Download Crucial Role of c-Jun Phosphorylation at Ser63/73 Mediated by

Published OnlineFirst October 3, 2014; DOI: 10.1158/1940-6207.CAPR-14-0233
Cancer
Prevention
Research
Research Article
Crucial Role of c-Jun Phosphorylation at Ser63/73 Mediated
by PHLPP Protein Degradation in the Cheliensisin A Inhibition
of Cell Transformation
Junlan Zhu1,2, Jingjie Zhang1, Haishan Huang1,2, Jingxia Li1, Yonghui Yu1, Honglei Jin1,2, Yang Li1,2,
Xu Deng3, Jimin Gao2, Qinshi Zhao3, and Chuanshu Huang1
Abstract
Cheliensisin A (Chel A), as a novel styryl-lactone isolated from Goniothalamus cheliensis Hu, has been
demonstrated to have an inhibition of EGF-induced Cl41 cell transformation via stabilizing p53 protein in a
Chk1-dependent manner, suggesting its chemopreventive activity in our previous studies. However, its
underlying molecular mechanisms have not been fully characterized yet. In the current study, we found
that Chel A treatment could increase c-Jun protein phosphorylation and activation, whereas the inhibition
of c-Jun phosphorylation, by ectopic expression of a dominant-negative mutant of c-Jun, TAM67, reversed
the Chel A inhibition of EGF-induced cell transformation and impaired Chel A induction of p53 protein and
apoptosis. Moreover, our results indicated that Chel A treatment led to a PHLPP downregulation by
promoting PHLPP protein degradation. We also found that PHLPP could interact with and bind to c-Jun
protein, whereas ectopic PHLPP expression blocked c-Jun activation, p53 protein and apoptotic induction
by Chel A, and further reversed the Chel A inhibition of EGF-induced cell transformation. With the findings,
we have demonstrated that Chel A treatment promotes a PHLPP protein degradation, which can bind to
c-Jun and mediates c-Jun phosphorylation, and further leading to p53 protein induction, apoptotic
responses, subsequently resulting in cell transformation inhibition and chemopreventive activity of Chel
A. Cancer Prev Res; 7(12); 1270–81. 2014 AACR.
Introduction
Cheliensisin A (Chel A), as a novel styryl-lactone isolated
from Goniothalamus cheliensis Hu, has been reported to
possess the potent chemoprevention effect (1–3). Our
published studies have demonstrated that chemopreventive
activity is mediated by its induction of apoptosis via triggering p53 protein expression and activation (4). The
pleckstrin homology domain leucine-rich repeat protein
phosphatase (PHLPP), including PHLPP1 and PHLPP2, are
protein phosphatases, which have been demonstrated to
specifically dephosphorylate the hydrophobic motif of Akt,
subsequently triggering apoptosis and suppressing tumor
1
Nelson Institute of Environmental Medicine, New York University School
of Medicine, Tuxedo, New York. 2Zhejiang Provincial Key Laboratory for
Technology and Application of Model Organisms, School of Life Sciences,
Wenzhou Medical University, Wenzhou, Zhejiang, China. 3State Key Laboratory of Phytochemistry and Plant Resources in West China and
Kunming Institute of Botany, Chinese Academy of Sciences, Kunming,
China.
J. Zhu, J. Zhang, and H. Huang contributed equally to this article.
Corresponding Authors: Chuanshu Huang, Nelson Institute of Environmental Medicine, New York University School of Medicine, 57 Old Forge
Road, Tuxedo, NY 10987. Phone: 845-731-3519; Fax: 845-351-2320;
E-mail: [email protected]; Qinshi Zhao, E-mail:
[email protected]; and Jimin Gao, E-mail: [email protected]
doi: 10.1158/1940-6207.CAPR-14-0233
2014 American Association for Cancer Research.
1270
growth (5). However, the following studies have found that
PHLPP could also act as a tumor suppressor in several types
of cancer due to its ability to block growth factor–induced
signaling in cancer cells (5, 6). Most recently, the studies
from our group have indicated that PHLPP1 downregulation serves as cell apoptosis controller by promoting p53
protein translation via activation of Akt/p70S6K cascade
(7). We found here that PHLPP was downregulated in cells
treated with Chel A, which mediated chemopreventive
activity of Chel A.
