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Introduction
Pseudolaric acid B (PAB; Figure 1) is a diterpene acid
isolated from the root and trunk bark of Pseudolarix
kaempferi Gordon (Pinaceae), known as "Tu-Jin-Pi" in
Chinese, which has been used to treat dermatological fungi
infections. PAB exerts potent antifungal,
antimicrobial[1],
antifertility[2,3], and cytotoxic activity
in vitro[4], and the mechanisms of PAB-induced cell death in human myelocytic
leukemia K562, human cervical carcinoma HeLa, and human
melanoma A375-S2 in vitro have been
reported[5_7]. However, the mechanism of PAB-induced cell death in human breast
cancer MCF-7 is not clear. In the present study, PAB is
shown to inhibit MCF-7 cell proliferation through apoptosis
and senescence.
Complex organisms have evolved at least 2 cellular
mechanisms to suppress cell proliferation at risk of oncogenic
transformation: apoptosis and cellular senescence in
particular. Apoptosis kills and eliminates potential cancer
cells, and cellular senescence irreversibly arrests their
growth[8]. Some chemicals, such as doxorubicin,
simultaneously induce apoptosis, senescence, and necrosis in a
concentration-dependent manner. Caspase-3 is mainly
responsible for apoptosis, while p53 for senescence. It has
been reported that the inhibition of caspases can switch
doxorubicin-induced apoptosis to
senescence[9]. MCF-7 cells are characterized by wild-type p53 and defective
caspase-3 functions, therefore, they were selected as the
senescence model.
At key transitions during eukaryotic cell cycle
progres-sion, signaling pathways monitor the successful completion
of upstream events before proceeding to the next phase.
These regulatory pathways are commonly referred to as cell
cycle checkpoints[10]. Cells are arrested at cell cycle
checkpoints temporarily to allow for: (i) cellular damage to be
repaired; (ii) the dissipation of an exogenous cellular stress
signal; or (iii) the availability of essential growth factors,
hormones, or nutrients. Checkpoint signaling may also
result in the activation of pathways leading to programmed
cell death, if cellular damage can not be properly
repaired[11]. Cell cycle arrest is necessary to senescence, because the
experimental inactivation of DNA-damage checkpoint
response abrogates oncogene-induced senescence.
DNA-damage checkpoint response is triggered by
oncogene-induced DNA
hyper-replication[12], hence cell cycle arrest
should happen after the S phase.
Cyclin/Cdk complexes play a key role in the cell cycle
progression. Cdk is negatively regulated by a group of
functionally-related proteins called Cdk inhibitors. p21Waf1/Cip1
belongs to Cip/Kip inhibitors, and the Cip/Kip family takes
charge of all phases of the cell
cycle[13]. Wild-type p53 is involved in essential functions, such as DNA repair,
transcription, genomic stability, senescence, cell cycle
control, and apoptosis. A major target gene which
participates in p53-mediated growth arrest is
p21Waf1/Cip1[14]. It is reported that in cell cycle progression, cyclin B1 (whose
levels rise during the S and G2 phases) then was transported
to nuclei, and peaks in mitosis[15]. Cyclin B1 in nuclei must
be destroyed before the cells escape from
mitosis[16]. Therefore, the expression of p21, p53, and cyclin B1 was
investigated in this study.
