Biochem. J. (2012) 444, 291–301 (Printed in Great Britain)
291
doi:10.1042/BJ20111855
JNK- and Akt-mediated Puma expression in the apoptosis
of cisplatin-resistant ovarian cancer cells
Zhiwei ZHAO*, Jingjing WANG*, Jingsheng TANG†, Xinyu LIU*, Qian ZHONG‡, Fang WANG*, Wenbin HU*, Zhu YUAN*1 ,
Chunlai NIE*1 and Yuquan WEI*
*The State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, 4# Keyuan Road, Hitech District, Sichuan University, Chengdu 610041, China, †ChongQing NewFine
Biology Technology, 8# Lingfang Road, Banqiao District, Chongqing 402460, China, and ‡Department of Gynecology and Obstetrics, West China Second Hospital, Chengdu, Sichuan
University, Chengdu 610041, China
BH3 (Bcl-2 homology domain 3)-only proteins have an important
role in the cisplatin resistance of cells. However, the effect of
BH3-only proteins on cisplatin-resistant ovarian cancer cells has
not been thoroughly elucidated. Our results from the present
study indicate that Puma plays a critical role in the apoptosis of
chemo-resistant ovarian cancer cells treated with BetA (betulinic
acid). The reduction of Puma expression inhibits Bax activation
and apoptosis. However, p53 gene silencing has little effect on
Puma activation. Further experiments demonstrated that Aktmediated FoxO3a (forkhead box O3a) nuclear translocation and
the JNK (c-Jun N-terminal kinase)/c-Jun pathway only partially
trigger Puma induction and apoptosis, whereas dominant-negative
c-Jun expression with FoxO3a reduction completely inhibits
Puma expression and cell death. Furthermore, our results suggest
that JNK regulates the Akt/FoxO3a signalling pathway. Therefore
the dual effect of JNK can efficiently trigger Puma activation
and apoptosis in chemoresistant cells. Taken together, our results
demonstrate the role of Puma in BetA-induced apoptosis and the
molecular mechanisms of Puma expression regulated by BetA
during ovarian cancer cell apoptosis. Our findings suggest that the
JNK-potentiated Akt/FoxO3a and JNK-mediated c-Jun pathways
co-operatively trigger Puma expression, which determines the
threshold for overcoming chemoresistance in ovarian cancer cells.
INTRODUCTION
mitochondrial pathway is involved in BetA-induced apoptosis.
This apoptosis pathway is closely related to the Bcl-2 family
of proteins. Anti-apoptotic members sequester multi-domain
pro-apoptotic proteins, thereby inhibiting their active role in
apoptosis. In contrast, BH3 (Bcl-2 homology domain 3)-only
proteins that are considered stress sensors can dissociate Baxlike proteins from their anti-apoptotic sequestrators, thus leading
to apoptosis [7].
The expression of Bcl-2 family proteins is regulated during
carcinogenesis [7], and the expression of both the Bcl-2 and
Bcl-xL anti-apoptotic proteins is associated with resistance to
antitumour agents such as cisplatin [8], most notably in ovarian
carcinoma [9,10]. In ovarian carcinoma, the inhibition of the
protective function of anti-apoptotic Bcl-2 members can either
restore the normal apoptotic process in cancer cells or circumvent
resistance to chemotherapy. In this regard, enhanced expression
of BH3-only proteins can effectively bind the anti-apoptotic
members and prevent the function of these proteins.
Some reports suggest that the BH3-only protein Puma has
important roles in p53-dependent and -independent apoptosis in
human cancer cells and mediates cell death through the Bcl-2
family proteins Bax/Bak and the mitochondrial pathway [11,12].
Experiments have revealed that Puma expression is up-regulated
in chemosensitive ovarian cells treated with cisplatin, but not in
chemoresistant cells treated with cisplatin. Thus the elevation of
Puma expression can overcome chemoresistance in ovarian cancer
cells [13].
Ovarian cancer is the most common cause of cancer death
from gynaecological tumours. Ovarian cancer patients initially
respond to standard treatments such as cytoreductive surgery and
subsequent cisplatin-based chemotherapy. However, there is a
distinct problem with this therapy in that ovarian cancer cells are
resistant to cisplatin, leading to a survival rate of less than 30 % in
patients with ovarian cancers [1]. Thus finding a new low-toxicity
and efficient drug to overcome cisplatin resistance would be a
major breakthrough.
BetA (betulinic acid) is a naturally occurring triterpenoid that
was initially reported to specifically kill melanoma cells by inducing apoptosis [2] and was subsequently shown to have efficacy
in neuroectoderm-derived tumours. The anticancer activity of
BetA against other types of cancers, such as leukaemia, prostate,
ovarian, breast, lung and colon cancer, has also been reported
[3]. Moreover, there have been promising studies indicating that
BetA not only has inherent single-agent tumoricidal activity
against different tumour cells, but also functions co-operatively
with other anticancer drugs to trigger apoptosis in chemoresistant
neuroblastoma cells [4,5]. These results indicate that BetA may
overcome some forms of drug resistance.
A previous study reported that the extrinsic death receptor
pathway does not participate in BetA-induced apoptosis [6]. A
decrease in the mitochondrial inner transmembrane potential is
associated with BetA treatment, which suggests that the intrinsic
Key words: Akt dephosphorylation, cisplatin-resistant, c-Jun
N-terminal kinase (JNK) activation, ovarian cancer, Puma.
Abbreviations used: BetA, betulinic acid; BH3, Bcl-2 homology domain 3; ChIP, chromatin immunoprecipitation; DMEM, Dulbecco’s modified
Eagle’s medium; ERK, extracellular-signal-regulated kinase; FBS, fetal bovine serum; FoxO3a, forkhead box O3a; GAPDH, glyceraldehyde-3-phosphate
dehydrogenase; GFP, green fluorescent protein; HA, haemagglutinin; JNK, c-Jun N-terminal kinase; Mcl-1, myeloid cell leukaemia sequence 1; MAPK,
mitogen-activated protein kinase; MOI, multiplicity of infection; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H -tetrazolium bromide; PARP, poly(ADPribose) polymerase; PKB, protein kinase B; RT, reverse transcription; siRNA, small interfering RNA.
1
Correspondence may be addressed to either of these authors (email [email protected] or [email protected]).
c The Authors Journal compilation c 2012 Biochemical Society
292
Z. Zhao and others
In the present study, we showed for the first time that
BetA can induce apoptosis in cisplatin-resistant ovarian cancer
cells. Expression of the BH3-only proteins Puma and Noxa
increased following cell death, whereas only Puma siRNA (small
interfering RNA) could effectively inhibit Bax translocation,
conformational change, oligomerization and the subsequent
apoptosis in cisplatin-resistant cells. Moreover, our results
demonstrated that Puma expression is independent of p53
regulation in apoptosis. Further experiments revealed that Aktmediated FoxO3a (forkhead box O3a) nuclear translocation and
the JNK (c-Jun N-terminal kinase)/c-Jun pathway co-operatively
modulate Puma expression. Furthermore, we found that JNK
mediates the Akt/FoxO3a pathway, indicating that JNK is an
upstream event of BetA treatment-induced apoptosis. These
results elucidated the mechanisms of Puma regulation in BetAinduced apoptosis and confirmed that Puma activation is a
major molecular event in mediating BetA-induced apoptosis in
chemoresistant ovarian cancer cells.