c-Jun, a member of the basic region leucine zipper
protein family of transcription factors, in combination
with itself or other proteins such as c-Fos, forms the
transcription factor activator protein 1 (AP-1). c-Jun protein consists of a C-terminal DNA-binding domain and
an N-terminal transactivation domain. The transcriptional
activity of c-Jun is increased by phosphorylation of serines
63 and 73 in the transactivation domain (8, 9). c-Jun
phosphorylation at Ser 63 and Ser 73 could be mediated
by activation of JNKs upon a large variety of external or
internal stimulations (10–12) or the inhibition of its phosphatase. However, to the best of our knowledge, phosphatase that targets phosphorylated c-Jun protein has not been
identified yet. Upon activation, c-Jun exerts various biologic
effects on cell proliferation, differentiation, cellular transformation, and apoptosis (10–12). It has been reported
that inhibition of c-Jun activation by expressing a c-Jun
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Published OnlineFirst October 3, 2014; DOI: 10.1158/1940-6207.CAPR-14-0233
Chel A Inhibits Cell Transformation via Degradation of PHLPP
dominant-negative mutant TAM67 inhibits apoptosis due
to survival signal withdrawal (11). In the current study, we
revealed that Chel A treatment resulted in PHLPP protein
degradation, which further mediated c-Jun phosphorylation at Ser 63 and 73 through JNK-independent manner.
Moreover, we found the downregulation of PHLPP and its
mediated activation of c-Jun were essential for the induction
of apoptosis as well as the inhibition of cell transformation
induced by EGF.
Materials and Methods
Reagents and plasmids
Chel A was isolated from Goniothalamus cheliensis by the
Kunming Institute of Botany, Chinese Academy of Sciences
(Kunming, Yunnan, China) as previously described (1, 3).
The chemicals cycloheximide and MG132 were purchased
from Calbiochem. Luciferase assay substrate and EGF
were from Promega. The antibodies specific against c-Jun,
c-Jun(D), p-c-Jun Ser63, p-c-Jun Ser73, p-AKT Ser473, pAKT Thr308, AKT, p-Erk1/2, Erk1/2, p-p38, p38, p-JNK1/2,
JNK1/2, PARP, cleaved PARP, caspase-3, cleaved caspase-3,
p53, p-p53 Ser15, GFP, and GAPDH were purchased from
Cell Signaling Technology. HA antibody was obtained from
Covance Inc.. Antibodies specific against PHLPP1 and
PHLPP2 were purchased from Bethyl Laboratories. Antibodies against b-actin and a-tubulin were bought from
Sigma. The plasmid, HA-PHLPP1 and HA-PHLPP2 were
from Addgene. The plasmids, AP-1-luciferase reporter,
dominant-negative c-Jun–mutant plasmid TAM67, and
GFP-c-Jun were used and are described in our previous
studies (13–15).
Cell culture and transfection
Normal mouse epidermal Cl41 cells, which have been
previously described (4, 16, 17), and their stable transfectants were maintained in 5% FBS Eagle’s minimum
essential medium (MEM), supplemented with 1% penicillin/streptomycin and 2 mmol/L L-glutamine(Life Technologies) at 37 C in 5% CO2 incubator that have been
described previously (4, 16, 17). PW cells have been
described previously (18), and 293T cells and their stable
transfectants were cultured in DMEM with 10% FBS. The
human colon cancer cell lines HCT116 cells and their
stable transfectants were cultured in McCoy’s 5A medium
(Invitrogen), supplemented with 10% FBS. Cl41 cells
stably transfected with AP-1 transactivation luciferase
reporter, TAM67, and their corresponding control vector
have been established in our previous studies (15). These
cells are all authenticated; the ATCC number of Cl41 cell
is CRL-2010; of 293T cell is CRL-11268; and of HCT116
cell is CCL-247.
Cl41 cells were transfected with HA-PHLPP1, HAPHLPP2, and their vector control (pcDNA3.0), HCT116
cells were transfected with HA-PHLPP1 and its vector control, 293T cells were transfected with HA-PHLPP2 and
its vector control, and 293T cells were transfected with
GFP-c-Jun together with HA-PHLPP1 or HA-PHLPP2, or
GFP-c-Jun, by using PolyJet DNA In Vitro Transfection
www.aacrjournals.org
Reagent (SignaGen Laboratories) following the manufacturer’s instructions. Their stable transfectants were established by G418-resistant selection. PW cells were transfected
with TAM67 or its corresponding vector control by using the
same method as described above, and the stable transfectants were selected by G418.
Anchorage-independent growth in soft agar
Soft agar colony formation assay was conducted as
described previously (4, 15, 16, 19). Briefly, 2.5 mL of
0.5% agar in basal modified Eagle medium (BMEM) supplemented with 10% FBS and 20 ng/mL EGF, as well as Chel
A at indicated concentrations, was layered onto each well of
6-well tissue culture plates. A total of 1 104 Cl41 cells, and
their stable transfectants, were mixed with 1 mL of 0.33%
agar BMEM (supplemented with 10% FBS with or without
20 ng/mL EGF, as well as with or without Chel A), and
layered on top of the 0.5% agar layer. The plates were
incubated at 37 C in 5% CO2 for 3 weeks. The colonies
were then counted under inverse microscopy. Colonies with
more than 32 cells were scored. Each experiment was done
at least three independent times. The results were presented
as colonies/104 seeded cells.
