Extract
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Introduction
Human hepatoma is one of the most frequent cancers in the world and it has the characteristics of secret coming on,
including rapid growth rate, strong malignancy, easy invasion, metastasis and bad prognosis, and the occurring of hepatoma
presented an ascending trend. However, the curative effect of current therapies in liver cancer is not
perfect[1]. Treatment is difficult if the cancer has spread beyond the liver. Therefore, it is very important to find new medicine to treat hepatoma.
Salvia miltiorrhiza has been used as a traditional medicine in China for centuries. It was commonly used for the treatment
of cardiovascular diseases, such as angina pectoris, myocardial infarction, and
stroke[2]. Some studies also indicated that the
extract of Salvia miltiorrhiza had antitumor
potential[3]. The aqueous extract of
Salvia miltiorrhiza could inhibit cell growth
and induce apoptosis in human hepatocellular carcinoma cell line HepG2
cells[4] and salvianolic acid B, a water-soluble
compound from Salvia miltiorrhiza that has anti-oxidative and
free radical scavenging effects[5]. It was recently reported
that it could effectively inhibit the proliferation of the chronic
myelogenous leukemia K562 cell line[6]. However, the
underlying anticancer mechanisms of salvianolic acid B and the
aqueous extract of Salvia miltior-rhiza are unclear.
Paeoniae radix is the root of Paeonia lactiflora
Pallas and is a crude drug used in many traditional prescriptions in
China and Japan[7]. Paeoniae
radix is a potential anti-aging and anticarcinogenesis agent as it has been reported to
inhibit oxidative DNA cleavage induced by various
oxidative DNA damage chemicals[8]. Paeoniflorin, a water-soluble
compound from Paeoniae radix, could induce apoptosis of
lymphocytes[9]. Lee et al showed that the water soluble
extract of Paeoniae radix (PRE) had an inhibitory effect on
the growth of both the HepG2 and Hep3B cell lines. The
induction of internucleosomal DNA fragmentation and
chromatin condensation appearance, and the accumulation of
the sub-G1 phase of cell cycle profile in PRE-treated hepatoma
cells showed that the cytotoxicity of PRE to hepatoma cells
was through the activation of apoptosis in a
p53-independent pathway. The underlying mechanism of PRE as an
anticancer agent has not been completely
defined[10].
Compounds that block or suppress the proliferation of
tumor cells by inducing apoptosis are considered to have
potential as antitumor agents[11]. In this study, Chi-Shen
extracts (CSE) were composed of the water-soluble extracts
of Salvia miltiorrhiza, mainly containing salvianolic acid B
and the glycoside extract of Paeoniae radix
containing paeoniflorin at a ratio of 7:3. The objective of this study was
to examine the in vitro antitumor activities of CSE on
hepatoma cell line HepG2, which was through the activation
of apoptosis, evidenced by the induction of internucleosomal
DNA fragmentation and the accumulation of the
sub-G1 phase of cell cycle profile. The activation of apoptosis by CSE was
dependent on the caspase pathways and probably through
altering the expression level of the Bcl-2 family and p53 as
identified by Western blotting analysis.
Materials and methods
Materials Slices of Salvia miltiorrhiza
and Paeoniae radix were purchased at a local market in Wuhan, a
well-known production area for Salvia miltiorrhiza
and Paeoniae radix in China. Methyl thiazolyl tetrazolium (MTT), acridine
orange (AO), ethidium bromide (EB), RNase A, propidium
iodide (PI), and trypsin were purchased from Sigma
Chemical Co (St Louis, MO, USA). Dulbecco's modified Eagle's
medium (DMEM), fetal bovine serum (FBS), low melting point
agarose, and normal melting point agarose were from GIBCO
(Grand Island, NY, USA). The apoptosis ladder detection kit
was from Wako Pure Chemical Industries (Osaka, Japan).
AMV reverse transcriptase was purchased from Promega
(Madison, WI, USA). The caspase colorimetric assay kit
was obtained from BioVision (Mountain View, CA, USA).