EXPERIMENTAL
obtained from patients who were refractory to platinum-based
combination chemotherapy [16].
For general transfection, cells were placed in 6-well plates and
then transfected with the appropriate plasmid DNA or siRNA
using the protocol described by the manufacturer. Typically,
1 μg of plasmid DNA or siRNA and 4 μl of DMRIE-C reagent
(Invitrogen) were used per coverslip. The cells were incubated
for 4 h in the transfection mixture, which was then replaced with
fresh culture medium. For stable Akt transfection, cells were
transfected with the constitutively active Akt1 construct HA–
PKB-T308D/S473D as described previously [14]. Positive clones
were selected with 1 mg/ml G418 for several weeks.
For adenoviral infection, cells were plated in 6-well plates
for 24 h and then infected with adenoviral wild-type p53 at a
MOI (multiplicity of infection) of 0, 5, 10 or 20. Infection with
an equivalent concentration (MOI) of LacZ was carried out as
a proper control. To test the changes in cisplatin sensitivity,
infected cells were treated with or without cisplatin (5 μg/ml;
24 h) after a 72-h infection period. The number of apoptotic cells
was determined by a Cell Death Detection ELISA (Roche). The
p53 protein content was assessed by Western blotting.
Reagents
Cisplatin, BetA, LY294001, SP600125, PD98059, SB203580,
Hoechst 33342, and the anti-Bax 6A7 monoclonal and
anti-actin antibodies were obtained from Sigma. Antibodies
against Lamin B1, Puma, Noxa, Bim, caspase-9, PARP
[poly(ADP-ribose) polymerase], p53, phospho-Akt (Ser473 ), Akt,
phospho-JNK (Thr183 /Tyr185 ), phospho-c-Jun (Ser73 ), phosphop38 (Thr180 /Tyr182 ), phospho-ERK (extracellular-signal-regulated
kinase) (Thr202 /Thr204 ) and cleaved caspase-3 were purchased
from Cell Signaling Technology. Antibodies against Mcl-1 (myeloid cell leukaemia sequence 1), Bcl-2, Bcl-xL, cytochrome
c and Bax were from Santa Cruz Biotechnology. Antibodies
against phospho-FoxO3a (Thr32 ) and FoxO3a were from Upstate
Biotechnology.
Gene silencing with siRNAs and plasmids
siRNA oligonucleotides were created with the following
sequences: Puma, 5 -UCUCAUCAUGGGACUCCUG-3 ; Noxa,
5 -CUUCCGGCAGAAACUUCUG-3 ; p53, 5 -CGGCAUGAACCGGAGGCCCAU-3 ; and FoxO3a, 5 -ACUCCGGGUCCAGCUCCAC-3 were purchased from Dharmacon. The constitutively active Akt1 construct [HA (haemagglutinin)–PKB
(protein kinase B)-T308D/S473D] was described previously [14].
The dominant-negative c-Jun plasmid (pGFP-TAM67; where
GFP is green fluorescent protein) was provided by Dr Jian Zhao
(Sichuan University, Chengdu, China). The adenoviral wild-type
p53 and LacZ constructs were provided by Dr Frank L. Graham
(McMaster University, Hamilton, ON, Canada).
Cell culture and transfection
Cisplatin-sensitive (A2780) and -resistant (A2780/CP) human
ovarian cancer cell lines were gifts from Dr Matshela Molepo
(Ottawa Regional Cancer Center, Ottawa, ON, Canada) and were
incubated in DMEM (Dulbecco’s modified Eagle’s medium)
supplemented with 10 % (v/v) FBS (fetal bovine serum) and
1 % penicillin/streptomycin [15]. The COC1/CP cells were also
obtained from Dr Molepo. The COC1 and OVCAR-3 cells were
obtained from A.T.C.C. No authentication of the cell lines was
performed by the authors. The COC1 and OVCAR-3 cells were
cultured in suspension with RPMI 1640 medium supplemented
with 10 % (v/v) FBS (Hyclone) and 1 % penicillin/streptomycin
at 37 ◦ C under 5 % CO2 . The OVCAR-3 cells were originally
c The Authors Journal compilation c 2012 Biochemical Society
Cell viability and apoptosis assay
Ovarian cell viability was assessed by the MTT [3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay in 12 replicates. This procedure has been described previously
[17]. The following four methods were used to assess induced
apoptotic cell death: detection of DNA fragmentation with the
Cell Death Detection ELISA kit; Western blot analysis of caspase
activation and PARP cleavage; measurement of apoptotic cells
by flow cytometry (Annexin V); and cell staining with Hoechst
33342. The cell death detection ELISA was performed to quantify
the apoptotic cells by detecting the histone-associated DNA
fragments (mono- and oligo-nucleosomes) generated by apoptotic
cells [14]. In brief, the ovarian cells were treated with the
experimental compounds and collected to prepare the cytosolic
fractions that contained the smaller fragments of DNA. Equal
volumes of these cytosolic fractions were incubated in antihistone antibody-coated wells (96-well plates), and the histones
of the DNA fragments were allowed to bind to the antihistone antibodies. The peroxidase-labelled mouse monoclonal
DNA antibodies were used to localize and detect the bound
fragmented DNA using photometric detection with 2,29-azinodi-(3-ethylbenzthiazoline sulfonate) as the substrate, according to
the manufacturer’s instructions.
Cell fractionation
Mitochondrial and cytoplasmic cell fractions were obtained
by differential centrifugation as described previously [18,19].
Briefly, cells were harvested and resuspended in 3 vol.
of hypotonic buffer (210 mM sucrose, 70 mM mannitol,
10 mM Hepes, pH 7.4, and 1 mM EDTA) containing 1 mM
PMSF, 50 mg/ml trypsin inhibitor, 10 mg/ml leupeptin, 5 mg/ml
aprotinin and 10 mg/ml pepstatin. After gentle homogenization
with a Dounce homogenizer, cell lysates were centrifuged
at 1000 g for 5 min to remove unbroken cells and nuclei.
The supernatant was collected and centrifuged at 10 000 g to
pellet the mitochondria-enriched heavy membrane fraction. The
supernatant was further centrifuged at 100 000 g to obtain a
cytosolic fraction.
Nuclear and cytoplasmic lysates were prepared according to a
protocol published previously [20]. Briefly, cells were lysed on
ice for 20 min in a buffer containing 10 mmol/l Hepes, pH 7.4,
JNK- and Akt-mediated Puma expression
10 mmol/l KCl, 0.01 mmol/l EDTA, 0.1 mmol/l EGTA, 2 mmol/l
dithiothreitol, 10 mmol/l NaF, 1 mmol/l sodium orthovanadate,
30 mmol/l sodium glycerophosphate and protease inhibitors.
Nuclei were sedimented by centrifugation, and the supernatant
(cytoplasmic fraction) was retained. Nuclei were lysed in the
same buffer containing 1 % (v/v) Nonidet P40 and 400 mmol/l
NaCl.
