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Introduction
Herbal medicine, Donglingcao (rabdosia
rubescens), has been traditionally used in China for the treatment of leukemia.
Oridonin is a diterpenoid compound isolated from
rabdosia rubescens. It has potent antitumor
functions[1] and has been used for the treatment of human cancers, especially
esophageal carcinoma[2]. This compound has been observed to
prevent mutation, decrease Na+-pump transportation
activity of cancer cells, and promote the efficiency of other
antitumor agents[3_5]. It induces apoptosis to inhibit cancerous
cell proliferation, as well as enhances the sensitivity of
drug-resistant cancer cell lines[6,7].
Apoptosis is an essential and highly conserved mode of
cell death that is important for normal development, host
defense, and the suppression of oncogenesis. Apoptosis
removes cancerous or virally-infected cells, and aberrant
apoptosis is the major cause for tumor development and
progression[8]. Among the numerous proteins and genes
involved, members of caspase family and the Bcl-2 family
play pivotal roles in modulating apoptosis. Apoptosis is
mediated by the activation of caspases, which amplify the
apoptotic signal and proteolytically process numerous
cellular target molecules with various
functions[9]. Bcl-2 family proteins are vital for the regulation of apoptosis by
controlling the mitochondrial membrane potential to release
cytochrome c and for activating caspase-9 and
-3[10,11]. The balance between pro-apoptotic proteins and anti-apoptotic
proteins determines the fate of cells.
The DNA damage response, caused by a variety of
stimuli, arrests the cell cycle to allow damage repair or direct
cell apoptosis[12]. An imbalance between DNA damage and
DNA repair activities may affect cell viability. After DNA
damage, the cell cycle is arrested at the transition from the
G1 to S phase or from the G2 to M phase of the cell cycle.
Increasing evidence indicates a central role for p53 in
mediating cell cycle arrest or
apoptosis[13,14].
In the present study, we demonstrated that activated p53
contributed to oridonin-induced cell cycle arrest and
apopto-sis, and mitochondrial alternations amplified the activation
of the caspase cascade; meanwhile, caspase-9, together with
calpain rather than caspase-3, led to oridonin-induced
apopto-sis in human breast MCF-7 cells.
Materials and methods
Chemicals Oridonin was obtained from Kunming
Institute of Botany (The Chinese Academy of Sciences, Kunming,
China), and the structure of oridonin was assigned by
comparing the chemical and spectral data
(1H-NMR, 13C-HMR) with those reported in other published
literature[15]. The purity of oridonin was measured by HPLC and determined to
be more than 99%. Oridonin was dissolved in dimethyl
sulfoxide (DMSO) to make a stock solution. DMSO
concentrations were kept at below 0.05% in all the cell cultures and did
not exert any detectable changes in cell growth or apoptosis.
Fetal bovine serum (FBS) was purchased from TBD
Biotechnology Development (Tianjin, China); propidium iodide (PI),
Rhodamine 123, and RNase A were purchased from Sigma
Chemical (St Louis, MO, USA). Thiazolyl blue (MTT) was
from Sino-American Biotechnology (Beijing, China);
Z-VAD-fmk and calpain inhibitor II were bought from Sigma
Chemical (St Louis, MO, USA); rabbit polyclonal antibodies against
Bax, p53, p-p53, p21, caspase-9 and -3, PARP, heat shock
protein (Hsp)90, the inhibitor of caspase-activated DNase
(ICAD), mouse polyclonal antibodies against Bcl-2, and
horseradish peroxidase-conjugated secondary antibody
(goat-anti-rabbit or goat-anti-mouse) were purchased from
Santa Cruz Biotechnology (Santa Cruz, CA, USA).
Cell culture The MCF-7 cell line (#CRL HTB-22) was
purchased from American Type Culture Collection (ATCC,
Manassas, VA, USA). The cells were cultured in RPMI-1640
medium (GIBCO,LA, NY, USA) supplemented with 10% FBS
and 0.03% L-glutamine (GIBCO, USA) and maintained at 37
°C with 5% CO2 in a humidified atmosphere.