Flow cytometry assay
Flow cytometry assay was conducted as described previously (4, 16, 20). Cl41 cells and their stable transfectants
were cultured in 6-well plates until they reached 70% to
80% confluence. Cell culture medium was replaced with
0.1% FBS medium for 36 hours. The cells were then treated
with EGF (20 ng/mL) with or without Chel A at indicated
concentrations in the medium containing 0.1% FBS. Cells
were harvested and fixed in ice-cold 70% ethanol. The cells
were stained with propidium iodide (PI) for 15 minutes and
then subjected to flow cytometry (Beckman Coulter) for
apoptotic analysis.
Western blotting
Cells were cultured using the same method described in
flow cytometry assay, followed by pretreated with Chel A for
30 minutes, and afterwards exposed to EGF as indicated.
The cells were subsequently washed on ice-cold PBS, and
then extracted with lysis buffer (10 mmol/L Tris–HCl,
pH 7.4, 1% SDS, 1 mmol/L Na3VO4, and proteasome
inhibitor). The cell extracts were subjected to the Western
blot analysis and the protein bands specifically bound to
antibodies were detected using alkaline phosphatase–
linked secondary antibody and ECF Western blotting system as described previously (4, 16).
Reverse transcription PCR
Total RNAs were extracted after treatment for the indicated time periods using TRIzol reagent (Invitrogen). Total
cDNAs were synthesized by using oligo (dT) 20 primer
by Superscript First-Strand Synthesis system (Invitrogen).
PHLPP1, PHLPP2, and b-actin mRNA presented in the
cells were determined by semiquantitative reverse
transcription (RT)-PCR assay. The primers for mouse
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Published OnlineFirst October 3, 2014; DOI: 10.1158/1940-6207.CAPR-14-0233
Zhu et al.
PHLPP1 (forward: 50 -ACACCGTGATTG CTCACTCC-30 , reverse: 50 -TTCCAGTCAGGTCTAGCTCC-30 ), mouse PHLPP2
(forward: 50 -AGGTTCCTGAGCATCTCTTC-30 , reverse: 50 -GTTCAGGCCCTTCAGTTGAG-30 ), and mouse b-actin (forward: 50 -ATATCGCTGCGCTGGTCGTC-30 reverse: 50 -AGGATGGCGTGAGGGAGAGC-30 ) were used to determine the
mRNA amount of PHLPP1, PHLPP2, and b-actin, respectively. The results were imagined with Alpha Innotech SP
image system (Alpha Innotech Corporation) as described
previously (15).
Luciferase assay
Cl41 cells stably transfected with AP-l luciferase reporter constructs were seeded into 96-well plates and cultured
until 70% to 80% confluent. The cells were treated with
various concentrations of Chel A in MEM medium containing 0.1% FBS for 12 hours and then lysed for luciferase assay using luciferase substrate as described previously (21, 22).
Immunoprecipitation
Stable transfectants of 293T cells, 293T (GFP-c-Jun,
pcDNA3.0), 293T (GFP-c-Jun, HA-PHLPP1), and 293T
(GFP-c-Jun, HA-PHLPP2), were cultured in 10-cm dishes
until 70% to 80% confluence. Culture medium was
replaced with DMEM containing 0.1% FBS for 12 hours,
and the cells were then lysed in cell lysis buffer (1% Triton X100, 150 mmol/L NaCl, 10 mmol/L Tris, pH 7.4, 1 mmol/L
EDTA, 1 mmol/L EGTA, 0.2 mmol/L Na3VO4, 0.5% NP-40,
and complete protein cocktail inhibitors from Roche) on ice.
Lysate was incubated with normal IgG/Protein A/G plusagarose or anti-GFP agarose (Santa Cruz Biotechnology, Inc.)
at 4 C for 12 hours. The agarose beads were collected by
centrifugation, followed by being washed three times with
cell lysis buffer, and the beads were extracted with Western
blot sample buffer and subjected to Western blot assay (23).
Stable transfectants of Cl41 cells, Cl41 (GFP-c-Jun/HAPHLPP1), and Cl41 (GFP-c-Jun/HA-PHLPP2), were cultured
in 10 cm dishes until 70% to 80% confluence. Culture
medium was replaced with MEM containing 0.1% FBS for
12 hours, followed by pretreatment with Chel A as indicated.
Then, the cells were lysed and extracted for Western blot assay
by using the same method as described above.
Statistical analysis
Student t test was employed to determine the significance of differences between the different groups in each
experiment. The differences were considered significant at
P < 0.05.
