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Introduction
Cervical cancer is the second most common cancer in
women worldwide, but it is the leading malignancy among
women in many developing countries[1]. Traditional herb
formulations have been re-evaluated by clinicians because
these medicines have fewer side-effects and are more
suitable for long-term use compared with chemically
synthesized medicines. New therapeutic strategies must be
evaluated to improve survival. Clinical responses can be improved
by identifying therapies that are particularly effective in
activating apoptosis in vivo among cervical carcinoma cells.
Oridonin, a traditional herb formulation, has been
successfully used for women's diseases. Oridonin is a natural,
biologically active substance isolated from Rabdosia
rubescens[2], one of several Chinese herbs in the once
popular dietary supplement called PC_SPES. PC_SPES, which
contains 7 Chinese and 1 North American herbal supplements,
has potent inhibitory activities against a variety of cancer
cells[3]. The use of the entire plant of this Chinese herb
purportedly offers digestive benefits, and anecdotal
evidence suggests its efficacy in the treatment of esophageal
carcinoma with little toxicity[4]. Oridonin, the main active
component of Rabdosia rubescens, is a tetracyclic diterpenoid
compound[5]. Tetracyclic diterpenoids are natural,
biologically-active substances with various physiological and
pharmacological properties, including antitumor
activities[6].
In recent years, apoptosis has emerged as the major
mechanism by which anticancer agents eliminate
preneo-plastic or neoplastic cells. Oridonin induces apoptosis in
neoplastic cells from the breast[7],
liver[8], lung[9]and
osteosarcoma[10]. Oridonin causes apoptosis of cancer cells by
inhibiting the activation of NF-κβ, signal transducer and
activator of transcription 3 and protein kinase B
(Akt)[11]. Oridonin also downregulates the expression of various genes
that NF-κβ regulates, including Bcl-2, cyclooxygenase-2
(COX-2), cyclinD1, and adhesion
molecules[12]. The phosphatidylinositol 3-kinase (PI3K)/Akt pathway plays an
important role in various cellular processes including cell
growth, survival, and motility[13]. In addition, the abnormal
expression of the PI3K/Akt pathway has been reported for
many human tumors[14]. Phosphatase and tensin homolog
deleted on chromosome 10 and Src homology 2-containing
5' phosphoinositol phosphatases, which serve as negative
regulators of the PI3K/Akt pathway, are frequently mutated
in a number of human cancers[15,16]. The principal target of
the PI3K pathway is the activation of Akt (protein kinase B),
which directly effects apoptosis by targeting the
pro-apoptotic Bcl-2-related protein Bad. In addition, PI3K plays
a role in regulating the proliferation of many cell
types[17]. Akt prevents apoptosis by generating anti-apoptotic
signals by phosphorylating Bad, glycogen synthase kinase 3
(GSK3), and caspase-9, and by activating transcriptional
factors, such as forkhead box class O (FOXO)-1 and
NF-κβ[18_22]. Therefore, the deregulated signaling functions in the
PI3K/Akt pathway make it a potential target for
chemotherapeutic agents.
Cervical carcinoma accounts for about 10% of all newly
diagnosed cancers in women worldwide[1]. Cervical
carcinoma in patients with a poor prognosis is characterized by
rapid cellular proliferation and strong expression of
anti-apoptotic genes. These features may be due to incomplete
cell cycle arrest and apoptosis resistance to conventional
therapies. The purpose of this study was to explore the
mechanism of the chemopreventive effects of oridonin in
the human cervical carcinoma HeLa cell line. We examined
the involvement of Akt and its substrates FOXO and GSK3
in HeLa cells, the role of members of the Bcl2 and inhibitor of
apoptosis protein (IAP) families, the effect of oridonin on
mitochondrial-mediated apoptosis involving
cytochrome c release, and the activity of caspases-3 and -9 in propagating
death signals.