Cell viability assay The cytotoxic effect of oridonin on
the MCF-7 cells was measured by MTT assay as described
in previous studies[16]. The cells were dispensed in 96-well,
flat bottom microtiter plates (NUNC, Roskilde, Denmark) at a
density of 1×104 cells per well. After 24 h incubation, they
were treated with various concentrations of oridonin,
followed by 12, 24, 36, and 48 h cell culture. 20 µL MTT
solution (5.0×103 mg/L) was added to each well 4 h before the end
of incubation. The effects of Z-VAD-fmk and
calpain inhibitor II on oridonin treated MCF-7 cells were also determined
by MTT assay, and both inhibitors were added into the cell
culture 1 h before oridonin administration, respectively. The
resulting crystals were dissolved in DMSO. Absorbance
was measured with an ELISA reader (TECAN SPECTRA, Wetzlar, Germany). The cytotoxic effect was expressed as a
relative percentage of inhibition calculated as follows:
Relative inhibition (%)=[(A 490
control - A 490
oridonin)/A 490 control]×100
Observation of morphological changes The MCF-7 cells
were plated in the wells of a 6-well plate at a density of
3×105 cells per well. After cultured for 24 h, the cells were treated
with 80 µmol/L oridonin and incubated for 12 or 24 h. The
cellular morphological changes were observed using phase
contrast microscopy (Leica, Nussloch, Germany).
Membrane leakage assay The integrity of the plasma
membrane of the MCF-7 cells was determined by monitoring
the cell lactate dehydrogenase (LDH) leakage, which was
accomplished by following the rate of conversion of NADH
to NAD+. LDH released into the extracellular medium was
expressed as a percentage of total
LDH[17,18]. In brief,
1.5×106 cells were cultured with oridonin for 0, 8, 16, 24, and 36 h.
Floating dead cells were collected from the culture medium
by centrifugation, and the LDH content from the pellets
lysed in 0.1% NP-40 for 15 min was used as an index of apoptotic
cell death (LDHp). The LDH released in the culture medium
[extracellular LDH (LDHe)] was used as an index of necrotic
cell death. The LDH released from adherent and viable cells
was expressed as intracellular LDH (LDHi). The substrate
reaction buffer of LDH was added. The A value at 490 nm of
reaction for 1 and 5 min was assayed. LDH
activity=(A5 min-A1
min)/4. The percentage of apoptotic and necrotic cell death
was calculated as follows:
% apoptosis=[LDHp/(LDHp+LDHe+LDHi)]×100
% necrosis=[LDHe/(LDHp+LDHe+LDHi)]×100
Mitochondrial transmembrane potential alternation
Mitochondrial transmembrane potential
(Δψmit) alternation was determined by Rhodamine 123 staining. For the
fluorescence observation, the MCF-7 cells were seeded into 6-well
plates and mounted on the coverslips. For the flow cytometric
analysis, the cells were harvested by tripsin. After culture
with oridonin for 12 h, the cells were removed from the
culture medium for staining. The culture medium was replaced
with phosphate buffered solution (PBS) and washed 3 times.
The cells were incubated in Rhodamine 123 staining stock
solution (5 g/L) for 20_30 min at 37 °C. The coverslips were
washed 3 times in PBS and mounted with the stained cells
for fluorescent microscopy. Mitochondrial transmembrane
potential changes were indirectly determined by measuring
Rhodamine 123 fluorescence variance using a cytoflowmeter
with an emission wavelength of 525 nm and an excitation
wavelength of 488 nm. The samples were quantified as quickly
as possible[19_21].
DNA extraction and detection of DNA
fragmentation The MCF-7 cells
(2×106 cells) were harvested with pancreatin and
centrifuged at 1000×g for 10 min. The cell pellets were
suspended in 10 mmol Tris-HCl (pH 7.4), 10 mmol edetic
acid 0.5% Triton X-100, and 40 µg/L proteinase K (Merck,
WS, NJ, USA) at 37 °C for 2 h. The lysate was extracted with
0.5% 5 mol/L NaCl and 50% 2-propanol and incubated
overnight at -20 °C, and then centrifuged at
7 000×g for 20 min. The supernatant was washed with 70% ethanol and
centrifuged at 7 000×g and the pellets were dried and resuspended
in10 mmol/L Tris-HCl (pH 7.4) and 1 mmol/L edetic acid. DNA
was incubated with 40 µg/L RNase A (Sigma, St Louis,
MO,USA) at 37 °C for 60 min, separated by 2 % agarose gel
electrophoresis at 100 V for 40 min, and stained with 0.1
mg/L ethidium bromide[22,23].