Results
Chel A treatment inhibited EGF-induced cell
transformation with induction of c-Jun
phosphorylation at Ser63/73 in Cl41 cells
Our most recent studies have indicated that Chel A could
act as a chemopreventive agent for inhibition of cell transformation (2, 4). Chel A treatment consistently showed the
1272
Cancer Prev Res; 7(12) December 2014
inhibition of EGF-induced cell transformation in mouse
epidermal Cl41 cells in a dose-dependent manner (Fig. 1A
and B). AP-1 is a transcription factor, and its inhibition has
been reported to be involved in chemopreventive effect in
previous studies (15, 24). Therefore, we determined whether Chel A treatment could inhibit AP-1 activation by utilizing Cl41 cells stably transfected with AP-1-luciferase reporter. Unexpectedly, the results indicated that Chel A treatment
induced AP-1–dependent transactivation in a dose-dependent manner (Fig. 1C). c-Jun is the most extensively
studied protein of AP-1 components and has also been
reported to be involved in the regulation of numerous cell
activities, such as proliferation, survival, tumorigenesis,
and apoptosis (24, 25). The transcriptional activation of
c-Jun depends on its phosphorylation at Ser 63 and 73 in
the transactivation domain (8, 9, 11). The phosphorylation of c-Jun has also been reported to play a role in the
mediation of apoptosis under withdrawal of survival
signaling (24). Therefore, the next experiment was carried
out to evaluate the effect of Chel A on c-Jun phosphorylation at Ser63/73. As shown in Fig. 1D, Chel A treatment alone induced c-Jun phosphorylation at Ser63/73 in
a time-dependent manner with maximum induction at 3
hours after treatment. Moreover, we found that cotreatment of cells with Chel A and EGF increased c-Jun
phosphorylation at Ser63/73 (Fig. 1E and F). These
results indicated that Chel A treatment led to c-Jun
phosphorylation at Ser63/73 and promoted EGF-induced
c-Jun phosphorylation in Cl41 cells.
c-Jun phosphorylation at Ser63/73 was crucial for the
Chel A inhibition of EGF-induced transformation in
Cl41 cells
To evaluate the potential role of c-Jun activation in Chel A
inhibition of cell transformation, Cl41 cells stably transfected with dominant-negative N-terminal–truncated
mutant of c-Jun, TAM67, and its parental vector control
plasmid (Cl41 vector) had been established and well characterized in our previous publications (15, 26, 27). Verification of TAM67 expression in Cl41 stable transfectant cells
was indicated in Fig. 2D. Our results revealed that ectopic
expression of TAM67 dramatically attenuated the inhibitory
effect of Chel A on EGF-induced cell transformation (Fig. 2A
and B); this strongly indicates that induction of c-Jun
phosphorylation at Ser63/73 is crucial for the chemopreventive activity of Chel A.
c-Jun exerted its chemopreventive effect via
upregulation of p53 protein expression and apoptosis
To elucidate the molecular mechanisms underlying
c-Jun–mediated chemopreventive activity of Chel A, the
flow cytometry assay was used to assess the effect of
TAM67 overexpression on cell-cycle progression and apoptotic responses, and western blot assay for p53 protein
expression due to Chel A treatment. As shown in Fig. 2C,
Chel A treatment led to a marked cell death in Cl41 (vector)
cells. Very interestingly, EGF cotreatment slightly increased
Chel A–induced cell death, rather than provide a protective
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Published OnlineFirst October 3, 2014; DOI: 10.1158/1940-6207.CAPR-14-0233
Chel A Inhibits Cell Transformation via Degradation of PHLPP
Vehicle
EGF+ChelA 0.5 μmol/L
EGF 20 ng/mL
B
D
0 1
3
EGF+Chel A 2 μmol/L
EGF+Chel A 4 μmol/L
6 12 24 36 48 Chel A (h)
p-c-Jun S63
*
20
*
15
*
10
5
*
*
4
25
*
*
0
3
2
*
1
0
0 0.5 1 2 4
Chel A (μmol/L)
p-c-Jun S73
c-Jun
Colonies (× 100) /104 cells
30
EGF+Chel A 1 μmol/L
C5
35
Relative AP-1 activity
A
0 1 2 4
Chel A (μmol/L)
F
– – – – – – + + + + + + Chel A
– + + + + + – + + + + + EGF
1.0 2.0 4.0 Chel A (μmol/L) 0 15 30 60 120 180 0 15 30 60 120 180 Time (min)
+
+
+ EGF
p-c-Jun S63
p-c-Jun S63
GAPDH
E
0
–
0
+
0.5
+
p-c-Jun S73
p-c-Jun S73
c-Jun
c-Jun
α-Tubulin
GAPDH
Figure 1. The inhibition on EGF-induced cell transformation and the induction of c-Jun phosphorylation and AP-1 transactivation by Chel A in Cl41 cells. A and
B, Cl41 cells were exposed to indicate concentrations of Chel A in combination with EGF for cell transformation assay in soft agar as described previously (4).