RNA isolation and RT (reverse transcription)–PCR
Ovarian cancer cells were treated as described in the Figure
legends, and total RNA was isolated with TRIzol® . The
oligonucleotides used for amplification were: p53, 5 -CCTACCAGGGCAGCTACGG-3
and
5 -GGAAGACTCCAGTGGTAATC-3 ; and GAPDH (glyceraldehyde-3-phosphate
dehydrogenase), 5 -GAAGGTGAAGGTCGGAGTCA-3 and
5 -CCGACTCTTGCCCTTCGAAC-3 . Total RNA (1 μg) was
used for RT–PCR with the Superscript One-step kit (Invitrogen)
(20 cycles for p53 and GAPDH). The PCR products, which
were 486 bp long for p53 and 260 bp long for GAPDH, were
fractionated on a 1 % agarose gel containing ethidium bromide
and visualized under UV light.
ChIP (chromatin immunoprecipitation) assay
The ChIP assay was performed using the Chromatin Immunoprecipitation Assay kit (Upstate Biotechnology) according to
the manufacturer’s protocol. ChIP was performed with 3 μg
of anti-c-Jun polyclonal antibody (Pierce) or anti-FoxO3a
antibody (Sigma) incubated with Protein G-coated magnetic
beads overnight with rotation at 4 ◦ C. The DNA fragments
that co-immunoprecipitated with the target proteins c-Jun or
FoxO3a were subjected to quantitative real-time PCR analysis
using various primer sets. The CT (threshold cycle) value of
each sample was normalized to the CT value obtained from the
PCR reaction using the corresponding input genomic DNA as
a template. Primer sequences for Puma and FoxO3a binding
were designed as described previously [21]. Primer sequences
were: Puma FoxO3a, 5 -GCCGCCACTGCAGTTAGAG-3 and
5 -AACAGCCGGTTATTGGCC-3 ; and Puma c-Jun, 5 -AGATTACCTGCATCTCTTGG-3 and 5 -CCCTGCTCTGGTTTGGTGAGT-3 .
SDS/PAGE and immunoblotting
SDS/PAGE and immunoblotting were performed as described
previously [18]. Briefly, whole cells or the membrane fractions
were resuspended in lysis buffer containing Nonidet P40 (10 mM
Hepes, pH 7.4, 2 mM EGTA, 0.5 % Nonidet P40, 1 mM NaF,
1 mM sodium orthovanadate, 1 mM PMSF, 1 mM dithiothreitol,
50 μg/ml trypsin inhibitor, 10 μg/ml aprotinin and leupeptin)
and placed on ice for 30 min. The lysates were centrifuged at
12 000 g for 12 min at 4 ◦ C, and the protein concentration was
measured. Equivalent samples (30 μg of protein) were subjected
to SDS/PAGE on 12 % gels. The proteins were then transferred
on to nitrocellulose membranes and probed with the indicated
antibodies followed by the appropriate secondary antibodies
conjugated to horseradish peroxidase (KPL). Immunoreactive
bands were visualized using enhanced chemiluminescence
(Pierce). The molecular sizes of the proteins detected were
determined by comparison with pre-stained protein markers
(Invitrogen).
Bax conformational change and oligomerization
Bax conformational change analysis was performed as described
previously [18]. Cytosolic or mitochondrial fractions were lysed
293
with CHAPS lysis buffer (10 mM Hepes, pH 7.4, 150 mM NaCl
and 1 % CHAPS) containing protease inhibitors as described
previously [18]. The cell lysates were normalized for protein
content, and 250 μg of total protein was incubated with 1 μg of
anti-Bax 6A7 monoclonal antibody (Sigma) in 500 μl of CHAPS
lysis buffer at 4 ◦ C for 3 h or overnight. Then, 25 μl of Protein G–
agarose was added, and the reactions were incubated at 4 ◦ C for
an additional 2 h. The beads were washed three times in CHAPS
lysis buffer, boiled in loading buffer and the conformationally
changed Bax protein in the immunoprecipitates was subjected to
SDS/PAGE and immunoblot analysis with anti-Bax polyclonal
antibody (Santa Cruz Biotechnology) as described above.
Bax oligomerization was detected as described previously
[18]. Cells were washed with conjugating buffer containing
150 mM NaCl, 20 mM Hepes (pH 7.2), 1.5 mM MgCl2 and
10 mM glucose. Disuccinimidyl suberate in DMSO was added
from a 10-fold stock solution to a final concentration of 2 mM
[22]. The samples were incubated at room temperature (25 ◦ C)
for 30 min with no reduced buffer, and the cross-linker was then
quenched by the addition of 1 M Tris/HCl (pH 7.5) to a final
concentration of 20 mM and incubation at room temperature for
15 min. The samples were then solubilized in 0.5 % Nonidet P40
lysis buffer without a reducing agent and centrifuged at 12 000 g
for 10 min. Bax was detected by Western blotting with an anti-Bax
polyclonal antibody (Santa Cruz Biotechnology).
Statistical analysis
Statistical analysis of the differences between the experimental
groups was performed using a Student’s t test. P < 0.05 was
considered statistically significant.
RESULTS AND DISCUSSION
BetA induces apoptosis in chemoresistant ovarian cancer cells
We first examined the apoptotic effect of cisplatin in various
cisplatin-sensitive (COC1 and A2780) and -resistant (A2780/CP,
COC1/CP and OVCAR-3) human ovarian cancer cells. Apoptosis
was detected by the Cell Death Detection ELISA kit with cisplatin
treatment (5 μg/ml) at different periods of time. As shown
in Figure 1(A), cisplatin caused apoptosis in chemosensitive
ovarian cancer cells, but not in chemoresistant cells. We
then investigated the antitumour effect of BetA at different
concentrations on ovarian cancer cells. BetA caused a dosedependent reduction in cell viability in cisplatin-sensitive and
-resistant cells, showing that the IC50 value was close to 10 μg/ml
(Figure 1B). To examine BetA-induced apoptosis, the cells were
treated with 10 μg/ml BetA and cell death was confirmed by
a DNA fragmentation ELISA assay at various time points. BetA
effectively induced apoptosis in all ovarian cancer cells, regardless
of their differences in chemo-sensitivity (Figure 1C). Western
blot analysis confirmed the cell death through the detection
of caspase-3 cleavage in chemoresistant cells (Figure 1D).
These results demonstrated that BetA could initiate cell death
in cisplatin-resistant ovarian cancer cells. Thus our findings
provided the first evidence that BetA can induce apoptosis in
ovarian cancer cells regardless of their chemosensitivity. We
also found that BetA enhanced the efficacy of cisplatin in
chemoresistant ovarian cancer cells (Supplementary Figure S1
at http://www.BiochemJ.org/bj/444/bj4440291add.htm). These
results suggest that, in chemotherapy-based regimens, BetA may
be used as a ‘sensitizer’ that enhances the efficacy of anticancer
agents in the inhibition of tumour growth [23].