Flow cytometric analysis After the oridonin treatment,
the sample preparation was performed as previously
described[24,25]. 1×106
MCF-7 cells were harvested and washed once in cold PBS. The cell pellets were fixed in 75% ethanol
at 4 °C overnight and washed in cold PBS. Then the pellets
were suspended in 1 mL of 50 mg/L PI solution, 0.1%
(w/v) sodium citrate, and 0.1% (v/v) Triton X-100. The cell samples
were incubated at 4 °C in the dark for at least 15 min, and
analyzed by a FACScan flowcytometer (Becton Dickinson,
Franklin Lakes, NJ, USA).
Western blotting 2×106 MCF-7 cells were pre-incubated
with or without 2 mmol/L 3-methyladenine (3-MA) for 1, then
treated with 64 μmol/L oridonin for 12, 24, 36, or 48 h. Both
adherent and floating cells were collected and Western
blotting was performed as previously
described[26]. Briefly, the MCF-7 cells were washed with ice-cold PBS and solubilized
with lysis buffer (1% SDS, 1 mmol/L phenylmethylsulfonyl
fluoride, 1 mmol/L EDTA, 2 mmol/L leupeptin, and 1 mmol/L
aprotinin). The protein concentration was determined by
the Bio-Rad DC protein assay (Bio-Rad Laboratories,
Hercules, CA, USA). The protein lysates were separated by
12% SDS-PAGE and transferred to a nitrocellulose membrane.
The membranes were soaked in blocking buffer (5% skimmed
milk in PBS), and then incubated overnight with primary
antibodies, followed by horseradish peroxidase-conjugated
secondary antibodies. The color was developed with
diaminobenzidine (DAB).
Statistical analysis All data represent at least 3
independent experiments and are expressed as the mean±SD unless
otherwise indicated. Statistical comparisons were made by
Students's t-test. P-values of less than 0.05 were
considered significant.
Results
Oridonin induced apoptosis in MCF-7 cells Oridonin
induced MCF-7 cell death in a time- and concentration-
dependent manner. The IC50 value of 24 h of oridonin
treatment for the MCF-7 cells was 84.18 µmol/L. The treatment of
the MCF-7 cells with 80 µmol/L oridonin for 24 h induced
approximately 48.3% of the growth inhibition (Figure 1).
Mean-while, marked morphological changes were observed
compared with the untreated control. The oridonin-treated
MCF-7 cells underwent retraction of cellular processes and
became round in shape at 12 h (Figure 2B). By 24 h, the
majority of the MCF-7 cells had shrunk in shape. Blebbing
nuclei and granular apoptotic bodies appeared, and some
cells were almost floating (Figure 2C). Untreated cells did
not show these apoptotic characteristics (Figure 2A). After
treatment with 80 µmol/L oridonin for 12, 24, and 36, h with
40, 80, and 120 µmol/L oridonin respectively the MCF-7 cells
began to generate typical DNA fragmentation, which is a
hallmark of apoptosis (Figure 4). The number of apoptotic
cells increased from 8.24% at 8 h to 52.74% at 36 h in the
presence of oridonin, while the necrotic cell proportion also
increased from 7.58% at 8 h to 19.24% at 36 h (Figure 3).
However, the percentage of necrotic cells was still negligible
compared with that of apoptotic cells. These results
demonstrated that treatment with oridonin induced the majority of
MCF-7 cell apoptosis.