The micro BCA protein assay kit was from Beijing Biosea
Biotechnical Corporation (Beijing, China). RNAzol was from
Omega Biotek (Doraville, GA, USA). Bcl-2, Bax, p53, and
β-actin antibodies were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA, USA). Other chemicals and
reagents used were obtained from Sigma (USA). All
chemicals and reagents were of analytical grade.
Preparation of CSE extracts The dried root slices
of Salvia miltiorrhiza were percolated with 30% ethanol at room
temperature. After evaporation of the solvent, the
concentrated aqueous extract was acidified with hydrochloric acid
(pH 2_3) and then extracted with acetic ether. The organic
phase was then concentrated and dried to yield the
water-soluble extract of Salvia
miltiorrhiza, which mainly contained salvianolic acid B.
The crude powder of Paeoniae radix was
extracted with 50% ethanol and then filtered. After
evaporation of the solvent, the crude extract was dissolved in water
and applied to a macroporous resin column that was washed
with water and 30% ethanol successively. The eluent was
then concentrated, dried to yield the glycoside extract
of Paeoniae radix, which mainly contained paeoniflorin. CSE,
which was composed of the water-soluble extract
of Salvia miltiorrhiza and the glycoside extract of
Paeoniae radix, at a ratio of 7:3, was dissolved in phosphate-buffered saline
(PBS) for storage at 4 oC and used in all subsequent
experi-ments. The final storage concentration was 100
mg/mL, and the concentration used in the experiment was based on the dry
weight of the extract (mg/mL).
HPLC assay The extract of Salvia
miltiorrhiza, which mainly contained salvianolic acid B used for HPLC analysis,
was performed in Agilent 1100 high-performance liquid
chromatograph (Agilent, Waldbronn, Germany), with a Kromasil
C18 column (Hichrom, Reading, UK; 5 µm, 4.6 mm×250 mm),
eluted with the mobile phase containing formic acid-0.2%
H3PO4 (17:83), at a flow rate of 1.0 mL/min and a detector
wavelength of 280 nm. The whole run time was 20 min, while
the retention time of salvianolic acid B was 15.460 min (Figure
1A). The extract of Paeoniae radix mainly containing
paeoniflorin used for the HPLC analysis was performed in a
Varian Prostar 210 high-performance liquid chromatograph
(Palo Alto, CA, USA), with a Zorbax Eclipse
XDB-C18 column (5 µm, 4.6×250 mm, Agilent, CA, USA), eluted with the
mobile phase containing acetonitrile-H2O (17:83), at a flow
rate of 1.0 mL/min and a detector wavelength of 230 nm. The
whole run time was 20 min, while the retention time of
paeoniflorin was 10.755 min (Figure 1B).
Cell culture The human hepatoma cell line (HepG2) was
obtained from the China Center for Type Culture Collection
(Wuhan, China). The cells were cultured with DMEM with
10% (v/v) heat-inactivated FBS, 100 µg/mL streptomycin, and
100 unit/mL penicillin in 100 mL culture flasks in a humidified
atmosphere at 37 oC with 5%
CO2. Single cell detachment was achieved with incubation at 37
oC with trypsin-EDTA.
Cell viability assay Cell viability was measured by
3-(4,5)-2,5-diphenyltetrazolium bromide (MTT) assay. In brief,
the HepG2 cells were treated with CSE at concentrations of
0.25, 0.5, 1.0, and 2.0 mg/mL for 12, 18, 24, and 36 h,
respec-tively, and the control group was treated with the same
amount of medium. 5-Fluorouracil (5-FU), an anticancer drug
inhibiting DNA and RNA synthesis, was used as a standard
control. After the MTT labeling reagent was added and
incubated for 4 h at 37 oC, they were incubated for 12 h
with the 10% sodium dodecyl sulphate (SDS) solution. The
absorbance (at a test wavelength of 540 nm and a reference
wavelength of 690 nm) was measured by a microtiter plate
reader (Molecular Devices, Silicon Valley, CA, USA). The
optical density (OD) was calculated as the difference
between the absorbance from the reference and test
wave-lengths. The percentage of viability was calculated as the
following formula: (viable cells) %=(OD of drug-treated
sample/OD of untreated sample)×100.