4
The colony formation was photographed (A), and the number of colonies was scored and presented as colonies/10 seeded cells (B). , a significant
decrease as compared with that of EGF treatment alone (P < 0.05). Each bar indicates the mean and SD of three independent experiments. C, Cl41 cells
3
(1 10 ) stably expressing AP-1-luciferase reporter were seeded into each well of a 96-well plate. After synchronization, cells were cultured in 0.1% FBS
medium for 48 hours and then treated with various concentrations of Chel A for 12 hours, and then extracted for determination of luciferase activity as
described previously (19, 41). , a significant increase in AP-1 activity (P < 0.05). Each bar indicates the mean and SD of three independent experiments.
D–F, Cl41 cells were seeded into each well of 6-well plates and cultured as described in ref. (4). Then, the cells were treated with EGF and Chel A at
different concentrations (E) or for different time periods (D and F). The cell extracts were subjected to Western blotting as described in Materials and Methods.
GAPDH and a-tubulin were used as a control for protein loading. The results shown are data represented from three independent experiments.
effect on cell death induced by Chel A in Cl41(vector) cells.
Importantly, Chel A–induced cell death was dramatically
attenuated by ectopic expression of TAM67 in Cl41 cells
(Fig. 2C), suggesting that c-Jun activation was crucial for
Chel A–induced cell death. Consistently, the results
obtained from flow cytometry also showed that sub-G1
DNA content (cell death peak) increased significantly
upon Chel A treatment in Cl41 vector cells, and stable
expression of TAM67 abolished Chel A–induced increase
in sub-G1 cells (Fig. 2E). Moreover, our results indicated
that TAM67 abrogated Chel A–induced cleaved PARP and
caspase-3 as demonstrated in Western blot analysis (Fig.
2F and G), clearly revealing that Chel A–induced cell
death was an apoptotic response. As our recent studies
have demonstrated that p53 protein induction was essential for Chel A–induced apoptotic responses (4), we assess
the relationship between c-Jun activation and p53 protein
expression by comparison of p53 protein expression due
to Chel A treatment between Cl41 (vector) and Cl41
(TAM67) transfectants. As indicated in Fig. 2D, the inhi-
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bition of c-Jun activation by ectopic expression of TAM67
blocked p53 protein expression in comparison with that
observed in Cl41 vector cells, demonstrating that c-Jun
activation mediated p53 protein expression.
Chel A treatment promoted PHLPP protein
degradation and such PHLPP protein degradation
mediated c-Jun phosphorylation at Ser63 and Ser73
due to Chel A treatment
MAPK, including the ERK1/2, JNK, and p38 have been
reported to be responsible for the regulation of various cell
functions in different experimental systems (28). Activated
MAPK causes phosphorylation and activation of transcription factors in the cytoplasm and/or nucleus (29). c-Jun has
been reported as one of the most important transcription
factors that can be phosphorylated at Ser63/73 and activated by MAPKs, especially JNKs. Thus, to elucidate the mechanism underlying c-Jun phosphorylation upon Chel A
treatment, we first examined whether Chel A treatment
could induce the activation of MAPKs. The results showed
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Published OnlineFirst October 3, 2014; DOI: 10.1158/1940-6207.CAPR-14-0233
Zhu et al.