Materials and methods
Materials PAB, which was purchased from the National
Institute for the Control of Pharmaceutical and Biological
Products (Beijing, China), was dissolved in DMSO to make a
stock solution. The DMSO concentration was kept below
0.01% in all the cell cultures, and did not exert any detectable
effect on cell growth or cell death. Propidium iodide (PI),
RNase A, proteinase K,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), DMF
(dimethylforma-mide), and acridine orange were purchased from Sigma (St
Louis, MO, USA). The senescence detection kit was
purchased from Calbiochem (La Jolla, CA, USA). Rabbit
polyclonal antibodies against cyclin B1, p21, p53,
caspase-3, Fas-associated death domain FADD, Fas L,
β-actin, and horseradish peroxidase-conjugated secondary antibodies
(goat-antirabbit) were obtained from Santa Cruz
Biotechnology (Santa Cruz, CA, USA). Agonistic anti-Fas
immunoglobulin M mAb (clone CH-11) and anti-Fas immunoglobulin G
(clone UB2) were purchased from Medical and Biological
Laboratories (Nagoya, Japan)
Cell culture Human breast cancer MCF-7 cells were
obtained from American Type Culture Collection (Manassas,
VA, USA) and were cultured in RPMI-1640 medium (Hyclone,
Logan, UT, USA) supplemented with 10% heat-inactivated
(56 °C, 30 min) fetal calf serum (Beijing Yuanheng Shengma
Research Institution of Biotechnology, Beijing, China), 2
mmol/L glutamine (Gibco, Grand Island, NY, USA), penicillin
(100 U/mL), and streptomycin (100 µg/mL), and maintained
at 37 °C with 5% CO2 in a humidified atmosphere.
Cell growth inhibition test The inhibition of cell growth
was determined by a MTT test. The MCF-7 cells
(1.5×104 cells/well) were seeded into 96-well culture plates (Nunc,
Roskilde, Denmark). After overnight incubation, various
concentrations of PAB, CH11, or UB2 were added to the
plates. Following incubation, cell growth was measured at
different time points by the addition of MTT at 37 °C for 3 h;
DMSO (150 µL) was added to dissolve the formazan
crystals. Absorbance was measured at 492 nm with an
ELISA plate reader (Bio-Rad, Hercules, CA, USA). The percentage
of inhibition was calculated as follows:
Cell death (%)=(A492
[control]-A492 [sample])/A492 [control]×100%.
Observation of morphological changes by light
microscopy The MCF-7 cells were treated with PAB (0 and 4
µmol/L) for 36 h. The morphological changes were observed by
phase contrast microscopy (Leica, Nusslich, Germany).
Nuclei alternation observed by acridine orange
staining The MCF-7 cells, which were incubated in RPMI-1640
containing 10% fetal calf serum, were seeded into 6-well plates
(Nunc, Denmark) with coverslips and cultured overnight.
The cells were treated with 0 and 4 µmol/L PAB for 36 h. The
morphological changes of the nuclei were observed by
acridine orange staining. The cells on the coverslips were rinsed
and stained with acridine orange (10 mg/L) at 37 °C for 30
min. After the coverslips were sealed, the samples were
observed by fluorescence microscopy (Leica, Germany).
Determination of DNA fragmentation by agarose gel
electrophoresis[17] The MCF-7 cells were treated with 0, 1, 2, 4,
and 10 µmol/L PAB for 36 h, and then both the adherent and
floating cells were collected by centrifugation at
1000×g for 5 min. The cell pellet was suspended in cell lysis buffer [10
mmol/L Tris-HCl (pH 7.4), 10 mmol/L EDTA acid (pH 8.0),
and 0.5%Triton-100], and kept at 4 °C for 30 min. The lysate
was centrifuged at 25 000×g for 20 min. The supernatant was
incubated with 20 g/L RNase A (2 µL) at 37 °C for 1 h, then
incubated with 20 g/L proteinase K (2 µL) at 37 °C for 1 h.
The supernatant was mixed with 5 mol/L NaCl (20 µL) and
isopropanol (120 µL) at -20 °C over-night, and then
centrifuged at 25 000×g for 15 min. After discarding the supernatant,
the DNA sediment was dissolved in 20 µL TE buffer [10
mmol/L Tris-HCl (pH 7.4) and 1 mmol/L EDTA (pH 8.0)], and
separated by 2% agarose gel electrophoresis at 100 V for 50
min.
