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Introduction
There is an increasing demand for natural compounds
that improve humans' health. Many nutritive and
non-nutritive phytochemicals with diversified pharmacological
properties have shown promising responses for the prevention
and/or intervention of various
cancers[1]. Aloe-emodin (1,8-dihydroxy-3-hydroxymethyl-9,10-anthracenedione) is a herbal
anthracenedione derivative from Rhei
rhizoma, a traditional Oriental herb commonly used in laxation, antivirus, and
hepatoprotection practice[2_4]. Recent reports have shown
that aloe-emodin possesses antiproliferation effects on some
types of cancer cells, such as lung squamous, glioma, and
neuroectodermal cancer cells[5_7]. The anticancer mechanisms
of aloe-emodin involve the induction of caspase-dependent
apoptosis, where the greater sensitivity of neuroectodermal
tumor cell lines could be related to an energy-dependent
pathway[5,7]. Furthermore, the antiglioma action of
aloe-emodin involves extracellular signal-regulated kinases (ERK) 1
and 2-independent induction of both apoptosis and
auto-phagy, as well as the ERK inhibition-mediated
differentiation of glioma cells[6]. The inhibitory effect of aloe-emodin
on the activity and gene expression of N-acetyltransferase,
which plays an initial role in the metabolism of arylamine
carcinogens, was found in human malignant melanoma
cells[8]. Recently, Lin et al found that aloe-emodin-induced apoptosis
in T24 human bladder cancer cells was mediated through the
activation of p53, p21, Fas/Apo-1, Bax, and
caspase-3[9]. However, the anticancer molecular
mechanisms of aloe-emodin are largely unclear, especially for cervical cancer cells.
Cervical cancer continues to be a major public health
problem in the world. Of all neoplasms found in females
around the world, cervical cancer has the third highest
incidence and is the number four cause of
death[10,11]. In this study, in order to probe the mechanisms underlying the
chemopreventive potential of aloe-emodin on cervical cancer,
its effects on cell growth, cell cycle, alkaline phosphatase
(ALP) activity, protein kinase C (PKC), and c-myc
expressions in HeLa cells were investigated. The results of the
present study demonstrated the ability and detail
mechanisms of aloe-emodin with the potential anticancer
therapeutic activity of cervical cancer.
Materials and methods
Materials and cell line Aloe-emodin
(No A7687), 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT,
No M5655), and crystal violet (No C3886) were purchased
from Sigma (St Louis, MO, USA). RPMI-1640 medium was
purchased from Life Technologies (Grand Island, NY, USA),
and human cervical cancer cell line HeLa was obtained from
Shanghai Institute of Biochemistry and Cell Biology,
Chinese Academy of Sciences (Shanghai, China).
Cell culture The HeLa cells were cultured in plastic flasks
or multi-well plates at 37 oC in a humidified atmosphere of 5%
CO2 with RPMI-1640 medium containing 10% fetal calf serum,
50 000 U/L penicillin, and 50 mg/L streptomycin. The
medium was changed every other day. Exponentially growing
cells were used in the experiments. For the quantitative
assays of proliferation, 1×104 cells were seeded in 96-well plates
in regular growth medium and incubated for 24 h. The cells
were then incubated in medium at different concentrations
of aloe-emodin dissolved in dimethylsulphoxide
(Me2SO). The concentrations of aloe-emodin used were 2.5, 5, 10, 20,
and 40 µmol/L, respectively. The cells were then treated for
1_5 d and monitored for cell growth using the MTT assay.
In all the assays, the vehicle (Me2SO) was present at less
than 0.1% and the controls with the vehicle (0.1%
Me2SO) were carried out in parallel.
MTT assay Sets of 12 wells were used for each dose in
this assay. In total, 30 µL MTT solution [2 g/L in
phosphate-buffered saline (PBS)] was added into each of the 96 wells.
After the cells were incubated at 37 oC for 4 h, the medium
was removed and 150 µL of Me2SO was added to solubilize
the formazan. The microplate was shaken on a rotary
platform for 10 min. Finally, the optical density
(OD) values were measured at 550 nm using a Wellscan reader (Labsystems,
Santa Fe, NM, USA). The inhibitive rate was used to
indicate the suppressive effect of aloe-emodin on the HeLa cells.
Growth inhibition was calculated as a percentage as follows:
([ODcontrol_ODexperiment
]/ODcontrol)×100%[12]
.