As intrinsic mitochondrial pathways play an important role
in apoptosis [18,22], we then determined whether mitochondria
c The Authors Journal compilation c 2012 Biochemical Society
294
Z. Zhao and others
Figure 1 BetA induces apoptosis in cisplatin-resistant human ovarian
cancer cells
(A) Analysis of cell apoptosis treated with cisplatin (CP). Cells were treated with cisplatin
(5 μg/ml) for the indicated times, and then collected to examine apoptosis. Cell apoptosis was
quantitatively detected by the Cell Death Detection ELISA kit as described in the Experimental
section. The graph shows the results of quantitative analyses (n = 3, means +
− S.D.). (B)
Dose-dependent effects of BetA in ovarian cell lines. Cells were exposed to BetA at the indicated
concentrations in DMEM or RPMI 1640 medium with 10 % (v/v) FBS in a 96-well plate for 48 h
and cell viability was assessed using the MTT assay, showing that the IC50 value was close
to 10 μg/ml. The graph shows the results of the MTT assay (n = 3, means +
− S.D.). (C) Cells
were treated with BetA (10 μg/ml) for different periods of time and cell apoptosis was detected
as described in (A). (D) Detection of caspase-3 activation. After BetA (10 μg/ml) treatment, cell
lysates were collected and immunoblotted with an antibody specific for caspase-3, which only
detected the cleaved caspase-3 fragments. β-Actin was used as a protein loading control. (E)
Analysis of cytochrome c (Cyt c ) release. After treatment with BetA for different periods of time,
cells were subjected to subcellular fractionation. The cytosolic or mitochondrial fractions were
immunoblotted (30 μg of protein/lane) with an antibody specific for cytochrome c . β-Actin and
COX IV were used as a protein loading control. The results are representative of at least three
independent experiments.
play an important role in BetA-induced apoptosis. The level
of cytochrome c release was determined by cell fractionation
analysis. Our results revealed that BetA induced cytochrome
c release in a time-dependent manner in chemoresistant cells
(Figure 1E), suggesting that BetA-induced apoptosis is associated
with mitochondrial dysfunction.
Expression changes of Bcl-2 family proteins are correlated with the
induction of cell death
Mitochondrial dysfunction plays an important role in ovarian
cell apoptosis, and the function of Bcl-2 proteins is related to
the mitochondrial apoptotic pathway. Previous studies reported
that Bcl-2 family proteins are associated with mitochondrial
dysfunction and the chemoresistance of cells [24,25], and that the
BH3-only proteins are important for chemoresistance in ovarian
cancer cells [26].
Several reports have also showed that BetA can modulate the
expression levels of Bcl-2 family proteins. For example, BetA
c The Authors Journal compilation c 2012 Biochemical Society
Figure 2
cells
BetA induces changes in Bcl-2 family proteins in ovarian cancer
(A) Time-dependent analysis of Bcl-2 family member’s expression levels in A2780/CP cells
treated with BetA (10 μg/ml) for the indicated times. (B) Detection of Bcl-2 family protein levels in
cells treated with or without BetA (10 μg/ml) for 24 h. Cells were treated and collected, then lysed
in Nonidet P40 buffer for Western blot detection. β-Actin was used as a protein loading control.
(C) Cells were treated with BetA as described in (B) and lysed for Western blot detection. Panel
a, to detect Bax translocation, cells were subjected to subcellular fractionation. The cytosolic
(Cyto) or mitochondrial (Mito) fractions were immunoblotted (30 μg of protein/lane) with an
antibody specific for Bax. Panel b, to detect Bax oligomerization, cells were treated with or
without BetA for different periods of time, and then the oligomerization of Bax was assessed by
cross-linking with disuccinimidyl suberate. The samples were then solubilized in Nonidet P40
lysis buffer, and Bax was detected by Western blotting with an anti-Bax polyclonal antibody.
Panel c, to detect Bax conformational change, cells were cultured in the presence or absence of
BetA for 24 h and then lysed in CHAPS buffer and subjected to immunoprecipitation (IP) with
the anti-Bax 6A7 antibody (Sigma), and then immunoprecipitated cell lysates were detected
with anti-Bax polyclonal antibody. A cell lysate obtained using the Nonidet P40 buffer was
used as a positive control. Bax total lysates were detected by Western blotting with an anti-Bax
polyclonal antibody (Santa Cruz). All results are representative of three independent experiments.
Molecular mass is given in kDa on the right-hand side. Ab, antibody.
treatment resulted in the high expression of the pro-apoptotic
Bcl-2 family protein Bax in neuroblastoma, glioblastoma and
melanoma cells [27]. Additionally, BetA triggered the upregulation of Mcl-1, another anti-apoptotic Bcl-2 family protein,
in melanoma cells, whereas no changes in Mcl-1 levels were
detected in squamous cell carcinoma cells [28,29]. These results
suggest that Bcl-2 family proteins have an important role in BetAinduced apoptosis. However, the function of BH3 proteins in
ovarian cancer cells treated with BetA is unclear. Therefore, we
examined the expression of Bcl-2 family proteins in cisplatinsensitive and -resistant cells after BetA treatment.
We used A2780 and A2780/CP cells as models to study
the function of BetA. As shown in Figures 2(A) and 2(B),
JNK- and Akt-mediated Puma expression
pro-apoptotic proteins, such as Bax and Bim, were not
significantly changed in all cells treated with BetA at different
time points. Anti-apoptotic Mcl-1, Bcl-2 and Bcl-xL proteins
also exhibited no major alterations. However, in contrast
with other proteins, the expression levels of Puma and Noxa
markedly increased after BetA treatment. Meanwhile, Puma
and Noxa expression levels were up-regulated in COC1 and
COC1/CP cells after BetA treatment (Supplementary Figure S2
at http://www.BiochemJ.org/bj/444/bj4440291add.htm)
At the same time, we observed a Bax conformational change,
translocation and oligomerization in ovarian cancer cells after
BetA treatment (Figure 2C), providing further evidence that Bcl-2
family proteins are involved in BetA-induced apoptosis in ovarian
cancer cells. Moreover, our experiments also suggest that Puma
and Noxa play important roles in BetA-induced apoptosis in
ovarian cancer cells.
Puma down-regulation by siRNA prevents apoptosis
To identify further the functions of Puma and Noxa, we decided
to knock down these proteins using siRNAs in A2780/CP cell
lines (Figure 3A). Puma siRNA was able to inhibit Puma
protein expression, but did not lead to cell death on its
own. As described above, a 12 h exposure to BetA induced
an obvious apoptotic response in A2780/CP cells. Whereas
control siRNA did not modify the response to BetA treatment,
we observed a strong decrease in the apoptotic response in
cell lines when BetA treatment was combined with Puma
siRNA (Figure 3B). Similar results were observed in A2780
cells (results not shown). Meanwhile, Puma siRNA treatment
decreased cell death in COC1/CP cells, showing the same
results observed in A2780/CP cells (Supplementary Figure
S3 at http://www.BiochemJ.org/bj/444/bj4440291add.htm). In
contrast, Noxa siRNA markedly inhibited the expression of
Noxa, but had little or no effect on the levels of apoptosis
induced by BetA in either cell line (Figures 3C and 3D, and
Supplementary Figure S3). These results indicated that Noxa
is dispensable for BetA-induced apoptosis. The results are in
agreement with some previous studies that demonstrated that
Noxa has an unimportant role in apoptosis. These studies revealed
that forced expression of Noxa was insufficient to trigger neuronal
cell apoptosis. Moreover, an experiment using Noxa-deficient
mice also indicated that it plays a minor role in the apoptotic
pathway [30,31]. In contrast, several studies revealed that Puma
induction strongly correlated with apoptosis, and the ectopic
expression of Puma was sufficient to induce apoptosis [30,31].