Lactate dehydrogenase activity-based cytotoxicity
assays[18] The cells were cultured with 4 µmol/L PAB for 12,
24, 36, and 48 h. Floating dead cells were collected from the
culture medium by centrifugation
(240×g for 10 min at 4 °C), and the lactate dehydrogenase (LDH) content from the
pellets lysed in 0.1% Nonidet P 40 (NP-40) for 15 min was used
as an index of apoptotic cell death (LDHp). The LDH
released into the culture medium [extracellular LDH (LDHe)]
was used as an index of necrotic cell death. The adherent
and viable cells were lysed in 0.1 % NP-40 for 15 min to
release LDH [intracellular LDH (LDHi)]. The substrate
reaction buffer of 0.5 mmol/L LDH (L [+]-lactic acid, 0.66 mmol/L
indonitrotetrazolium, 0.28 mmol/L phenazine methosulfate,
1.3 mmol/L nicotinamide adenine dinucleotide in pH 8.2
Tris-HCl) was added. The A value at 490 nm of the reaction for 1
and 5 min was assayed and the formula was as follows:
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.
SA-β-galactosidase
detection[19] The MCF-7 cells
(1.5×105 cells/well) were seeded into 24-well culture plates
(Nunc, Denmark). After overnight incubation, 4 µmol/L PAB
was added to the plates. After 3 d of incubation, the
surviving MCF-7 cells were cultured for 5 d in fresh medium.
Following the protocol of the senescence detection kit, the
culture medium were removed and the cells were washed once
with 1 mL phosphate-buffered saline (PBS), then the cells
were fixed with 0.5 mL of fixative solution at room
temperature for 10_15 min. The staining solution mix was prepared
in a polypropylene plastic tube. For each well, 470 µL
staining solution, 5 µL staining supplement, and 25 µL 20 mg/mL
X-gal in DMF were added. The cells were rinsed twice with
1 mL PBS; 0.5 mL of the staining solution mix was added to
each well and then incubated at 37 °C without
CO2 overnight. The cells were observed under a microscope for the
development of a blue color, and the number of blue-stained cells
in 100 cells was calculated.
Flow cytometric analysis[20] The MCF-7 cells were
harvested and rinsed with PBS. The cell pellets were fixed in
70% ethanol at 4 °C overnight. After washing twice with
PBS, the cells were stained with 1.0 mL PI solution
containing 50 mg/L PI, 1 g/L RNase A, and 0.1% Triton X-100 in 3.8
mmol/L sodium citrate, followed by incubation on ice in the
dark for 30 min. The samples were analyzed by a FACScan
flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).
Western blot analysis of protein
expression[21] The MCF-7 cells were treated with 4 µmol/L PAB for the
indicated times. Both adherent and floating cells were
collected and frozen at -80 °C. A Western blot analysis was
performed for the total proteins as follows. Briefly, the
cell pellets were resuspended in lysis buffer, including 50
mmol/L 4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid (HEPES) (pH 7.4), 1% Triton-X 100, 2 mmol/L
sodium orthovanadate, 100 mmol/L sodium fluoride, 1
mmol/L EDTA, 1 mmol/L egtazic acid (EGTA), 1 mmol/L
phenyl-methylsulfonyl fluoride (PMSF), 0.1
g/L aprotinin, and 0.01 g/L
leupeptin, then lysed at 4 °C for 1 h. After
13 000×g centrifugation for 10 min, the protein content of the
supernatant was determined using Bio-Rad protein assay reagent
(Bio-Rad, USA). The protein was loaded in each lane, then
separated by 12% SDS_PAGE, and blotted onto a
nitrocellulose membrane. The protein expression was detected using
primary polyclonal antibody (1:200_1000) and secondary
polyclonal antibody (1:500) conjugated with peroxidase. For
the cytoplasmic and nuclear proteins, the protein content
was determined as follows: the cell pellets were resuspended
in 60 µL lysis buffer, including 20 mmol/L HEPES, 10 mmol/L
KCl, 1.5 mmol/L MgCl2, 1 mmol/L EDTA, 1 mmol/L EGTA, 1
mmol/L dithiothreitol (DTT), and 1 mmol/L PMSF (pH 7.5),
then lysed on ice at 4 oC for 1 h. After
16 000×g centrifugation for 20 min, the supernatant for the cytoplasm proteins was
collected. The pellet was resuspended in 15 µL lysis buffer,
including 20 mmol/L HEPES, 25% glycerol, 420 mmol/L NaCl,
1.5 mmol/L MgCl2, 0.2 mmol/L EDTA, 0.5 mmol/L DTT, 0.5
mmol/L PMSF, and 5 mg/L leupeptin, and lysed at 4 °C for 15
min on ice. After 12 000×g centrifugation for 10 min, the
nuclear proteins were contained in the supernatant.