Materials and methods
Reagents The Kunming Institute of Botany, the
Chinese Academy of Sciences (Kunming, China) provided
oridonin (purity 99.7%) isolated from the aerial parts of
Rabdosia rubescens. The method for isolation of oridonin
was the same as that described
previously[23]. Stock solution for oridonin (10 mmol/L) was prepared in DMSO and
protected from light at 4 oC, then diluted to a appropriate
concentration with medium immediately before use.
Bisbenzi-mide (Hoechst H33342), ethidium bromide, rhodamine,
monoclonal mouse anti-β-actin antibody, and heat-inactivated
fetal bovine serum were purchased from Sigma-Aldrich (St
Louis, MO, USA). Z-D(OMe)-E(OMe)-V-D(OMe)-FMK (z-DEVD-fmk), (EMD Chemicals, Inc. San Diego, CA, USA)
were prepared in 100 mmol/L in DMSO stock solution.
Propidium iodide was purchased from Bio Basic (Toronto,
Canada). L-Glutamine (200 mmol/L) and a mix of
penicillin-streptomycin (10000 IU/mL and 10000 mg/mL, respectively)
were purchased from Mediatech (Herdon, VA, USA). Antiphospho-Akt antibodies
(Ser473, Thr308),
anti-poly-adenosine diphosphate_ribose polymerase (PARP) and
anti-caspase-3 antibodies were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA, USA). Anti-phospho-GSK3,
anti-phospho-Forkhead Drosophila homolog
rhabdomyosarcoma like 1 (FKHRL1), anti-cleaved caspase-3, anti-cIAP1,
anti-X-linked inhibitor of apoptosis protein (XIAP), and
antisurvivin antibodies were purchased from Cell Signaling
Technologies (Beverly, MA, USA). The anticaspase-9
antibody was obtained from R&D Systems (Minneapolis, MN,
USA). Secondary horseradish peroxidase-labeled goat
antimouse and antirabbit antibodies were obtained from
Santa Cruz Biotechnology (Santa Cruz, CA, USA). The
Polyvinylidene fluoride membrane was purchased from
Millipore (Billerica, MA, USA). Plastic dishes were obtained
from Corning-Costar (Cambridge, MA, USA), and all other
tissue culture plastics were from Falcon Plastics (Los
Angeles, CA, USA).The Bio-Rad protein assay based on
the Bradford dye-binding procedure was obtained from
Bio-Rad Laboratories (Hercules, CA, USA). The enhanced
chemiluminescence detection system was purchased from
Amersham (Piscataway, NJ, USA). All other reagents were
of analytical reagent quality.
Cell culture and treatment The clonal human cervical
carcinoma HeLa cells, obtained from American Type Culture
Collection (ATCC, Manassas, VA, USA), were
grown in Dulbecco's modified Eagle's medium supplemented with
10% (v/v) fetal calf serum, 100 µg/mL streptomycin, and 100 U/mL
penicillin. Cultures were maintained at 37
oC in a humidified incubator in an atmosphere of 5%
CO2. The cells were exposed to oridonin at different concentrations and for
different times. The cells grown in medium containing equivalent
amounts of DMSO without oridonin served as the controls.
Cell viability assay The viability of the HeLa cells was
assessed by using an
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay, which was based on
the reduction of MTT by the mitochondrial dehydrogenase
of intact HeLa cells to a purple formazan product. A total of
2×104 HeLa cells were plated on a 96-well plate. After
incubation for 24 h, they were treated with oridonin at different
concentrations for different intervals. After treatment, the
medium containing oridonin were carefully aspirated. 100
µL of 0.5 mg/mL MTT was added to the cell culture medium
in the wells and incubated for 4 h, as described
previously[24]. We added 100 µL of 1% SDS to each well after 4 h incubation.
The plates were covered with aluminum foil and kept in an
incubator for 12 h to allow the formed formazan crystals to
dissolve. An ELISA reader was used to measure the
absorbance at 570 nm, and the half maximal inhibitory
concentration (the concentration that caused 50% inhibition of cell
proliferation) was assessed by using the Bliss
method[9].