Medium control
EGF
EGF+Chel A 2 μmol/L
EGF+Chel A 4 μmol/L
Medium control
EGF
EGF+Chel A 2 μmol/L
EGF+Chel A 4 μmol/L
C
Cl41(TAM67)
Cl41(vector)
Medium
EGF
Medium
EGF
0.6
0.35
0.25
0.4
Vehicle
Cl41 (vector)
Chel A 4 μmol/L Chel A 2 μmol/L
0
2
0
0
Cl41 (vector)
Cl41 (TAM67)
Cl41 (TAM67)
4
0
2
4 Chel A (μmol/L)
c-Jun
TAM67
p53
p-p53
Ser15
α-Tubulin
Cl41 (TAM67)
Ap
G0–G1
Ap
S
G0–G1
Ap
S
2
0.48
71.97
16.57
6.86
21.12
62.11
7.00
5.50
G0–G1
Ap
G2–M
Ap
G0–G1
G2–M
S
2
26.41
48.70
10.91
8.10
G0–G1
S
G2
Ap
Ap
G0–G1
G2–M
S
G
G2–M
10.56
61.14
20.23
5.82
G0–G1
Ap
S
4 Chel A (μmol/L)
PW (vector)
0
4
Ap
0.43
G0–G1 77.57
G2–M 11.70
S
4.99
S
Ap
G0–G1
G2–M
S
3.75
66.93
15.84
8.32
S
G2–M
G2–M
PW (TAM67)
0
4
Chel A (μmol/L)
Caspase-3
Caspase-3
Cleaved
Caspase-3
Cleaved
Caspase-3
PARP
Cleaved
PARP
β-Actin
Cancer Prev Res; 7(12) December 2014
Ap
S
M
0
G0–G1
G2–M
Cl41 (TAM67)
4
G0–G1
Ap
EGF
Ap
0.54
G0–G1 82.46
G2–M 10.77
S
3.11
G2–M
Cl41 (vector)
0
Ap
G0–G1
G2–M
S
S
G2–M
Ap
G0–G1
G2–M
S
Medium
EGF
0.51
Ap
G0–G1 89.92
G2–M 7.26
1.13
S
G0–G1
Vehicle
0.70
* 0.63
*
0.8
0.2
Cl41 (vector)
Chel A 4 μmol/L
1.0
1.0
1.0
D
Medium
1274
Medium
EGF
EGF + ChelA 2 μmol/L
EGF + ChelA 4 μmol/L
0
E
F
B
Relative colony formation
Cl41 TAM67
Cl41 Vector
A
PARP
Cleaved
PARP
β-Actin
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Chel A Inhibits Cell Transformation via Degradation of PHLPP
A
0
1
2
4 Chel A (h)
p-Erk1/2
C
– + + + +
+ −
– – – – –
– +
0 15 30 60 120 180 0
Erk1/2
+ + + + + EGF
+ + + + + Chel A
15 30 60 120 180 Time (min)
p-Erk1/2
p-p38
Erk1/2
p38
p-p38
p-JNK1/2
p38
p-JNK1/2
JNK1/2
JNK1/2
PHLPP1
PHLPP2
D
β-Actin
B
–
0
+
0
+ EGF
+
+ +
0.5 1.0 2.0 4.0 Chel A (μmol/L)
p-Erk1/2
Erk1/2
p-p38
E–
–
–
0
0
+ + +
– + +
– – –
0 1 2
1
2
3 Chel A (h)
β-Actin
phlpp1
β-Actin
phlpp2
+ + +
+ + +
– – +
3 6 1
+
+
+
2
+
+
+
3
+ MG132 (10 μmol/L)
+ CHX (50 μg/mL)
+ Chel A (4 μmol/L)
6 Time (h)
p38
PHLPP1
p-JNK1/2
PHLPP2
JNK1/2
β-Actin
Figure 3. Chel A decreased PHLPP1 and PHLPP2 protein expression by inducing PHLPP1 and PHLPP2 protein degradation. A and B, Cl41 cells were exposed
to Chel A for indicated time periods or concentrations, and the cell extracts were applied to Western blotting for determination of the protein expressions using
specific antibodies. b-Actin was used as a protein loading control. The result represents one of three independent experiments. C, Cl41 cells were treated with
EGF and/or Chel A (4 mmol/L) for indicated time periods, and the cell extracts were applied to Western blotting for determination of the protein expressions
using specific antibodies as indicated. D, Cl41 cells were exposed to Chel A (4 mmol/L) for indicated time periods and PHLPP1 and PHLPP2 mRNA was
determined by RT-PCR. E, Cl41 cells were treated with MG132 for 4 hours, followed by exposure with cycloheximide (CHX) combined with Chel A or
cycloheximide alone as indicated. Then cell extracts were subjected to Western Blotting and b-actin protein expression was used as a protein loading control.
The result represents one of three independent experiments.
that Chel A treatment alone only induced a slight increase in
the activation of ERKs, p38, and JNKs at high dose (4 mmol/
L) and did not promote kinase activation in dose–response
studies (Fig. 3A and B). In contrast, to increase cell death in
cotreatment of Cl41 cells with Chel A and EGF, as compared
with treatment of cells with either one alone, Chel A
cotreatment of cells with EGF slightly inhibited EGFinduced activation of ERKs and JNKs 15 minutes after
treatment, as compared with EGF treatment alone (Fig.
3C). Thus, the results obtained from determination of ERKs,
p38, and JNKs activation were completely inconsistent with
c-Jun phosphorylation at Ser63/73 (Fig. 1D–F), suggesting
that MAPK activations were not major mediators responsible for c-Jun activation by Chel A treatment. Thus, we
anticipated that phosphatases might play a major role in
c-Jun phosphorylation following Chel A treatment.