Morphological examination The cells were harvested
and washed 3 times with PBS after being incubated with
different concentrations of CSE for 12, 18, 24, and 36 h
respectively, and were stained with 10 µg/mL AO/EB for 5
min. Apoptotic morphology was observed by a fluorescence
microscope (BX51, Olympus, Shinjuku-ku, Tokyo, Japan) after
AO/EB staining.
Analysis of DNA fragmentation The cells were harvested
and washed 3 times with PBS after being incubated with
different concentrations of CSE for 24 h. DNA was extracted
with the apoptosis ladder detection kit, analyzed by 1.0%
agarose gel electrophoresis, and then stained with EB.
DNA content and cell cycle analyzed by flow
cytometry The DNA content and the cell cycle of HepG2 cells were
determined by flow cytometry. Briefly, at the end of the
various designated treatments, the cells were scrapped,
washed, and fixed with 80% ice-cold ethanol at -20
oC overnight. The cells were incubated with freshly prepared
PI staining buffer containing 0.25 mg/mL RNase A, 2
mmol/mL EDTA, and 0.1 mg/mL PI in PBS. After incubation in dark
conditions at 37 oC for 30 min, the fluorescence of 10 000
cells was analyzed using a flow cytometer with an argon ion
laser (488 nm) as the excitation source and Cell Quest
software (Becton Dickinson, San Jose, CA, USA). The
percentage of degraded DNA was determined by the number of
hypodiploid DNA cells, and the change of cell cycle was
determined at the same time.
Caspase activity assay Caspase activation was measured
using a caspase colorimetric assay kit as described by the
manufacturer. Briefly, after being treated with 1.0 mg/mL
CSE for the indicated periods of time, the cells were harvested,
pelleted, and frozen on dry ice. Cell lysis buffer was added
to the cell pellets, and protein concentration was determined
by a micro BCA kit. Then, 100 µg protein was suspended in
50 µL cell lysis buffer for each assay,
and 50 µL of 2× reaction buffer with 10 mmol/L dithiothreitol (DTT) was added
and incubated at 4 oC. The substrates of Ile_Glu_Thr_Asp
conjugated to p-nitroanilide (IETD_pNA),
Leu_Glu_His_Asp conjugated to p-NA
(LEHD_pNA), and Asp_Glu_Val_Asp conjugated to
p-NA (DEVD_pNA) were added into the tubes, respectively. Formations of
p-NA were measured by an ELISA micro-plate reader (Thermo, Vantaa, Finland)
at a wavelength of 405 nm after the samples were
incubated for 1.5 h at 37 oC. The activation of caspases-8, -9, and
-3 were analyzed in parallel.
RNA purification and RT-PCR The expression of
apoptosis-related genes, such as Bcl-2, Bax, and p53 were
determined by RT-PCR. Total RNA was isolated with RNAzol
according to the manufacturer's instruction. Single-strand
cDNA was synthesized from 2 µg total RNA using AMV
reverse transcriptase. Bcl-2, Bax, p53, and GAPDH (an
internal standard) cDNA were amplified by PCR with specific
primers (Table 1). The annealing temperature was 58
oC for Bcl-2, 55 oC for Bax, 55
oC for p53, and 58 oC for GAPDH. The
amplified fragment sizes of Bcl-2, Bax, p53, and GAPDH were
301, 114, 316, and 387 bp, respectively. The RT-PCR
products were electrophoresed on the 1.0% agarose gel and
visualized by staining with EB.