Crystal violet assay The intensity of crystal violet
staining is directly proportional to the number of adherent
cells[13]. In order to observe the long-term antiproliferation effect,
the HeLa cells were seeded in flat-bottom 6-well plates at
1×104 cells/well and treated with various concentrations of
aloe-emodin for 8 d. Then the medium was removed very
carefully by mild suction, and 2 mL/well of 1% glutaraldehyde
solution in PBS was added. The plates were incubated for 15
min at room temperature to fix the cells. The fixative was
removed and replaced by the same amount of PBS. The PBS
was removed and the same amount of 0.02% aqueous
solution of crystal violet was added. After incubation at room
temperature for 30 min, the crystal violet solution was poured
and the plates were washed gently with water. Then 2 mL
70% ethanol was used to release crystal violet. Finally, the
absorbance was measured at 570 nm using a Wellscan reader.
Cell cycle analysis and apoptosis measurement
A total of 1×106 HeLa cells were treated with various concentrations
of aloe-emodin for 1_5 d. The cells were harvested with
0.25% trypsin and sedimented by centrifugation at
937×g for 5 min at room temperature. After the supernatant was
removed, ice-cold 70% ethanol was added. Finally, the cell
cycle was analyzed with a Coulter flow cytometer (Beckman
Coulter, Miami, FL, USA). The cell cycle distribution was
estimated according to standard
procedures[14]. The percentage of cells in the different cell cycle phases
(G0/G1, S, or G2/M phase) was calculated using Coulter Epicx XL-MCL
DNA analysis software (Beckman Coulter, USA). The
sub-G1 peak was considered a measure of
apoptosis[15,16].
Determination of relative ALP activities
The HeLa cells were seeded at a density of
1×104 and treated with 2.5, 5, 10,
20, and 40 µmol/L aloe-emodin for 1_5 d before being
assayed for ALP activity. The cells were then dissolved with
0.25% sodium deoxycholate. Finally, the ALP activities were
measured by dynamics assay with a Screen Master 3000
semi-automatic biochemistry analyzer (Hospitex Diagnostics,
Florence, Italy). The relative enzyme activity was expressed
as U/g[17,18]. Six wells of a 12-well plate were used for each
dose and treatment time. Three independent experiments
were performed in this analysis.
Western blotting Total cell lysates from
2×105 cells were prepared by lysing the washed cell pellet directly in
radioimmunoprecipitation buffer. The lysates were clarified
by centrifugation at 13 000×g for 15 min at 4 °C. The lysates
were boiled and separated by SDS_PAGE in 10%
polyacrylamide gels, blotted onto a polyvinylidene fluoride membrane
(Millipore, Bedford, MA, USA) and analyzed by Western
blotting with the antibodies from Boster Bioengineering
(Wuhan, China).
Statistics The statistical analysis was performed using
SPSS version 10.0 (SPSS, Chicago, IL, USA). The Student's
t-test was used to make a statistical comparison between the
groups. The level of significance was set at
P<0.05.
Results
Growth inhibitory effect of aloe-emodin on human
cervical cancer cells The effect of aloe-emodin on cell growth
was evaluated by MTT assay (Figure 1A). Aloe-emodin
inhibited the growth of HeLa cells in a time- and dose-dependent inhibitory manner at concentrations of 2.5_40
µmol/L (at 2.5 µmol/L_20 µmol/L,
P<0.01; at 40 µmol/L, P<0.001).
Next, to observe the long-term effect, the crystal violet
assay was used. The results showed that the number of
adherent cancer cells was decreased by aloe-emodin
(P<0.001, Figure 1B). These data imply that aloe-emodin has a significant
growth inhibitory effect on HeLa cells in
vitro.
Effect of aloe-emodin on the cell cycle progression of
HeLa cells In order to decipher the suppressive
mechanisms of aloe-emodin on HeLa cells, changes in the cell cycle
distribution were monitored by flow cytometry. The
treatment of aloe-emodin resulted in a time-dependent increase
in the distribution of cells at the G2/M phase (Table 1).
Furthermore, the sub-G1 peak (apoptosis peak) was not
observed (data not shown). By using DNA fragmentation
analysis, the DNA ladder was not obviously observed (data
not shown). Next, the levels of the cell cycle-associated
proteins were determined by Western blotting. As shown in
Figure 2, aloe-emodin decreased the abundance of cyclin A
and cyclin-dependent kinase (CDK) 2, while increased cyclin
B1 and CDK1 in the protein levels in a dose-dependent manner.
Such data were consistent with arresting cells in the
G2/M boundary. Cyclins function as regulators of CDK. Cylin A
binds and activates CDK2, and thus promotes both cell cycle
G1/S and G2/M
transitions[19]. Cyclin B1 binds and activates
CDK1, and is expressed predominantly during the
G2/M phase[20]. The results obtained from this study suggest that
one of the mechanisms of the growth inhibitory effect of
aloe-emodin on HeLa cells is through cell cycle arrest, but
not by apoptosis induction, at least at the concentrations
observed.