Hoechst 33342 staining Apoptotic morphology was
studied by staining the cells with Hoechst 33342. The HeLa
cells were seeded on coverslips on a 6-well plate and treated
with 25 µmol/L oridonin. After 24 h, the cover glasses were
carefully washed with phosphate-buffered saline (PBS) and
stained with 20 µg/mL Hoechst 33342 for 10 min. Thereafter,
the HeLa cells were washed in PBS and observed under a
fluorescence microscope (Leica Microsystems AG, Wetzlar,
Germany).
Flow cytometry analysis Flow cytometry (FCM) was
used to quantitatively assess the rates of apoptosis. A total
of 1×106 HeLa cells were plated onto 10 cm tissue culture
dishes 1 d before treatment. They were then treated with 25
µmol/L oridonin for 6, 12, and 24 h. At the indicated time,
floating and attached HeLa cells were harvested, washed
with PBS, fixed in 70% ethanol overnight at 4
oC, and stained with 50 mg/mL propidium iodide. The
sub-G1 peak (DNA content <2 N) was measured by means of FCM (FACScan;
Becton Dickinson and Company, Franklin Lakes, NJ USA)
and analyzed by using CellQuest software (BD, Franklin
Lakes, NJ USA, USA).
Assay for cytochrome c release
The release of cytochrome c from mitochondria was assayed as described
elsewhere[4]. In brief, the HeLa cells were treated with and
without oridonin and centrifuged at
1000×g. The cell pellets were resuspended in 5 volumes of a hypotonic buffer containing
20 mmol/L HEPES-KOH (pH 7.5), 10 mmol/L KCl, 1.5 mmol/L
MgCl2, 1 mmol/L ethylenediamine tetraacetic acid, 1 mmol/L
ethylene glycol tetraacetic acid, 1 mmol/L DL-Dithiothreitol
(DTT), 20 µg/mL leupeptin, 10 µg/mL aprotinin, and 250
mmol/L sucrose, and incubated for 15 min on ice. The HeLa cells
were homogenized by passing them 15_20 times through a
22 gauge, 1.5 inch-long needle. The lysates were
centrifuged at 1000×g for 5 min at 4
oC to create a pellet of nuclei and unbroken cells. The supernatants were collected and
centrifuged at 12 000×g for 15 min. The resulting
mitochondrial pellets were resuspended in lysis buffer. The
supernatants were transferred to new tubes and centrifuged again at
12 000×g for 15 min, and the resulting supernatants, which
represented cytosolic fractions, were separated. We
analyzed 20_25 µg of protein from the cytosolic fraction of each
sample by performing immunoblotting using an antibody
against cytochrome c.
Measurement of mitochondrial membrane
potential After a fluorescent probe was incorporated, the HeLa cells were
incubated for up to 4 h with or without 25 µmol/L oridonin.
At first, 1×106 HeLa cells/mL was incubated with 10 µmol/L
rhodamine 123 for 10 min at 37 oC. At the end of the incubation,
the cells were washed twice with PBS, harvested by means
of centrifugation, and resuspended in 1.5 mL PBS. The
fluorescent intensity of each cell suspension was measured at
an excitation wavelength of 480 nm and an emission
wavelength of 530 nm in a L15B fluorescence spectrophotometer
(Perkin-Elmer, Waltham, MA, USA). The fluorescence
intensity was recorded in arbitrary units representing the
mitochondrial transmembrane potential.
Western blot analysis The HeLa cells were treated with
oridonin and lysed, as previously
described[25]. In brief, the cell pellets were resuspended in phosphorylation lysis buffer
(0.5%_1.0% TritonX-100, 150 mmol/L NaCl, 1 mmol/L
ethylenediamine tetraacetic acid, 200 µmol/L
sodium orthovanadate, 10 µmol/L sodium pyrophosphate, 100 µmol/L sodium
fluoride, 1.5 mmol/L magnesium chloride, 1 mmol/L phenylmethylsulfonyl fluoride, and 10 µg/mL aprotinin).