Western blotting For Western blotting, the cells were
first treated for 24 h with different concentrations of CSE,
2×106 cells were washed twice with ice-cold PBS, lysed for 30
min at 4 oC, and then the debris was removed by
centrifugation for 15 min at 12 000×g at 4
oC. The equivalent amount of protein (20 µg) were separated by 10% SDS-PAGE and
transferred onto nitrocellulose membranes. The membranes were
first stained to confirm uniform transfer of all samples and
then incubated in blocking solution for 2 h at room
temperature. The filters were hybridized first with
monoclonal antibody (anti-Bcl-2, anti-Bax and anti-p53,
respectively) at a dilution of 1:1000 for 2 h, followed by
extensive washes with PBS twice and TBST (Tris Buffered
Saline supplemented with 0.1% Tween-20) twice. The
membranes were then incubated with horseradish
peroxidase-conjugated secondary antibody at a dilution of 1:1000 for
1 h, and washed with TBST. As a loading control,
β-actin was also detected. The immunoreactive proteins were
detected using an ECL Western blotting detection system
(Beyotime Institute of Biotechnology, Haimen,
China).
Statistical analysis Data are presented as mean±SD. The
differences among different groups were analyzed using
one-way ANOVA with Scheffe's test. A P-value less than 0.05
was considered statistically significant.
Results
Effect of CSE on viability in HepG2 cells The potential
effect of CSE was investigated on the viability of a human
hepatoma cell line (HepG2). MTT assay was performed to
evaluate the cell viabilities (Figure 2). Compared to the
untreated cells (taken as 100% viable), different
concentrations of CSE or 5-FU showed a time- and dose-dependent
inhibition of HepG2 cell proliferation. No significant decrease
in cell viability was observed when HepG2 cells were treated
with 0.25 mg/mL CSE for 36 h. However, exposure of HepG2
cells to CSE at 1.0 and 2.0 mg/mL caused a marked decrease
in overall viability from 100% to 33% and 19% of untreated
control levels, respectively.
CSE treatment caused apoptosis To determine whether
the cell growth arrest caused by CSE treatment was related
to the induction of apoptosis, morphological observation
with AO/EB staining was detected by fluorescence
micro-scopy. The HepG2 cells treated with 1.0 mg/mL CSE for 24 h
were stained and observed. Green live cells with normal
morphology were seen in the control group (Figure 3A), while
early apoptotic cells with nuclear margination and chromatin
condensation, and later apoptotic cells with fragmented
chromatin, were orange in the experimental group treated
with 1.0 mg/mL CSE (Figure 3B). The results suggested that
CSE induced marked apoptotic morphology in HepG2 cells.
The effect of CSE on the intranuclear DNA fragmentation
was further examined. After CSE treatment (1.0 and
2.0 mg/mL) for 24 h, DNA fragmentation was observed (Figure
4); no DNA ladder appeared in the control group. Apoptosis
was further quantified by flow cytometry. Based on a
previous report, cells with sub-G1 DNA content were scored as
apoptosis. Apoptosis could result in the progressive
generation of particles corresponding to hypodiploid DNA
content, which reflects DNA
fragmentation[12]. In our study, CSE treatment resulted in an increment of the
sub-G1 fraction (M1 fraction of Figure 5) in HepG2 cells, which indicated that
apoptosis induced by CSE treatment was in a
dose-dependent manner. These results were in accordance with those
obtained from the DNA fragmentation assay.
Activation of caspase by CSE To identify whether
caspases are involved in the mechanism of apoptosis, we
measured the catalytic activity of caspases-3, -8, and -9. The
results showed that caspases-3 and -9 were activated and
peaked at 6 h of CSE treatment (Figure 6A,6B). However, the
results indicated that activated caspase-8 did not change
significantly (Figure 6C).
Effect of CSE on cell cycle distribution To test whether
there was an induction of cell growth arrest of CSE treatment
in HepG2 cells, cell cycle analyses were
performed. HepG2 cells treated with the rising CSE concentration series
(0.25_2.0 mg/mL) for 24 h had a dose-dependent growth arrest, as
did the cells in the S phase. The cell proportion which
decreased in the G2/M phase (Figure 7). The S-phase arrest of
HepG2 cells was significant at above 1 mg/mL of CSE. These
data indicated that CSE attracted cell cycle to the S phase in
HepG2 cells.