Oridonin induced disruption of mitochondrial integrity
Rhodamine 123 was first used to measure the
Δψmit in intact cells, both as a microscopic stain and by cytoflowmeter by
monitoring the increase in fluorescence due to its
electrophoretic accumulation in mitochondria. In isolated
mitochondria, energization induced a red shift and extensive
quenching of Rhodamine 123 fluorescence; therefore, dye
accumulation could be suggested as a sensitive and
specifically fluorescent potentiometric probe of
Δψmit of mitochondria in living
cells[27]. The oridonin-treated MCF-7 cells
displayed a specifically lower Rhodamine fluorescent density
(Figure 5A) and lower transmembrane potential (Figure 5B)
compared with the untreated cells, indicating that oridonin
damaged the mitochondrial respiratory chain and triggered
apoptosis. Moreover, the alternations of fluorescence and
transmembrane potential were both in a dose-dependent
manner in oridonin-challenged MCF-7 cells.
Oridonin induced S phase arrest and the upregulation
of p53 and p21 To investigate further features of cell growth
inhibition by oridonin, a flow cytometric analysis was
performed. After treatment with oridonin for 12 h, the cells
were accumulated in the S phase. The
Sub-G0/G1 peak
denoting apoptosis was clearly observed (Figure 6).
Simul-taneously, aneuploid-presented tumor cells were declined
after oridonin treatment (Table 1). Meanwhile,
phosphorylated p53 and p21 proteins were also both upregulated at 12
h, while non-phosphorylated p53 remained the same (Figure 7).
Both caspases and calpain facilitated cell death
Caspases are unique cysteine proteases that are synthesized as
inactive precursors and are activated during apoptosis. Among
them, caspase-3 is a common downstream apoptosis effector,
which is processed and activated by caspase-9 or -8 and
digested into the heterodimeric form (17_12 kDa) by
mitochondrial pathways or through the activation of death-
domain containing receptors[28]. Calpain, like caspase-3, is a
cytosolic cysteine protease, but requires
Ca2+ for its activity. Ubiquitous calpain exists in resting
cells, but it is activated by
Ca2+ and triggers autolytic processing; it is also activated
in some apoptosis systems[29].
To investigate the influences of caspases and calpain on apoptosis, we applied their
inhibitors into experiment systems. 10 µmol/L pan-caspase
inhibitor Z-VAD-fmk and/or 20 µmol/L calpain inhibitor
¢ò was introduced into the cell culture, then after 1 h, 80 µmol/L
oridonin was added. Both inhibitors exerted protective
effects on cell growth, indicating that caspases and calpain
accelerated oridonin-induced MCF-7 cell death. Thus, we
further investigated whether the combined use of these two
inhibitors in the cells could be sufficient to block the cell
death. The resulting data showed that the inhibitory ratio
further declined, compared with single inhibitor
administration of each inhibitor, but was not as low as the control level,
indicating that there might be some other factor(s)
influencing cell death (Figure 8).
Effects of oridonin on the expression of Bax, Bcl-2,
caspase-9, and Hsp90 Since anti- and pro-apoptotic
members of the Bcl-2 family arbitrate the survival-or-death
decision, we detected the expressions of Bcl-2 and Bax by
Western blot analysis. After the oridonin treatment, the
expression of Bcl-2 decreased; on the contrary, the
expression level of Bax increased in a time-dependent manner.
Caspase-9 was a pivotally effective apoptotic protease in
the postmitochondrial pathway. In the present study, under
the condition of oridonin employment, procaspase-9 was
cleaved, and activated caspase-9 was enhanced in a
time-dependent manner. Hsp90 displayed protective functions
in many cell lines[14]. Here, when the MCF-7 cells were
exposed to oridonin, the Hsp90 expression was significantly
downregulated (Figure 9).
Expressions of caspase-3, PARP, and ICAD
ICAD was a classical substrate of caspase-3. After the oridonin treatment,
the expression of ICAD was unchanged (Figure 10), which
seemed to be consistent with the viewpoint that apoptosis
in the MCF-7 cells was not through
caspase-3[30]. To further confirm this hypothesis, we detected the expressions of
caspase-3 and PARP. In the MCF-7 cells, procaspase-3 was
not cleaved into the activated form. However, PARP was
activated and formed the 85 kDa isoform which was digested
from its 116 kDa precursor in a time-dependent manner,
indicating that other enzymes activated PARP. According to
this speculation, we examined a more promising candidate,
calpain, since it was reported that PARP was a calpain
substrate[36]. However, unexpectedly, under calpain inhibitor II
application, the cleavage of the PARP precursor was
partially blocked. 116 kDa PARP precursors were almost not
discerned disparity, and 85 kDa PARP protein expressions
decreased, suggesting that calpain partially participated in
PARP activation, inducing apoptosis, and bypassing
caspase-3.