Increase of ALP activity by aloe-emodin In order to
determine whether aloe-emodin is capable of affecting ALP
activity in HeLa cells, a dynamics assay was used. The ALP
activity was increased by aloe-emodin and reached its peak
level at 5 d (Figure 3A). The statistical significance between
the ALP of the control and treated cells was observed (5
µmol/L, P<0.05; 10, 20, and 40 µmol/L,
P<0.01). As the proliferating cell nuclear antigen (PCNA) is a commonly used
marker of both cell proliferation and
differentiation[21], its changes in the protein level was studied by Western blotting.
As shown in Figure 3B, aloe-emodin decreased the
abundance of PCNA.
Aloe-emodin decreases PKCα and c-myc in HeLa cells
As both the PKC pathway and c-myc participate in a wide
range of cellular programs controlling proliferation,
differentiation, and survival, the influence of aloe-emodin
on the activation of these important signaling molecules in
HeLa cells was examined. Western blotting results showed
that the PKCα and c-myc protein levels in the control group
was high, while after treatment with aloe-emodin, both
decreased (Figure 4). A clear dose-dependent decrease was
found in cancer cells treated by aloe-emodin (Figure 4).
Discussion
Phytochemicals present in medicinal herbs and dietary
plants are one of the most attractive approaches in cancer
chemotherapy. Aloe-emodin, a hydroxyanthraquinone from
Rhei rhizoma leaves, has been found to have anticancer
effects in several cancer cell
lines[5_7]. More importantly, aloe-emodin was found to have no appreciable toxic effects
in vivo[7]. Until now, the effect of aloe-emodin on cervical
cancer has been largely unknown.
The results of the present study clearly demonstrate the
anticancer activity of aloe-emodin on human cervical cancer
HeLa cells. Aloe-emodin inhibited the growth of HeLa cells
in a dose-dependent manner from 2.5 µmol/L to 40 µmol/L
(Figure 1A). At the same time, crystal violet assays
indicated that aloe-emodin has a durable growth inhibition on
cervical cancer cells (Figure 1B).
To examine the mechanism responsible for cell growth
inhibition, cell cycle distribution was evaluated using flow
cytometry. The loss of the proliferative capacity of
cervical cancer cells treated by aloe-emodin was associated with the
G2/M phase arrest (Table 1). At the same time, the cell
cycle-associated proteins were obviously involved (Figure 2).
Similar to our observations, several other studies found that
aloe-emodin blocked human glioma U251 cells and promyelocytic
leukemia HL60 cells at the G2/M
phase[6,22], and hepatoma cells at the
G0/G1 phase[23]. These suggest that multiple
mechanisms may be responsible for the anticancer effects of
aloe-emodin on different types of cancers.
Cellular ALP are increasingly recognized as important
markers for monitoring tumor cell behavior in human
malignancies. The measurement of ALP activity is usually
used to determinate the effect of inducing the
differentiation of anticancer
reagents[24]. Early in vitro studies showed that
some drugs, such as Ara C, peptichemio, or hydrocortisone,
inhibited HeLa cell growth with the elevation of ALP
activity[25]. In this study, the ALP activity in HeLa cells treated by
aloe-emodin increased in a time-and dose-dependent manner
(Figure 3A). This is one of the first studies to focus on the
expression of ALP in human cervical carcinomas cells treated
by aloe-emodin.
Cell signal pathways have been become the targets for
many drugs. Among them, the PKC pathway is gaining more
and more attention for cancer chemotherapy. Several
studies have shown that PKCa plays a role in tumor proliferation
and survival[26,27]. In this study, we found that
PKCα was suppressed by aloe-emodin (Figure 4). These data strongly
suggest that PKCα is one of the key targets of the antitumor
action of aloe-emodin. To test this hypothesis, we observed
the expression change of c-myc, a target gene of
PKCα[28]. As shown in Figure 4, c-myc was also decreased by
aloe-emodin. As we know, c-myc is an immediate early gene
encoding transcription factors expressed in the
G1 phase of the cell cycle and has a DNA-binding property. Furthermore,
the inhibition of c-myc by antisense oligomers has been
shown to inhibit cell proliferation[29]. From this experiment, it
is well established that aloe-emodin has a downregulatory
effect on the expression of c-myc in human cervical cancer
cells (Figure 4). Since c-myc is the downstream target of the
PKC pathway, this effect may be through the PKCα pathway.
Taken together, aloe-emodin can affect cell growth, cell
cycle, and ALP activity of human cervical cancer
cells in vitro.
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