Effects of CSE on the transcription of Bcl-2, Bax, and
p53 in HepG2 cells The transcription of Bcl-2, Bax, and p53
was detected by RT-PCR analysis. The results revealed that
the transcription of Bcl-2 was downregulated, while that of
Bax and p53 was considerably upregulated when the HepG2
cells were treated with CSE for 24 h (CSE over 0.25 mg/mL) in
dose-dependent manner (Figure 8). This suggested that the
downregulation of Bcl-2 and the upregulation of Bax and
p53 may play an important role in CSE-induced apoptosis in
HepG2 cells.
Effects of CSE on the expression of Bcl-2, Bax, and p53
in HepG2 cells The results of the Western blot analyses of
Bcl-2, Bax, and p53 in HepG2 cells with or without CSE are
shown in Figure 9. CSE stimulated the expression of Bax and
p53 in a dose-dependent manner (Figure 9B, 9C). The
expression of Bcl-2, an anti-apoptotic protein, decreased in a
dose-dependent manner (Figure 9A).
Discussion
In a previous study, we verified that the antifibrotic and
antitumor activities of CSE were stronger than that of single
extract. The best proportion of the water-soluble extract of
Salvia miltiorrhiza and the glycoside extract of
Paeoniae radix was at a ratio of 7:3. The CSE capsule for relieving
hepatic fibrosis has been applied in clinical studies in China.
In the present study, the main aim of the investigation was
to explore the antihepatoma potential of CSE and its
mechanisms. Many investigations have been carried out
worldwide to discover naturally-occurring compounds which
can suppress or prevent the process of
carcinogenesis[10,13]. Natural compounds fit into a mechanism-based approach
that targets whole pathways and sets of intracellular events
rather than a single enzyme, as do many synthetic drugs.
This offers a less specific, but perhaps more effective strategy,
for cancer therapy by inducing the combination of effects
that may counteract the metabolic alterations related to
cancer promotion[14]. For example, a capsicum and green tea
mixture has been reported to exhibit 100 times greater
potency with respect to anticancer activity in a number of
cell lines, than that of green tea alone on a weight
basis[15]. Early research reported that the extracts of
Salvia miltiorrhiza and Paeoniae radix
both have been documented to posses antitumor
potential[6,10]. Therefore, in this study, CSE was
mainly composed of salvianolic acid B and paeoniflorin, which
are water-soluble extracts from Salvia miltiorrhiza
and Paeoniae radix, respectively. Here, we showed that CSE
was able to inhibit the growth of the human hepatoma cell
line (HepG2) in a dose-dependent manner, which exhibited a
similar potency as the commercial anticancer agent 5-FU
(Figure 2).
Hepatocellular carcinomas (HCC) with poor prognosis
are characterized by rapid cell proliferation and strong
expression of anti-apoptotic genes[16], which suggests that they
are mainly due to incomplete cell cycle arrest and apoptosis
resistance under conventional
therapies[17]. Recent data have shown that apoptosis, especially the
caspase-mediated cell death, plays an important role in the etiology,
pathogenesis, and therapy of a variety of human
malig-nancies, such as human HCC, and the cytotoxic effects of
many antihepatocellular carcinoma drugs are based on the
induction of apoptosis[18]. Agents that can induce apoptosis
in cells are considered to be potentially useful for the
management and treatment of cancer[13]. Apoptosis is
characterized by cell shrinkage, chromatin condensation, DNA
fragmentation, sub-G1 DNA peak, and the activation of
specific cysteine proteases known as
caspases[12]. In the present study, DNA fragmentation with a ladder pattern
characteristic of apoptosis was observed in HepG2 cells treated with
CSE (1.0 and 2.0 mg/mL) for 24 h (Figure 4). We found that
the growth inhibitory activity of CSE was associated with
the induction of apoptosis in HepG2 cells. Data from an
apoptosis assay showed that CSE induced obvious apoptosis in hepatoma HepG2 cells, presenting a dose-
dependent manner of apoptosis-specific morphological
changes (Figure 3) and sub-G1 peak (Figure 5). A noticeable
phenomenon was that the apoptotic rate contrarily increased
to 64.15% after treatment with 2.0 mg/ml CSE (Figure 5). This
showed that CSE inhibited the proliferation of HepG2 cells
through inducing cell apoptosis.