The recent discovery of the PHLPP Ser/Thr phosphatases
added a new player to the cast of phosphate-controlling
enzymes in cell signaling responses (30). PHLPPs, consisting of PHLPP1 and PHLPP2, catalyzes the dephosphorylation of a conserved regulatory motif, (the hydrophobic
motif) on the AGC kinases Akt, PKC, and S6 kinase, as well
Figure 2. Expression of TAM67 reversed the biologic effect of Chel A in Cl41 cells. A–C, effect of Chel A on EGF-induced cell transformation in Cl41 TAM67 and
Cl41 vector cells was determined in soft agar assays. The colony formation was observed under inverted microscope and photographed (A). The numbers of
colonies were scored and presented as colonies per 10,000 seeded cells (B). , a significant increase in Cl41 TAM67 cells compared with Cl41 Vector cells
(P < 0.05). Each bar indicates the mean and SD from three independent experiments. C, the Cl41 (TAM67) transfectant and its scramble vector transfectant,
Cl41 (vector), were treated with Chel A and EGF as indicated. Images were taken under microscopy at 48 hours after Chel A treatment. D, the stable
transfectant of Cl41 cells transfected with TAM67 and its scramble vector were treated with Chel A as indicated, and the cell extracts were applied to Western
blotting for the determination of the protein expressions using specific antibodies. a-Tubulin was used as protein loading control. E, Cl41 cells and Cl41
5
TAM67 stable transfected cells (2 10 ) were seeded into each well of 6-well plates and cultured the same as those for Western blot analysis, whereas cells
were treated with various concentrations of EGF and Chel A as indicated, for 48 hours, and then were fixed and stained with PI as described in Materials and
Methods. Cell apoptosis was determined by flow cytometry. The result shown represents one of three independent experiments. F and G, Cl41 TAM67 stably
transfected cells and its vector control cells, PW TAM67 stable transfected cells and its vector control cells were seeded into 6-well plates. The cells were
treated with Chel A for indicated concentrations for 48 hours and the cell extracts were applied to Western blotting to determine the expression of cleavages of
PARP and caspase-3. b-Actin was used as a protein loading control. The result represents one of three independent experiments.
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Zhu et al.
as an inhibitory site on the kinase Mst1, to inhibit cellular
proliferation and induce apoptosis (30). Our most recent
studies demonstrated that PHLPP1 inhibited cell apoptosis
via downregulation of p53 translation (7). Hence, we first
determined the effect of Chel A on PHLPP protein expression in Cl41 cells. The results showed that Chel A treatment
markedly attenuated the expression of PHLPPs in a timedependent manner (Fig. 3A). To elucidate the mechanisms
underlying Chel A downregulation of PHLPPs protein
expression, we first examined the effect of Chel A on phlpp1
and phlpp2 mRNA levels by RT-PCR assay. The results
showed that Chel A treatment had no observable effect on
mRNA levels of either phlpp1 or phlpp2 (Fig. 3D), excluding the possibility of Chel A affecting phlpp gene transcription or mRNA stability. Thus, we further assessed the
potential effect of Chel A on PHLPPs protein degradation.
To test whether Chel A treatment was able to promote
PHLPPs protein degradation, Cl41 cells were first pretreated
with proteasome inhibitor MG132 to accumulate PHLPPs
protein. The MG132 was then removed from cell culture
medium and the protein synthesis inhibitor cycloheximide
was added to the cells alone or in combination with Chel A.
The effect of Chel A on the dynamics of PHLPP protein
degradation was determined during the indicated time
periods. As shown in Fig. 3E, the PHLPP1 and PHLPP2
protein degradation rates were markedly increased when
cells were coincubated with Chel A plus cycloheximide in
comparison with the cells incubated with cycloheximide
alone. Those results strongly indicated that Chel A treatment decreased PHLPP expression via promoting PHLPP
protein degradation.
A
Cl41
HCT116
1
P1
PP
LP
r
HL tor
H
o
t
P
P
c
c
Ve
Ve
HA
HA
HA
To evaluate the role of PHLPPs protein degradation in
c-Jun activation upon Chel A treatment, we tested whether
the overexpression of HA-PHLPP proteins could regulate cJun phosphorylation at Ser63/73 in Cl41 cells. The results
revealed the c-Jun protein phosphorylation at Ser63/73 was
abolished upon ectopic expression of PHLPP1 in either
transfectants of Cl41 HA-PHLPP1, Cl41 HA-PHLPP2, or
HCT116 HA-PHLPP2 (Fig. 4A). To further buttress this
notion, Chel A–induced c-Jun phosphorylation was compared among the transfectants of Cl41 Vector, Cl41 HAPHLPP1, and Cl41 HA-PHLPP2. As anticipated, either overexpression of HA-PHLPP1 or HA-PHLPP2 blocked c-Jun
phosphorylation at Ser63/73 due to Chel A treatment as
comparison with Cl41 Vector cells (Fig. 4B). It was noted
that basal level of c-Jun phosphorylation at Ser63/73
was not inhibited in Cl41 HA-PHLPP1 and HA-PHLPP2
transfectants as compared with Cl41 (vector) transfectant
(Fig. 4B), which is different with the results observed in Fig.