Protein concentrations were assessed using the Bradford
assay before the samples were loaded. Equal amounts of
proteins were separated by means of SDS-PAGE and
transferred to a polyvinylidene difluoride membrane.
Immuno-blotting was performed with different antibodies and
visualized using an enhanced chemiluminescence method.
Statistical analysis Data were expressed as mean±SD.
Statistical analysis was performed by applying a
one-way ANOVA followed by the Student's
t-test. P-values of less than 0.05 indicated statistical significance.
Results
Cytotoxicity of oridonin in HeLa cells and apoptosis of
HeLa cells When we investigated the effect of oridonin on
HeLa cell survival, the cells were treated for 24 h in medium
containing various oridonin concentrations (5_50 µmol/L).
The cells were counted by performing MTT studies.
Oridonin had a potent cytotoxic effect in HeLa cells in a
dose- and time-dependent manner and was expressed as a
percentage of cell viability (Figure 1A). The survival rate of
the HeLa cells treated with 25 µmol/L oridonin started to
decrease when treated for 6 h and sharply dropped after 12 h
incubation (Figure 1B).When we examined several hallmarks
of apoptosis (nuclear chromatin condensation and
fragmentation of DNA by Hoechst 33342), oridonin induced
apoptosis in human HeLa cells. In contrast to the control
cells, the cells exposed to 25 µmol/L oridonin provoked nuclear
chromatin condensation and fragmentation (Figure 2). On
morphological examination, the oridonin-treated cells had
changes typical of apoptosis, such as cell shrinkage, nuclear
fragmentation, and formation of apoptotic bodies. When we
quantified the apoptotic cells after oridonin treatment with
FCM and propidium iodide staining, we identified apoptotic
nuclei in the subdiploid region (sub-G1 peak) of the
histo-grams. Figure 3 shows the rates of apoptosis in the control
group compared with the cells treated with 25 µmol/L oridonin
for 6, 12, and 24 h. These results suggested that oridonin
treatment may suppress the growth of cervical carcinoma
cells by inducing apoptosis.
Induced dephosphorylation of Akt and its substrates in
HeLa cells Several lines of evidence appear to suggest that
Akt may be a critical target for the discovery of novel
anticancer drugs[26]. Oridonin inhibits the activation of Akt in
other cell systems[27]. When we determined the expression
and phosphorylation level of Akt in cervical carcinoma cells
after treatment with various concentrations of oridonin, Akt
was constitutively activated in HeLa cells, as determined by
the expression of phosphorylated Akt at
Ser473 and Thr308 (Figure 4A). Oridonin treatment dephosphorylated Akt at
both sites. These findings suggested that Akt plays an
important role in the survival of cervical carcinoma HeLa cells
and their inactivation lead to their growth inhibition and
apoptosis.
The forkhead family of transcription factors is reported
as a downstream target of Akt-mediated apoptosis in other
systems. Active transcription factors in the FKHR family
promote the transcription of genes in cell cycle arrest and
apoptosis[28]. One mechanism by which Akt promotes cell
survival is by phosphorylating the transcription factors of
the FKHR family, inactivating them, and preventing
apoptosis. Because forkhead proteins are implicated as both
Akt targets and a potential regulator of Fas
ligand[29], we studied the status of forkhead phosphorylation in
oridonin-treated and non-treated cervical carcinoma cell lines by
performing Western blotting. The constitutive
phosphorylation of FKHR was seen in the HeLa cell line (Figure 4B).
Treatment with oridonin inhibited this phosphorylation in a
dose-dependent manner.
We further investigated the effect of oridonin on the
activation of GSK3 in the HeLa cell line. GSK3 is reported as a
target of Akt and is involved in the promotion of cell
survival[30]. The cervical carcinoma HeLa cell line treated with oridonin
showed dephosphorylation of GSK3 (Figure 4C). These
results suggested that HeLa cells expressed a high level of
constitutive Akt and its downstream targets FOXO
transcription factors and GSK3. The treatment of oridonin
inactivated Akt and its downstream targets and thereby inhibited
HeLa cell growth.
