are a family of
cyseine proteases in mammalian cells that cleave
following an aspartic acid residue [23]. Caspases can
be divided into initiator and effector caspases.
Initiator caspases (e.g. caspase-1, -2, -8, -9 and -10)
containing a long prodomain involve in early stages of
the proteolytic cascade, whatever effector caspases
(e.g. caspase-3, -6, and -7) containing a short
prodomain act downstream in cleavage of specific
intracellular substrates (e.g. poly-ADP-ribose polymerase, focal adhesion kinase) resulting programmed
cell death [23–25]. PS externalization is an early
feature if apoptosis and can be detected by the binding
of annexin V to PS on the cell surface [12]. In our
results, BS-AE can cause externalization of PS and
activate caspase-3 and -9. Elevation of caspase-3 and
-9 by BS-AE was initiated since 6 h of BS-AE
exposure and maintained to 48 h. Otherwise, our
results also showed that BS-AE-induced apoptosis on
A549 cells could be inhibited after broad caspase
inhibitor z-VAD-fmk pretreated. It indicated that
BS-AE-induced apoptosis is mediated through both
caspase-dependent pathways.
Disturbance of the cancer cell cycle is one of
therapeutic targets for development of new anticancer
drugs [26]. The result of cell cycle analysis evaluated
by flow cytometry analysis showed that BS-AE can
induce G2/M arrest on A549 cells. Targeting
regulatory molecules involving G2/M transition (e.g.
cdc2, cyclin A or B), or interfering with cytoskeletal
function could be contributed this effect [26,27]. Our
results also demonstrated that BS-AE can cause
polymerization of tubulin and, thus, interferes with
mitotic spindle formation. This plays a major role in
the molecular basis of BS-AE-induced cell cycle
arrest in G2/M. These effects resemble those of the
microtubule-stabilizing cancer chemotherapy agents,
such as Taxans and epothilone B, but the detailed
molecular mechanisms need for further evaluated.
192
Y.-L. Cheng et al. / Cancer Letters 222 (2005) 183–193
Using interference with tubulin’s biological functions
causing mitotic arrest and apoptosis in cancer cells
was one of the strategies for anticancer drug discovery
[27]. Our results showed that BS-AE can cause
tubulin polymerization which interrupts cell mitosis
and commits the cell to apoptosis, and might play a
critical role in BS-AE-induced cell cycle arrest as well
as apoptosis.
Saikosaponins were reported as the major components of B. scorzonerifolium [28]. Most saikosaponins isolated from Bupleuri Radix have been reported
to have pharmacological activities involving antiinflammatory, antiviral or antihepatotoxic activity
[29,30]. The antiproliferative effect in cancer cells of
saikosaponins was not valid. The components in BSAE belonged to non-polar characteristics, but saikosaponins were more polar compounds [31]. The
molecular mechanisms responsible for the anticancer
effects of BS-AE may encourage us for further
evaluation and application of BS-AE and elucidate
its chemical composition profile to identify its active
anticancer components.
In summary, our study showed that BS-AE could
proliferation of A549 cells via causing cell cycle
arrest in G2/M phase, inducing apoptosis, and
increasing microtubule stabilization. BS-AE also
exhibited antitumor activity in vivo. B. scorzonerifolium was widely used clinically in other diseases
rather than antitumor application. The in vitro and in
vivo antitumor effects were reported here, and it is
valuable for further investigation including elucidation of active components.
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
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