As previous studies revealed that BH3 proteins can directly
or indirectly induce Bax activation in apoptosis [7,11,12], we
also determined whether the reduction of Puma and Noxa
affected Bax activation in apoptosis. Gene silencing of Puma
obviously inhibited Bax conformational change, translocation
and oligomerization, whereas knockdown of Noxa could not
prevent Bax activation (Figures 3E and 3F). These results further
demonstrated that Noxa was not involved in regulating Bax
activation and apoptosis. Moreover, these results revealed that
Puma mediates Bax activation and the subsequent mitochondrialdependent apoptotic pathway in BetA-treated ovarian cancer cells.
p53 is not involved in Puma activation
We then examined which factor mediates Puma expression. As
a previous report indicated that p53 expression is enhanced
and p53 transcriptionally triggers Puma expression after stress
induction [32], we wanted to assay whether PUMA is induced
by BetA through a p53-dependent mechanism. However, A2780
295
cells express wild-type p53, whereas A2780/CP cells have mutant
p53 [33,34]. We have detected a single pair missense mutation
(G to T) at codon 172 in A2780/CP p53 by PCR amplification
and sequence analysis (results not shown). This mutation may
lead to a loss of protein function and thereby contribute to
cisplatin resistance. Therefore we first detected the status of
p53 in A2780 and A2780/CP cells with cisplatin treatment. Our
results demonstrated that the expression levels of p53 in A2780
cells increased with cisplatin treatment, whereas p53 induction
was unchanged in A2780/CP cells after treatment (Figure 4A,
panels a and b), as described previously [33]. Moreover, cisplatin
significantly induced apoptosis in A2780 cells, but not in
A2780/CP cells (Figure 1A). However, overexpression of wildtype p53 in A2780/CP cells resulted in a concentration (MOI)dependent increase in cisplatin-induced apoptosis (Figure 4B,
panel a), whereas LacZ expression in A2780/CP cells produced
little change in cisplatin-induced apoptosis (Figure 4B, panel a).
Western blot analysis also showed that p53 expression increased
in wild-type p53-infected cells (Figure 4B, panel b). Thus the p53
mutation in A2780/CP cells did contribute to cisplatin resistance
in the present study.
We then detected p53 expression in cells after treatment with
BetA. Western blotting showed that the expression level of p53
had minor changes in all cells regardless of their chemosensitivity
(Figure 4C). Meanwhile, RT–PCR also revealed that the mRNA
levels of p53 changed minimally at different periods of time after
BetA treatment (Figure 4D).
To further confirm the effect of p53 in Puma expression, we
then knocked down p53 with siRNA (p53 siRNA) in ovarian
cancer cells. The siRNA was able to inhibit p53 expression,
but did not induce caspase-3 cleavage on its own (Figures 4E
and 4F, and Supplementary Figure S4 at http://www.BiochemJ.
org/bj/444/bj4440291add.htm). We observed that BetA combined
with p53 siRNA could not prevent caspase-3 activation in ovarian
cancer cells. Importantly, up-regulated Puma expression was
invariant in p53 siRNA-transfected chemoresistant cells treated
with BetA (Figure 4F and Supplementary Figure S4). These
results demonstrate that p53 is not involved in regulating Puma
expression even in A2780 cells, which express the wild-type
p53. Our results also indicate that BetA-induced apoptosis is
independent of p53 activation. In fact, these results are as
expected. First, the p53 mutant may lead to inactivity of p53 in
apoptosis. Secondly, BetA induced-apoptosis is not associated
with the accumulation of wild-type p53 protein [27]. Thus
there are other mechanisms that function in modulating Puma
expression in ovarian cancer cells.
The Akt pathway does not completely regulate Puma expression
A previous report revealed that Akt dephosphorylation can
mediate Puma expression through FoxO3a transcriptional
regulation [35]. Moreover, BetA induces Akt dephosphorylation
in melanoma cell apoptosis [36]. We speculate that Akt is probably
involved in regulating Puma expression in ovarian cancer cells.
Therefore, we measured whether Akt was involved in BetAinduced apoptosis in ovarian cancer cells. As shown in
Figure 5(A), BetA induced the down-regulation of phosphoAkt at Ser473 in cisplatin-sensitive A2780 cells in a timedependent manner, whereas the expression level of total Akt
protein experienced little or no change. Similarly, BetA induced
a decrease in phospho-Akt in other cells despite their differences
in chemosensitivity.
The decrease in phospho-Akt leads to dephosphorylation
and activation of FoxO3a. Active FoxO3a can initiate Puma
expression through transcriptional regulation [35]. FoxO3a
c The Authors Journal compilation c 2012 Biochemical Society
296
Figure 3
Z. Zhao and others
Puma is major molecular determinant in BetA-treated cell apoptosis
Legend can be found on page 297.
c The Authors Journal compilation c 2012 Biochemical Society
JNK- and Akt-mediated Puma expression
Figure 4
297
p53 activation is not involved in Puma up-regulation
(A) Panel a, Western blotting to detect p53 expression in cells. A2780 or A2780/CP cells were
treated with cisplatin (CP) (5 μg/ml) for the indicated times, and then collected to detect p53
expression. β-Actin was used as a protein loading control. Panel b, grey scanning of p53 levels.
p53 levels in cells were measured by densitometric analysis of the Western blots and compared
with actin levels in cells. The relative amount of p53 from untreated cells was considered as 1.
(B) A2780/CP cells were infected with adenoviral LacZ or p53 (MOI 0, 5, 10 or 20; 72 h), and
then cells were treated with cisplatin (5 μg/ml) for 24 h. Apoptosis is shown in panel a. Panel
b shows representative Western blots for p53. (C) Western blotting to detect p53 expression in
cells treated with BetA (10 μg/ml). A2780 and A2780/CP cells were treated at indicated time
and lysed. β-Actin was used as a protein loading control. (D) As described in (C), cells were
treated and lysed to detect p53 RNA levels with RT–PCR. GAPDH was used as a protein loading
control. (E) Cells were transfected with p53 siRNA or control (Ctrl) siRNA for 48 h and then
treated with BetA for 24 h. Cells were lysed and determined by immunoblot analysis. (F) As
described in (E), cells were transfected for 48 h and treated with BetA for 24 h. Western blotting
was performed to detect caspase-3 cleavage and Puma expression. Representative results of
three experiments with consistent results are shown.
phosphorylation decreased during the time course with BetA
induction, whereas the expression level of total FoxO3a
was unchanged (Figure 5B). We then determined whether
FoxO3a regulates Puma expression in apoptosis. We used
the ChIP assay to detect the interactions between FoxO3a
and the Puma promoter, as described previously [35].