Statistical analysis All data represent at least 3
independent experiments and are expressed as mean±SD. Statistical
comparisons were made using Student's t-test.
P-values of less than 0.05 represented a statistically significant difference.
Results
Cytotoxic effect of PAB on cell growth To detect the
growth inhibition of PAB-exposed MCF-7cells, the cells were
treated with various doses of PAB, ranging from 0.6 to 80
µmol/L for 12, 24, 36, and 48 h, and the half maximal inhibitory
concentration IC50 values were 682.2, 165.8, 3.4, and 1.35
µmol/L at 12, 24, 36, and 48 h, respectively. PAB showed a slow
but potent suppressive effect on the MCF-7 cells. The
cytotoxicity was increased in a time- and dose-dependent
manner (Figure 2). In the following experiments, we adopted 4
µmol/L at 36 h as the best concentration. At 12 and 24 h, the
death ratio was low after 4 µmol/L PAB treatment.
Effects of PAB on morphological changes and DNA
fragmentation Morphological changes were observed by phase
contrast microscopy and fluorescence microscopy. After 36
h with 4 µmol/L PAB treatment, we observed a decrease in
the total cell number, an increase in floating cells, and the
appearance of apoptotic bodies (Figure 3B). Nuclear changes
were also detected by acridine orange staining. In the
control group, the MCF-7 cells were intact in shape and stained
green homogeneously (Figure 3C). After 36 h with PAB 4
µmol/L treatment, bright green nuclei blebbing and DNA
fragmentation were observed (Figure 3D). DNA fragmentation
became obvious after 4 and 10 µmol/L PAB treatments for 36
h on agarose gel electrophoresis (Figure 4). Taken together,
the major cause of cell death induced by PAB was through
the apoptotic pathway.
Characteristics of cell death identified by the LDH
release assay It was reported that the rate of LDH released
from viable cells, floating dead cells, and the culture
medium was used to distinguish the proportion of apoptotic
and necrotic cells[17]. The number of apoptotic MCF-7 cells
increased from 3% at 0 h to 39% at 36 h in the presence of 4
µmol/L PAB. However, the percentage of necrotic cells was
still negligible (Figure 5). These results were consistent with
morphological changes and DNA fragmentation (Figure 4),
suggesting that the major cause of PAB-treated MCF-7 cell
death was apoptosis at 36 h. At 12 h, the apoptotic ratio was
very low after 4 µmol/L PAB treatment.
Detection of senescence Mammalian cells express
lysosomal β-galactosidase activity, which is measured at pH 6.0.
SA-β-galactosidase has become widely accepted as an
important biomarker for senescence[19]. MCF-7 cells were
cultured for 5 d in fresh medium after 3 d of incubation with 4
µmol/L PAB, then the surviving cells were detected. In total,
96% of the cells were positive for
SA-β-galactosidase blue staining. Cell morphology showed gross enlargement,
flattening, and accumulation of granular cytoplasmic
inclusion (Figure 6).