Changes of mitochondrial membrane potential and
release of cytochrome c from mitochondria
Previous studies have demonstrated that Akt activity inhibits the release of
cytochrome c from mitochondria after UV
irradiation[25]. Therefore, we sought to determine whether oridonin
treatment causes the release of cytochrome
c from mitochondria to the cytosol. This release was examined with Western blot
analysis in HeLa cells after treatment with 25 µmol/L oridonin
for 24 h. Cytosolic-specific, mitochondria-free lysates were
prepared. Equal amounts of protein from the control and
oridonin-treated samples were separated on SDS-PAGE and
immunoblotted with an antibody against cytochrome
c.
Oridonin induced the translocation of cytochrome
c from the mitochondria to the cytosol in the HeLa cells (Figure
5A). When we further tested the effect of oridonin on the
mitochondrial membrane potential in the HeLa cells, oridonin
induced mitochondrial transmembrane depolarization, which
was represented as a decrease in the potential.
Oridonin-induced cytochrome c release was also observed in the HeLa
cells; this was represented as a significant increase in the
cytosolic cytochrome c concentration (Figure 5B).
Cytochrome c release and mitochondrial membrane potential was
analyzed spectrophotometrically. The data suggested that
a loss of mitochondrial membrane potential may be required
for oridonin-induced cytochrome c release into the cytosol,
which later triggered the cleavage and activation of
mitochondrial downstream caspases and the onset of apoptosis.
Activation of caspases-9 and -3 and PARP cleavage in
HeLa cells We investigated whether the oridonin treatment
of HeLa cells activates caspases-9 and -3 and promotes
cleavage of PARP further downstream in the apoptotic pathway.
Oridonin treatment activated caspases-9 and -3 in HeLa cells
(Figure 5). On caspase-3 activity assay after treatment for 24
h, 25 µmol/L oridonin activated caspase-3 in HeLa cells.
These results were consistent with the data on cytochrome
c release and indicated that the activation of effector caspases
participate in oridonin-induced apoptosis in HeLa cells.
When we used the cell permeable caspase-3 inhibitor
(z-DEVD-fmk), oridonin activated caspase-3, and cleaved PARP.
However z-DEVD-fmk pretreatment abolished this induced
activation and cleavage (Figure 6).
Modulation of the IAP protein family in oridonin-induced
apoptosis in the HeLa cell line IAP are physiological
substrates of Akt that are stabilized to inhibit programmed cell
death. IAP have direct effects on caspases-9 and
-3[31]. Therefore, we also determined whether oridonin induced cell
death by modulating the expression of IAP family members
that ultimately determine the cellular response to apoptotic
stimuli. The HeLa cells lines were treated with 25 µmol/L
oridonin for 24 h, and the expression of cIAP1, XIAP, and
survivin were determined by Western blotting. Oridonin
treatment caused the downregulation of cIAP1, XIAP, and
survivin (Figure 7). These results suggested that Akt may
modulate these survival proteins for the survival of HeLa
cells.
Discussion
Apoptosis plays an important role in the maintenance of
tissue homeostasis by selectively eliminating excessive cells.
At present, many researchers focus on the ability of natural
and dietary agents to induce cancer cell apoptosis to seek
new anticancer drugs. In the present study, we demonstrated
that oridonin significantly inhibited HeLa cell growth and
induced apoptosis in a time- and dose-dependent manner.
HeLa cells treated with oridonin exhibited characteristic
morphological features of apoptosis, such as membrane
shrinkage, chromosomal condensation, and increased
caspase-3 activity. Moreover, the pre-incubation of cells
with z-DEVD-fmk, a specific caspase-3 inhibitor, effectively
inhibited caspase-3 activity and prevented oridonin-induced
cell death. Oridonin induced apoptosis in HeLa cells in a
caspase-dependent apoptotic pathway.