Our results revealed that FoxO3a can act on the Puma
Figure 5
Akt partially regulates Puma expression
(A) Western blot analysis of total Akt and phospho-Akt (p-Akt) (Ser473 ) levels in ovarian cells
treated with BetA (10 μg/ml). A2780 and A2780/CP cells were treated with BetA for different
periods of time, then lysed to detect total Akt and phospho-Akt levels. β-Actin was used as a
protein loading control. (B) Detection of FoxO3a and phospho-FoxO3a (p-FoxO3a) levels in cells
treated with BetA. Cells were treated as described in (A) and subjected to immunoprecipitation
with anti-FoxO3a or anti-[phospho-FoxO3a (Thr32 )] antibodies. (C) Cells were transfected with
control (Ctrl) or FoxO3a siRNA for 48 h, and then treated with BetA for 24 h. Panel a, treated cells
were lysed to detect protein level using Western blotting analysis. Panel b, treated cells were
used to detect apoptosis with the Cell Death Detection ELISA kit as described in the Experimental
section (n = 3, means +
− S.D., *P < 0.05). (D) Panel a, A2780/CP cells were treated with or
without BetA (10 μg/ml) and/or LY294002 (25 μM) for 24 h, and then lysed to detect protein
expression. Panel b, A2780/CP cells were transfected with control and Akt1 vector for 48 h and
treated with BetA for 24 h. Treated cells were lysed for detection. For FoxO3a nuclear translocation
analysis, cells were incubated with BetA and subjected to subcellular fractionation as described
in the Experimental section. C-FoxO3a, cytosolic FoxO3a; N-FoxO3a, nuclear FoxO3a. Lamin
B1 was used as a nuclear marker. Representative results of three experiments with consistent
results are shown.
Down-regulation of Puma by siRNA decreases apoptosis in A2780/CP cells. (A) Cells were transfected with either control siRNA or Puma siRNA for 48 h and then treated with BetA (10 μg/ml) for
12 h. Cells were collected and lysed for immunoblotting with an antibody specific for Puma. β-Actin was used as a protein loading control. (B) Detection of cell apoptosis. DNA content, nuclear
morphology and viability were assessed after a 12 h exposure to BetA in transfected A2780/CP cells. Nuclear morphology was detected by microscopy. Arrows indicate the condensed fragmented
brightly stained nuclei, which are the hallmark of apoptosis. DNA content and cell viability was examined by flow cytometry with Annexin V staining. (C) Down-regulation of Noxa by siRNA does not
decrease apoptosis in cisplatin-resistant cells. Cells were transfected with either control siRNA or Noxa siRNA for 48 h, and then transfected cells were detected by Western blotting in the presence
of BetA for 12 h. Representative results of three experiments with consistent results are shown. (D) Graphs showing results of quantitative analyses of transfected apoptosis in A2780 and A2780/CP
cells. Cells were transfected as described in (C), then treated with BetA for 12 h. Cell apoptosis was examined as described in the Experimental section. (n = 3, means +
− S.D., **P < 0.01). (E) Cells
were transefected with the control or Puma siRNA vector for 48 h, and then treated with BetA for 24 h. Cells were collected and lysed for Western blot detection. Bax translocation, conformational
change and oligomerization were examined as described in the Experimental section. β-Actin was used as a protein loading control. (F) Cells were transfected with control or Noxa siRNA for 48 h,
and treated and detected as described in (E). All data are representative of three independent experiments. CF, cleaved fragments; Ctrl, control; IP, immunoprecipitation.
c The Authors Journal compilation c 2012 Biochemical Society
298
Z. Zhao and others
promoter after BetA treatment (Supplementary Figure S5
at http://www.BiochemJ.org/bj/444/bj4440291add.htm) and indicated that FoxO3a can regulate Puma expression. To
confirm the function of FoxO3a in Puma expression, gene
silencing of FoxO3a by siRNA was performed. Our experiment
demonstrated that Puma expression is up-regulated in control
siRNA cells, whereas ablation of FoxO3a decreases Puma
induction (Figure 5C, panel a, and Supplementary Figure
S6A at http://www.BiochemJ.org/bj/444/bj4440291add.htm).
Simultaneously, apoptosis also decreased with FoxO3a siRNA
transfection. However, significantly, the reduction of FoxO3a
could not completely inhibit Puma expression and apoptosis
(Figure 5C, panel b, and Supplementary Figure S6B). These
results suggest that FoxO3a may not be the sole transcription
initiation factor responsible for Puma expression.
To confirm that FoxO3a is a mediator of Akt-dependent
Puma-induced apoptosis, it was necessary to detect the nuclear
translocation of FoxO3a in chemoresistant cells. Our results
showed that FoxO3a translocated from the cytosol to the
nucleus and was accompanied by a decrease in phospho-Akt
and an increase in Puma expression after BetA treatment.
LY294002, the PI3K (phosphoinositide 3-kinase) inhibitor,
obviously enhanced nuclear translocation of FoxO3a in cells
treated with BetA (Figure 5D, panel a). Further experiments
indicated that overexpression of Akt1 increased the level of Akt
and phospho-Akt (Figure 5D, panel b, and Supplementary Figure
S6C), but inhibited nuclear FoxO3a translocation (Figure 5D,
panel b). These results indicated that FoxO3a is a mediator of
Akt-dependent Puma expression. It should be noted that the
increased protein level of Akt only partially prevented Puma
induction. Combined with the results displayed in Figure 5(C),
we speculate that other factors also participate in regulating Puma
induction.
JNK potentiates the Akt pathway to regulate Puma expression
To determine which factors within the Akt/FoxO3a pathway
mediate Puma expression, we first assayed the activation of
some important protein kinases, such as JNK, p38-MAPK
(mitogen-activated protein kinase) and ERK, which belong to
the MAPK family. These MAPK family proteins have been
reported to mediate the Akt/FoxO3a pathway [37,38]. As shown
in Figure 6(A), the level of phospho-JNK1/2 increased at
different periods after BetA treatment with Puma overexpression
and dephosphorylation of FoxO3a. The levels of activated
p38 (phospho-p38) were also elevated after BetA treatment
throughout the time course. A similar profile was found for
activated ERK1/2. Western blotting results also indicated that
the levels of total JNK, p38 and ERK exhibited no change
(results not shown). These results suggest that these kinases are
involved in regulating Puma expression and apoptosis after BetA
treatment.
To determine which of the kinases activated by BetA affects
ovarian cancer cell survival, we treated cells with SP600125,
SB203580 and PD98059, which are inhibitors of JNK1/2,
p38 and MEK1 (MAP/ERK kinase 1; the upstream activator
of ERK1/2) respectively. As shown in Figure 6(B), treatment of
cells with the p38-MAPK inhibitor SB203580 or ERK1
inhibitor PD98059 alone did not elicit cell death at 24 h.
Furthermore, PD98059 and SB203580 failed to protect the
cells against BetA-induced apoptosis. However, when cells were
incubated with SP600125 and BetA, the percentage of cells
undergoing apoptosis was dramatically lower than those incubated
with BetA alone, indicating that inhibition of JNK1/2 activation
protects against the cytotoxic effects of BetA.
c The Authors Journal compilation c 2012 Biochemical Society
Next, we investigated the consequences of JNK inhibition
on Puma expression and FoxO3a activation. Western blotting
results revealed that the activation of JNK (phosphoJNK) and its downstream target (phospho-c-Jun) upon BetA
treatment was abolished in the presence of SP600125.