Effect of PAB on the cell cycle To further investigate
the mechanism of cell growth inhibition by PAB, a flow
cytometric analysis was performed. After 4 µmol/L PAB
treatment for 0, 12, 24, and 36 h, the DNA amount was doubled
compared with the control group (Figure 7), indicating the
PAB-treated cells might be arrested at the
G2 or M phases. The samples were analyzed by Cell Quest software (Becton
Dickinson, USA), which determined the percentage of cells
at different phases of the cell cycle.
No procaspase-3 degradation in PAB-induced MCF-7 cell
death MCF-7 cells express wild-type p53, but are absent of
the functional caspase-3 protein[22]. As shown in Figure 8,
there was constant expression of non-functional
procaspase-3 after 4 µmol/L PAB treatment at 0, 24, 36, and 48 h, but no
expression of active caspase-3 (17 kDa). At different time
points, the expression of procaspase-3 was not altered,
indicating that PAB exerted no effect on caspase-3 activity
(Figure 8).
Apoptosis induced by PAB was independent of death
receptor pathways Anti-Fas agonistic antibody CH11
activated the Fas receptor resulting in apoptosis, but
anti-Fas antagonistic antibody UB2 can block this pathway and
prevent cell death. At 36 h, the inhibitory ratio of the PAB
group was 50.4%, the CH11+PAB group was 80.9%, and the
UB2+PAB group was 48.7% (Figure 9A). The result from the
Western blot analysis showed that the expression of FADD
and Fas L was stable at different time points (Figure 9B).
Involvement of p21 and p53 in the cycle arrest of MCF-7
cells When exposed to 4 µmol/L PAB, the expression of p21
and p53 was upregulated from 12 h and the expression levels
were stable for a further 12 h (Figure 10).
Translocation of cyclin B1 participation in mitotic
arrest of MCF-7 cells after PAB treatment After PAB
treatment, the amount of DNA was doubled, but the arrested
phase, G2 or M, was not confirmed. Then, the cyclin B1
expression was compared in the cytoplasm and nuclei. After
PAB treatment, total cyclin B1 was increased from 12 to 36 h,
but after 48 h it began to decrease. The expression of cyclin
B1 in nuclei was increased at 12 and 24 h, and then remained
stable. However, its expression in the cytoplasm was
significantly upregulated at 12 h, and then decreased at 24 h
(Figure 11).
Discussion
Apoptosis and senescence, which have different characteristics, exert anticancer effects. As a mechanism of
cell death, apoptosis is an important process for normal
development and suppression of oncogenesis. Apoptosis
is characterized by a series of typical morphological events,
such as cell shrinkage, DNA fragmentation, and
fragmentation into membrane-bound apoptotic bodies and rapid
phagocytosis by neighboring cells[24]. PAB obviously
inhibited MCF-7 cell growth in a time- and dose-dependent manner,
and had a potent suppressive effect on the MCF-7 cells until
36 h treatment. The appearance of apoptotic bodies and
DNA fragmentation were observed after 4 µmol/L
PAB treatment. Apoptotic bodies and DNA fragmentation are
regarded as the apoptotic hallmarks. The LDH
activity-based cytotoxicity assay also demonstrated that in MCF-7
cells, the apoptotic ratio was dominant at 36 h after 4 µmol/L
PAB treatment. Therefore, PAB induced the death of
MCF-7 cells mainly through apoptosis. Senescent cells displayed
morphological features, such as enlargement, flattening, and
positive SA-β-galactosidase staining. The surviving
MCF-7 cells, which were cultured for 5 d in fresh medium after 3 d of
incubation with 4 µmol/L PAB, possessed these typical
senescent phenotype. Therefore, senescence was also
induced by the same concentration of PAB in a different
manner compared with apoptosis in the MCF-7 cells.