Signal transduction pathways involved in growth and
apoptosis are potential targets for chemopreventive agents.
Oridonin, which is potently antitumorigenic, has
well-documented pro-apoptotic properties in a variety of cell
types[6_9]. The oridonin inhibition of
NF-κβ activity is accompanied by apoptosis in various types of cancer
cells[13]. Apoptosis is a multistep process, and an increasing number of genes
involved in the control or execution of apoptosis has been
identified. Our data showed that oridonin-induced HeLa cell
growth inhibition was accompanied by the inhibition of Akt
activity. Oridonin inhibited the Akt phosphorylation at
Ser473 and Thr308. For the full activation of Akt, cytokines and
growth factors must phosphorylate Akt at these
sites[32]. This observation further supports our hypothesis that the
inhibition of Akt activity by oridonin plays a critical role in
inducing growth inhibition by means of apoptosis. Studies have
also demonstrated that Akt activity inhibits the release of
cytochrome c from mitochondria after UV
irradiation[25]. In addition, constitutively activated Akt significantly protected
distal hereditary motor neuronopathy (HMN1) cells from
apoptosis induced by C2-ceramide[26].Our results are in
agreement with the finding that oridonin inhibited the
constitutive activity of Akt in HeLa cells accompanied with the
release of cytochrome c from mitochondria into the cytoplasm.
This result suggested a link between mitochondria and
oridonin-induced cell growth inhibition of cervical carcinoma
HeLa cells.
On exposure to oridonin, we also observed a
progressive decrease in the mitochondrial membrane potential and
release of cytochrome c into the cytosol in HeLa cells. In
many in vitro systems, apoptosis is associated with a loss
of mitochondrial membrane potential, which may correspond
to the opening of an outer membrane pore. Therefore, some
have suggested that this event is responsible for the
cytosolic release of cytochrome c from
mitochondria[33]. In this study, the accumulation of cytosolic cytochrome
c in oridonin-induced human HeLa cells was probably the
consequence of the loss of mitochondrial membrane potential,
which finally led to cell death. Members of the Bcl-2
homology family of proteins facilitate mitochondrial-mediated
apoptosis[34]. Reduced mitochondrial membrane potential
leads to the release of intermembrane proteins, such as
cytochrome c and apoptosis-inducing factors into the cytosol
and induces apoptosis[35].
In the cytosol, cytochrome c binds to Apaf-1 and
triggers Apaf-mediated caspase-9 activation, which activates
the death signal by activating caspase-3 and other
caspases[36]. Our data demonstrated that oridonin-mediated cytochrome
c release activated caspases-9 and -3 and cleaved PARP.
The expression of IAP is correlated with resistance to
apoptosis in transformed cell types and in a variety of
human tumors cells[37,38]. Akt regulates the expression
of many survival genes, including those encoding
IAP[39]. Our results showed that cervical carcinoma cells expressed IAP,
including cIAP1, XIAP, and survivin, and that oridonin
treatment decreased the expression level of these molecules. This
finding suggested that the downregulation of IAP may be
involved in the activation of caspases-9 and -3 in
oridonin-induced apoptosis.
In conclusion, oridonin induced apoptosis in cervical
carcinoma HeLa cells. The treatment of HeLa cells with
oridonin activated a cell-death pathway that regulated
mitochondrial membrane permeability by downregulating Akt
kinase signaling, triggering the release of
cytochrome c from the mitochondria into the cytosol. Loss of mitochondrial
membrane potential may be required for oridonin to induce
the release of cytochrome c, which led to the activation of
the downstream caspases-9 and -3 and the downregulation
of the survival proteins (cIAP1, XIAP, and survivin),
resulting in the death of cervical carcinoma cells. Taken together,
our data suggests that oridonin may be a promising
candidate as an antitumor agent against human cervical carcinoma.
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