Discussion
In this study, we have demonstrated that oridonin
inhibited cell growth, arrested the cell cycle, and induced apoptosis
in MCF-7 cells.
These biochemical events were possibly associated with
the p53 tumor suppressor gene. The p53 protein displayed a
key role of p53 in the G1/S checkpoint in response to DNA
damage[13] as a regulator of cell cycle progression and a
mediator of apoptosis in many cell lines. In response to
various types of DNA damage, the cell cycle checkpoints
and cell death signals are activated to stop cell growth and
to eliminate multiplication of the genetically-altered cells.
Damaged cells stop DNA replication at the
G1 or G2 phase, presumably allowing the repair systems to function before
the next cell cycle. Apoptosis is also triggered in response
to various DNA damage. The activation of the apoptotic cell
death pathway is a safeguard in removing irrepairably
damaged cells. Several cellular effector molecules, including p53,
are involved in arresting damaged cells at these checkpoints
and inducing apoptosis. The upregulation of the p53
protein is a common cellular response in many cell types
expos-ed to various DNA damaging
agents[14,31].
The cell cycle progression was decelerated by p53 and
cdk inhibitors, including p21[32]. The cyclin dependent
kinase inhibitor, p21, is a multifunctional protein involved in
coordinating the cellular response to negative growth signals.
Induced by cellular damage under the transcriptional
control of the p53 tumor suppressor protein, p21 interfaces with
a number of cellular proteins involved in growth control.
p21 plays an essential role in growth arrest after DNA damage,
and their overexpression leads to G1 and
G2 phase arrest[33]. Therefore, the activation of this signaling pathway has been
considered to be important for the efficacy of antitumor
agents, and direct transactivation of the p21 gene by
passing p53 can serve as a novel strategy for treating cancers
that are insensitive to classical antitumor agents. Our
results show that oridonin caused cell cycle arrest in the S
phase through the upregulation of p53 and p21. Here,
activated p53, after DNA damage by oridonin, might either
trigger the onset of DNA repair, leading to the completion of the
cell cycle, or lead to the exit from the cell cycle, apoptosis via
mitochondria.
Hsps are a large family of highly-conserved proteins
broadly categorized according to their size and functions,
which expressed under stress, usually confer survival
protection to the cell or interruption in the apoptotic pathways.
Among the Hsps, the Hsp90 family is ubiquitously expressed,
and is one of most abundant cytoplasm proteins. Hsp90 can
physically interact with either the mutant or the wild type
p53 in vivo[34], to partially block the apoptotic progression.
On the other hand, Hsp90 forms a cytosolic complex with
Apaf-1 and thereby inhibits the formation of the active
Apaf-1_caspase-9 apoptosome complex to negatively control
apoptosis[35]. Our findings demonstrate that Hsp90 declined
with oridonin application, and further confirmed that p53
exerted a double function on both cell cycle arrest and
apoptosis in oridonin-treated MCF-7 cells. On one hand,
p53 through p21 provoked cell cycle arrest at the S phase to
repair DNA damage by oridonin treatment in the MCF-7 cells;
on the other hand, p53 promoted apoptosis via Bax/Bcl-2 to
clean irrepairable cells from cell cycle arrest, which might be
an explanation as to why the oridonin-treated MCF-7 cells
did not arrest the cell cycle and apoptosis at the same time in
the present experiment system.
Caspases play a crucial role in the apoptotic progression,
morphological changes, and DNA fragmentation
suggesting that oridonin-induced MCF-7 cell death was involved in
a mechanism of apoptosis. One of the major pathways for
caspase activation involves the participation of mitochondria.
Bcl-2 inhibits the apoptotic process and promotes cell
survival, and Bax acts in the mitochondria to cause the
release of cytochrome c, leading to the activation of
caspase-9, and the subsequent activation of
caspase-3[36]. Moreover, Bax expression is regulated by p53 and the protein products
of the target genes of p53, including Bcl-2, are involved in
this process[37]. In the present study, oridonin decreased
the Bcl-2 expression and activated Bax, suggesting that the
mitochondria was involved in oridonin-induced apoptosis.