Apoptotic processes led to the activation of caspase-3
in a variety of cells which induced proteolytic cleavage of its
substrates, including the DNA fragmentation. MCF-7 cells
with wild-type p53, are absent of caspase-3 activity;
however, inactive procaspase-3 was expressed in the MCF-7
cells. The appearance of the active form of caspase-3 was not observed,
and the expression of procaspase-3 was unchanged with the
time-course in MCF-7 cells at our laboratory. The result is
consistent with that of Cui et
al[25]. The appearance of procaspase-3 may be the result of the mutation in MCF-7
cells, but MCF-7 cells in this study did not have functional
caspase-3. It is reported that DNA fragmentation
independent of caspase-3 is induced by some factors, such as
apoptosis-inducing factor AIF[26], therefore in this study,
the appearance of the DNA ladder is independent of
caspase-3, but the detailed mechanism remains to be elucidated. In
apoptosis, after the binding of death ligands, such as Fas L
or agonistic antibodies to their cognate receptors, a
cytosolic adaptor protein FADD is recruited to the cytoplasmic
domain of the Fas to trigger the death receptor
pathway[27]. In this study, the CH11 agonistic Fas antibody induced the
increasing death of PAB-treated cells, but the UB2
antagonistic antibody-treated group had no such effect on the death
ratio, indicating that there was another pathway, but not
death receptor pathway, to take effect in the apoptosis of
MCF-7 cells. The Western blotting analysis further
indicated that the stable expression of FADD and Fas L was not
involved in apoptosis. The detailed apoptotic pathway
induced by PAB remains to be elucidated.
Cell cycle arrest may be attributed to the activation of
pathways leading to programmed cell death and senescence.
Cycle arrest contributes to
apoptosis[11], but only cell cycle arrest which happens after the S phase is in charge of
senescence[11,19]. In this study, PAB induced obvious mitotic arrest,
which was preceded by apoptosis and senescence in time,
suggesting that PAB-induced apoptosis and senescence
might be caused by mitotic arrest in MCF-7 cells. It has been
reported that p21 is responsible for all cell cycle
arrest[13]. PAB 4 µmol/L treatment for
12 h could activate p21. Since p53 resides at the upstream of
p21[28-30], and 4 µmol/L PAB treatment for 12 h could induce the expression of p53, PAB
might increase the expression of p21 by upregulating the
expression of p53. It has been reported that cyclin B1
increases in mitosis[16,23]. In the present study, 4 µmol/L PAB
increased the expression of total cyclin B1. It is found that
cyclin B1 transports into nuclei in mitosis, and in nuclei it
must be destroyed before cells escape from
mitosis[16]. After 4 µmol/L PAB treatment for 12 h, nuclear cyclin B1
was increased and not degraded. The cytoskeleton mainly
consists of microfilaments and microtubules that form a
dynamic framework that maintains the cell
shape[31]. Microtubules are highly dynamic polymers that switch between
phases of extension and shortening at individual
microtubule ends[32]. This process, known as microtubule dynamics,
is particularly important for mitosis when the rapid
reorganization of microtubules is required for the alignment of
chromosomes, as well as for subsequent chromosome
separation[33,34]. One of the key characteristics of antimicrotubule
agents is the induction of mitotic arrest of the cell
cycle[35]. As a microtubule-disrupting agent, it is reported that PAB
exerts its effect by directly interacting with tubulin,
disrupting the formation of mitotic spindles and
microtubules[36,37]. Taken together, these data showed that PAB treatment did
not activate the G2 checkpoint, and suggested that
PAB-treated cells were probably arrested at this mitotic phase of
the cell cycle.
Taken together, we conclude that PAB could induce
mitotic arrest, apoptosis, and senescence in MCF-7 cells.
PAB induced apoptosis through a pathway other than death
receptor pathway. Mitotic arrest was associated with the
upregulation of p21, p53, and cyclin B1. PAB promoted the
transportation of cyclin B1 from the cytoplasm to nuclei, and
inhibited cyclin B1 degradation in nuclei.
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