Furthermore, SP600125 markedly attenuated the drop in
phospho-FoxO3a levels (Figure 6C, panel a) and Puma
expression (Figure 6C, panel a, and Supplementary Figure
S7 at http://www.BiochemJ.org/bj/444/bj4440291add.htm) upon
24 h of treatment with BetA. Meanwhile, nuclear FoxO3a
translocation decreased following SP600125 and BetA treatment.
The phosphorylation status of Akt mirrored that of FoxO3a, and
SP600125 obviously abolished the decline in the phospho-Akt
levels upon BetA treatment (Figure 6C, panel a). However, the
p38-MAPK inhibitor SB203580 and the ERK1 inhibitor PD98059
had little effect on Puma expression and FoxO3a activation
(Figure 6C, panels b and c). These results confirm that JNK can
regulate the Akt/FoxO3a pathway and induce Puma expression
through the Akt/FoxO3a pathway.
Our results indicated that JNK and Akt co-operatively
regulate FoxO3a transcriptional activation. However, FoxO3a
was obviously not the sole transcription factor to regulate
Puma expression. Therefore, another transcriptional factor
must participate in triggering the induction of Puma. The
JNK downstream target c-Jun is a transcriptional factor that
was shown to trigger induction of Puma RNA levels [21].
Thus c-Jun might induce Puma expression by transcriptional
regulation in ovarian cancer cells treated with BetA. Our
results indeed indicated that c-Jun can act on the Puma
promoter as described previously (Supplementary Figure S8
at http://www.BiochemJ.org/bj/444/bj4440291add.htm) [21]. To
further confirm that the JNK/c-Jun pathway also mediates Puma
activation and subsequent apoptosis, we utilized a dominantnegative mutant of c-Jun, TAM67, which was cloned into the
pGFP-C2 plasmid. An immunoblot assay demonstrated that
the location of GFP–TAM67 was in the nucleus of transfected
cells (Figure 6D, panel a), consistent with the location of c-Jun
in cells, as described previously [39]. Transfection of GFP–
TAM67 indeed reduced Puma expression and caspase cleavage
in cells treated with BetA (Figure 6D, panel b). Nevertheless,
the reduction of c-Jun activity also partially prevented Puma
expression. The inhibition of FoxO3a and c-Jun activation
prevented Puma expression and apoptosis in cells treated with
BetA (Figure 6D, panels b and c). Therefore our results suggest
that c-Jun (regulated by JNK) and FoxO3a (regulated by Akt and
JNK) co-ordinately mediate Puma expression and apoptosis in
ovarian cancer cells treated with BetA.
At the same time, we also determined whether FoxO3a and cJun can act on the Noxa promoter in our system. We knocked down
FoxO3a or c-Jun with siRNA to examine Noxa expression. The
reduction in FoxO3a and c-Jun had little effect on Noxa expression
(results not shown). These results are in agreement with previous
reports [40–42]. Valis et al. [41] demonstrated that FoxO3a has
little effect on Noxa expression. Another previous study revealed
that Noxa expression is also independent of c-Jun [42]. Eferl et al.
[40] even suggested that c-Jun antagonizes Noxa expression. Of
course, we still need to perform further experiments to study
the mechanism of Noxa up-regulation and the reasons why
Noxa expression is not involved in apoptosis induced by BetA
treatment.
Concluding remarks
Our findings can explain how BetA can induce apoptosis in
chemoresistant ovarian cancer cells. Our previous study showed
JNK- and Akt-mediated Puma expression
Figure 6
299
JNK and Akt pathway co-ordinately regulates Puma expression
(A) Western blot analysis of protein levels [phospho-JNK (p-JNK), phospho-c-Jun (p-c-Jun), etc.] in ovarian cancer cells treated with BetA (10 μg/ml) for different periods of time. Treated cells
were lysed to detect the indicated protein levels with specific antibodies as described in the Experimental section. β-Actin was used as a protein loading control. (B) A2780/CP cells were treated
with BetA (10 μg/ml), one of the following chemical kinase inhibitors: 20 μmol/l SP600125, 30 μmol/l PD98059 or 30 μmol/l SB203580, or a combination of BetA and a chemical inhibitor. Cells
were collected at 24 h after treatment, and the nuclear fragment was analysed using the Cell Death Detection ELISA kit as described in the Experimental section. (n = 3, means +
− S.D., **P < 0.01).
(C) Changes in the signal transduction pathway induced by BetA in the presence and absence of kinase activity. Cells were treated as described in (B), and then lysed to detect individual proteins
with their respective specific antibody. C-FoxO3a, cytosolic FoxO3a; N-FoxO3a, nuclear FoxO3a; p-, phospho-. Panel a, cells were treated with or without BetA and/or SP600125 for 24 h, and then
cells were lysed for detection. Panel b, cells were treated with or without BetA and/or PD98059 for 24 h, and then cells were lysed for detection. Panel c, cells were treated with or without BetA
and/or SB203580 for 24 h, and then cells were lysed for detection. (D) Panel a, cells were transfected with pEGFP-C2 or GFP–TAM67 for 48 h, and then lysed to detect protein level. Transfected
or untransfected cells were treated as described in the Experimental section for detection of protein nuclear translocation with the respective specific antibody. C, cytosolic fraction; CF, cleaved
fragments; N, nuclear fraction. Lamin B1 was used as a nuclear marker. Lane 1, pEGFP-C2 transfected cells; lane 2, untransfected cells; and lane 3, GFP–TAM67 transfected cells. Panel b, cells were
treated with either BetA (10 μg/ml), GFP–TAM67 and BetA, or a combination of BetA and two factors: FoxO3a siRNA and GFP–TAM67. Cells were collected at 24 h and analysed using Western
blotting. Panel c, the nuclear fraction was analysed using the Cell Death Detection ELISA kit as described in the Experimental section. (n = 3, means +
− S.D. **P < 0.01).
that JNK is an upstream event of apoptosis [22]. The experiments
described in the present study demonstrate that JNK is also an
early response to BetA treatment. JNK can not only mediate
the Akt/FoxO3a pathway, but also activate c-Jun in apoptosis.
This dual effect of JNK can efficiently trigger Puma activation
and subsequent apoptosis in chemoresistant cells (Figure 7). The
present study also indicates further the importance of BH3-only
proteins in chemoresistance, as our and other studies revealed that
Bim is a major determinant of cisplatin resistance in cancer cells
[22,43].