Upon cytochrome c release into the cytoplasm, Apaf-1 is
activated and triggers the caspase cascade. One of the key
events in this pathway is the caspase-3-mediated cleavage
of the ICAD, which allows caspase-activated DNase to
enter the nucleus and causes oligonucleosomal DNA fragmen-tation. In this study, ICAD was left unaffected due
to no active form of caspase-3 rather than lack of capase-3
expression, which was different from the view that the
MCF-7 cell line was deficient in procaspase-3 according to previous
studies[38], but DNA fragmentation was induced in this study.
More recently, it has been discovered that in response to
apoptotic stimuli, mitochondria can also release
caspase-independent cell death effectors such as apoptosis
inducing factor (AIF). AIF could induce nuclear apoptosis in a
variety of cell types and this effect was not inhibited by
pharmacological caspase inhibitors such as zVAD,
indicating that AIF can trigger nuclear apoptosis in a
caspase-independent manner. AIF binds to DNA in a
sequence-independent manner, which determines the entry of this complex
into the nucleus. It recruits or activates an endonuclease to
facilitate DNA fragmentation and chromatin
condensation[39]. Thus, it was probable that DNA was fragmented by AIF in
our experiment system. Meanwhile, capase-9 was
upregulat-ed; we could speculated that after the mitochondrial and
postmitochondrial caspase-9-dependent pathways were
activated, other effective caspases such as caspases-6 or -7,
but not caspase-3, might take responsibility for apoptotic
signal transduction. In addition, it was worth noting the
participation of calpain, a Ca2+-dependent intracellular
cysteine protease in oridonin-treated MCF-7 cell death. Calpain
is activated in various necrotic and apoptotic conditions,
while caspase-3 is only activated in apoptosis. Caspases
and calpains share several substrates, including PARP, and
during apoptosis, the 116 kDa PARP is degraded by
caspase-3 to distinct 89 and 27 kDa fragments; however, recently it has
been found to be cleaved by calpain at alternative sites,
generating fragments from 70 to 40 kDa in size during
necrosis[40]. Furthermore, growing evidence suggests that calpain
may play a central role in the execution of apoptosis. Our
results show that calpain inhibitor II could partially rescue
oridonin-induced MCF-7 cell death and PARP cleavage,
indicating that calpain contributes to cell death and replaces
caspase-3 to execute PARP activation in part. All data here
allowed us to speculate that other enzymes existed to induce
the apoptotic signals. Caspase-7 might substitute for
capase-3 in most cell types and
tissues[41], since it is highly homologous to caspase-3 and has very similar substrate specificity.
Further studies determining the factor(s) prohibiting
caspase-3 cleavage remain to be conducted.
Maintenance of a significant electrical potential
difference across biological membranes is crucial for a variety of
cellular functions, including development, signaling,
movement, energy balance, and apoptosis. Intracellular
organelles such as mitochondria possess function-related
membrane potentials far exceeding that of the plasma
membrane. The dissipation of the inner mitochondrial
transmembrane potential marks the point of no return during the
apoptotic program and occurs prior to DNA fragmentation.
Thus, the evaluation of mitochondrial transmembrane
potential depolarization is of critical importance for the
assessment of apoptosis[42,43]. As our results demonstrated,
exposure to oridonin caused an emission of Rhodamine
fluorescence which represented the declined mitochondrial
membrane potential, inferring that oridonin enhanced apoptosis
concomitantly with a decrease in Δψmit.
In summary, oridonin inhibited MCF-7 cell growth and
arrested cell cycle through the activation of p53 in response
to DNA damage, and decreased cdk activities by p21 was
involved in the apoptotic progression. Simultaneously,
oridonin induced cell apoptosis mediated by p53, through
the upregulation of Bax and the downregulation of Bcl-2 and
Hsp90, which contributed to the activation of caspase-9,
leading to the activation of downstream caspases in the
process. Moreover, calpain bypassed caspase-3 to partially
contribute to oridonin-induced MCF-7 cell death.
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