We investigated the effect of CSE on the levels of 3
members of the Bcl-2 family, the pro-apoptotic Bax and the
anti-apoptotic Bcl-2, which regulate mitochondrial
apoptosis[19]. When Bax was overexpressed in cells, apoptotic death in
response to death signals was accelerated, earning its
designation as a death agonist. When Bcl-2 was overexpressed,
it heterodimerized with Bax and death was repressed, thus
the ratio of Bcl-2 to Bax is important in determining
susceptibility to apoptosis[20]. Following the exposure to CSE, we
found an increase of Bax mRNA and protein level (Figures
8A, 9A) in the HepG2 cells, paralleled by a downregulation
of Bcl-2 mRNA and protein level (Figures 8B, Figure 9B).
This indicates that the mitochondria is an important target
for CSE actions. These findings were similar to data
obtained with hepatoma carcinoma cells exposed to gefitinib as
pro-apoptotic agent[21]. Furthermore, caspases are a
ubiquitous family of cysteine
proteases[22]. DNA fragmentation, a hallmark of apoptosis, can be achieved, at least in part,
through the intrinsic and extrinsic pathways. The activation
of caspases-9 and -3 is the result of the induction of the
intrinsic pathway, while in the extrinsic pathway, caspase-8
and then caspase-3 were activated[23]. In both pathways, the
initiator caspase cleaves and activates downstream effector
caspases, such as caspase-3[12]. It could be concluded that
CSE induces apoptosis in HepG2 cells through the intrinsic
pathway of the caspase cascade as only caspases-9 and -3,
but not through the extrinsic pathway.
In general, there is a close correlation between apoptosis
and the cell cycle. If the cell cycle is blocked in a phase,
apoptosis appears in the phase in which the cell cycle is
blocked[1]. In the present study, an accumulation of cells in
the S phase was observed immediately after the treatment of
CSE (Figure 7). Our findings indicate that CSE inhibits or
arrests DNA replication in HepG2 cells in the S phase and
then signals for apoptosis before the cells enter the
G2 phase. Cyclins and their partners, cyclin-dependent kinases (CDK),
constitute the basis of these molecular mechanisms. CDK2
is activated by cyclin E and allows progression into the S
phase. CDK2 is also activated by cyclin A to allow
progression into the S phase[24]. The effect of CSE on the activation
of CDK2 and the activity of cyclin A in human hepatoma
carcinoma HepG2 cells requires further investigation. The
tumor suppressor protein p53 is a crucial protein in cellular
stress responses. Upon DNA damage, p53 protein
expression increases, and then transactivates its downstream
target genes, inducing cell cycle arrest, DNA repair, and
apoptosis[25,26]. It was reported that ethylnitrosourea induces
neural progenitor cell apoptosis after S-phase accumulation
in a p53-dependent manner[27]. Here, we showed that in
HepG2 cells possessing wild-type p53, CSE-induced S-phase
arrest and apoptosis were accompanied by the upregulation
of p53 mRNA and protein level in a dose-dependent manner
(Figures 8C, 9C). These results indicated that the activation
of the p53 pathway may be involved in CSE-induced cell
cycle arrest and apoptosis in HepG2 cells.
Cell apoptosis is a programmed death process, which is
induced and controlled by many complicated factors, such
as blockage of the cell cycle, changes of expression of
correlative apoptosis genes, and the elevation of caspase
activity[1]. Many anticancer drugs perform their curative effect
by inducing apoptosis of tumor cells through those pathways.
In conclusion, the data reported here indicate that CSE
inhibits growth and proliferation by arresting the cell cycle at
the S phase, and induces apoptosis of human hepatoma
HepG2 cells. This apoptosis was mediated by the
activation of intrinsic caspase cascade, the downregulation of the
Bcl-2 level, and the upregulation of Bax and the p53 level
in HepG2 cells. The results of the present study provide
supportive data for the anticancer potential of CSE.
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