In conclusion, we have elucidated the molecular mechanisms
of BetA-induced apoptosis in cisplatin-resistant ovarian cancer
cells and demonstrated that Puma plays a critical role in cisplatin
resistance. We provide the first evidence that c-Jun (regulated
by JNK) and FoxO3a (regulated by Akt and JNK) co-ordinately
mediate Puma expression and apoptosis in chemoresistant ovarian
c The Authors Journal compilation c 2012 Biochemical Society
300
Z. Zhao and others
Figure 7 Signalling pathway for BetA-mediated Puma activation and
apoptosis in ovarian cancer cells
BetA-induced JNK pathway activation. On one hand, activated JNK regulated Puma activation
directly. On the other hand, JNK activated the Akt/FoxO3a pathway, which can also mediate
Puma expression. p53 was not involved in regulating Puma activation. BetA may also induce
the Akt/FoxO3a pathway directly (broken lines).
cancer cells. Hence, the ability of ovarian cancer to induce Puma
expression under a treatment regimen may well provide useful
prognostic markers to guide antitumour therapy.
AUTHOR CONTRIBUTION
Zhiwei Zhao and Chunlai Nie conceived and designed the experiments. Jingjing Wang and
Jingsheng Tang contributed to the RNA interference assay and data interpretation. Xinyu
Liu and Qian Zhong participated in the initial planning of the project and the experimental
approach. Fang Wang and Wenbin Hu contributed to the ChIP assay. Zhiwei Zhao, Zhu
Yuan, Chunlai Nie and Yuquan Wei interpreted the results and wrote the paper. Chunlai
Nie conceived the project.
ACKNOWLEDGEMENT
We thank Yong-qiu Mao for her expertise in flow cytometry.
FUNDING
This work was supported by the Natural Science Foundation of China (NSFC) [grant
numbers 81071817/H1609, 81001010 and 30901595].
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Received 24 October 2011/7 March 2012; accepted 7 March 2012
Published as BJ Immediate Publication 7 March 2012, doi:10.1042/BJ20111855
c The Authors Journal compilation c 2012 Biochemical Society
Biochem. J. (2012) 444, 291–301 (Printed in Great Britain)
doi:10.1042/BJ20111855
SUPPLEMENTARY ONLINE DATA
JNK- and Akt-mediated Puma expression in the apoptosis
of cisplatin-resistant ovarian cancer cells
Zhiwei ZHAO*, Jingjing WANG*, Jingsheng TANG†, Xinyu LIU*, Qian ZHONG‡, Fang WANG*, Wenbin HU*, Zhu YUAN*1 ,
Chunlai NIE*1 and Yuquan WEI*
*The State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, 4# Keyuan Road, Hitech District, Sichuan University, Chengdu 610041, China, †ChongQing NewFine
Biology Technology, 8# Lingfang Road, Banqiao District, Chongqing 402460, China, and ‡Department of Gynecology and Obstetrics, West China Second Hospital, Chengdu, Sichuan
University, Chengdu 610041, China
Figure S1 Effect of BetA plus cisplatin on apoptotic death in cisplatinresistant ovarian cancer cells
We used A2780/CP and COC1/CP cells as a model of cisplatin-resistant ovarian cells. Cells
were cultured in the presence or absence of BetA (10 μg/ml) and cisplatin (5 μg/ml) over
different periods of time. The graph shows the results of quantitative analyses of cell apoptosis
in cisplatin-resistant cells (n = 3, means +
− S.D.).
Figure S3
Figure S2 Effect of BetA on the expression of the Bcl-2 family of proteins
in COC1 and COC1/CP cells
Cells were treated with or without BetA (10 μg/ml) for 24 h and collected, then lysed in Nonidet
P40 buffer for Western blot detection. β-Actin was used as a protein loading control. All results
are representative of three independent experiments.
1
The effect of Puma and Noxa down-regulation on apoptosis
(A) Cells were transfected with either control (Ctrl) siRNA or Puma siRNA for 48 h and then
treated with BetA (10 μg/ml) for 12 h. Cells were collected and lysed for immunoblotting with
an antibody specific for Puma. β-Actin was used as a protein loading control. (B) Cells
were transfected with either control siRNA or Noxa siRNA for 48 h, and then transfected
cells were detected by Western blotting in the presence of BetA for 12 h. Representative results
of three experiments with consistent results are shown. (C) Graph showing the results of
quantitative analyses of transfected apoptosis in COC1 and COC1/CP cells. Cells were transfect
as described in (B), then treated with BetA for 12 h. Cell apoptosis was examined as descried in
the Experimental section of the main text. (n = 3, means +
− S.D., **P < 0.01).
Correspondence may be addressed to either of these authors (email [email protected] or [email protected]).
c The Authors Journal compilation c 2012 Biochemical Society
Z. Zhao and others
Figure S4 The effect of p53 down-regulation on Puma expression and
caspase-3 activation
Cells were transfected with p53 siRNA or control siRNA for 48 h and then treated with BetA for
24 h. Cells were lysed and analysed using Western blotting to detect caspase-3 cleavage and
Puma expression. Representative results of three experiments with consistent results are shown.
Figure S5
Quantification of FoxO3a association with the Puma promoter
ChIP was performed with anti-FoxO3a polyclonal antibody incubated with Protein G-coated
magnetic beads at 4 ◦ C overnight with rotation. DNA fragments co-immunoprecipitated with
the target protein FoxO3a were subjected to quantitative real-time PCR. Quantitative real-time
PCR assays were conducted from cells that were either left untreated (con) or treated with BetA.
Numbers on the y axis represent the levels of FoxO3a association with the Puma promoter
region after normalizing to C T values from input samples. The results shown are the means +
−
S.D. from three independent experiments.
c The Authors Journal compilation c 2012 Biochemical Society
Figure S6
Effect of Akt-FoxO3a pathway on Puma expression and apoptosis
(A) Cells were transfected with control (Ctrl) or FoxO3a siRNA (RNAi) for 48 h, and then treated
with BetA for 24 h. Treated cells were lysed to detect protein levels and analysed using Western
blotting. Representative results of three experiments with consistent results are shown. (B) Cells
were treated as described in (C) and then treated cells were used to detect apoptosis using a
Cell Death Detection ELISA kit as described in the Experimental section of the main text (n =
3, means +
− S.D., **P < 0.01). (C) Cells were transfected with control or Akt1 vector for 48 h
and treated with BetA for 24 h. Treated cells were lysed for detection of proteins using Western
blotting. β-Actin was used as a protein loading control.
JNK- and Akt-mediated Puma expression
Figure S7
Effect of JNK pathway on Puma expression
Cells were treated with BetA (10 μg/ml) with or without 20 μmol/l SP600125, and then cells
were collected at 24 h after treatment for detection by Western blotting. β-Actin was used as a
protein loading control. p-, phospho-.
Figure S8
Quantification of c-Jun association with the Puma promoter
ChIP was performed with anti-c-Jun polyclonal antibody incubated with Protein G-coated
magnetic beads at 4 ◦ C overnight with rotation. DNA fragments co-immunoprecipitated with
the target protein c-Jun were subjected to quantitative real-time PCR. Quantitative real-time
PCR assays were conducted from cells that were either left untreated (con) or treated with BetA.
Numbers on the y axis represent the levels of c-Jun association with the Puma promoter region
after normalizing to C T values from input samples. The results shown are the means +
− S.D.
from three independent experiments.
Received 24 October 2011/7 March 2012; accepted 7 March 2012
Published as BJ Immediate Publication 7 March 2012, doi:10.1042/BJ20111855
c The Authors Journal compilation c 2012 Biochemical Society