4A. This could be caused by various cell culture conditions
in two experiments. The cell culture medium used in Fig. 4A
is 5% FBS MEM, whereas the cell culture medium used for
experiment shown in Fig. 5B is 1% FBS MEM. It is well
known that low serum of cell culture causes cell stress
responses. We therefore anticipated that low concentration
of serum for experiment shown in Fig. 4B might cause cell
stress, which could increase the basal level of c-Jun phosphorylation through activation of stress kinases, such as
JNKs. These results demonstrated that PHLPP1/2 could
repress c-Jun phosphorylation at Ser63/73, which might be
responsible for the effect of Chel A on apoptosis and cell
transformation in Cl41 cells.
Cl41
P2
LP
H
r
P
o
ct HAVe
HA
B
Cl41
HA-PHLPP1 HA-PHLPP2
Vector
0
6
0
6
0
6 Chel A (h)
HA
HA-PHLPP1
HA-PHLPP2
p-c-Jun S73
p-c-Jun S73
p-c-Jun S63
p-c-Jun S63
c-Jun
c-Jun
p-c-Jun S73
p-AKT S473
p-AKT S473
p-c-Jun S63
p-AKT T308
p-AKT T308
AKT
AKT
β-Actin
β-Actin
HA-PHLPP1
HA-PHLPP2
c-Jun
β-Actin
Figure 4. PHLPP1 and PHLPP2 are crucial for inhibition of c-Jun phosphorylation by Chel A treatment. A, Cl41 cells were stably transfected with HA-PHLPP1
and HA-PHLPP2; HCT116 cells were stably transfected with HA-PHLPP1; the cell extracts were applied to Western blotting to determine the expression
of c-Jun and its phosphorylation. B, the stable transfectant of Cl41 cells transfected with HA-PHLPP1, HA-PHLPP2, and its scramble vector were
treated with Chel A for 6 hours, and the cell extracts were applied to Western blotting for determination of the protein expressions using specific antibodies.
b-Actin was used as protein loading control.
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Chel A Inhibits Cell Transformation via Degradation of PHLPP
Vehicle
Chel A (2 μmol/L)
Chel A (4 μmol/L)
B
Vector
Vector
A
0
HA-PHLPP1
2
4
0
4
2
HA-PHLPP2
0
2
4 Chel A (μmol/L)
Caspase-3
PARP
Cl41
HA-PHLPP1
Cleaved
Caspase-3
Cleaved PARP
p-p53 S15
HA-PHLPP2
p53
Medium control
EGF
EGF+Chel A 4 μmol/L
D
35
Vector
C
GAPDH
30
Medium
EGF
EGF+ChelA 4 μmol/L
25.2
Medium control
Medium control
EGF
EGF
EGF+Chel A 4 μmol/L
EGF+Chel A 4 μmol/L
Colonies (×100)/104 cells
HA-PHLPP2
HA-PHLPP1
25
20.1
19.1
20
11.8
*
15
11.9
*
10
5.1
5
0
0
0
0
Control
HA-PHLPP1
HA-PHLPP2
Figure 5. HA-PHLPP1 and HA-PHLPP2 overexpression blocked the biologic effect of Chel A in Cl41 cells. A, Cl41 cells were stably transfected with
HA-PHLPP1 and HA-PHLPP2, Cl41 vector were treated with indicated Chel A. The cultured cells showed a declined tendency to apoptosis by Chel
A. B, Cl41 HA-PHLPP1 cells, Cl41 HA-PHLPP2 cells, and Cl41 vector cells were treated with Chel A for indicated concentrations and the cell extracts were
applied to Western blotting to determine the expression and cleavages of PARP and caspase-3, p53, and phosphorylation at Ser15. b-Actin was used as a
protein loading control. The result represents one of three independent experiments. C and D, effect of Chel A on EGF-induced cell transformation in Cl41 HAPHLPP1 cells, Cl41 HA-PHLPP2 cells, and Cl41 vector cells was determined by soft agar assay. The colony formation was observed under inverted
microscope and photographed (C). The numbers of colonies were scored and presented as colonies per 10,000 seeded cells (D). , a significant increase in
Cl41 HA-PHLPP1 cells and Cl41 HA-PHLPP2 cells as compared with those in Cl41 vector cells in response to Chel A (P < 0.05). Each bar indicates the mean
and SD from three independent experiments.
PHLPP protein degradation contributed to apoptotic
response, p53 induction, and the inhibition of EGFinduced cell transformation by Chel A treatment
To evaluate whether PHLPP downregulation contributed
to Chel A–induced apoptotic induction, Cl41 HA-PHLPP1
and Cl41 HA-PHLPP2 were employed to compare their
apoptotic responses due to Chel A treatment. As shown
in Fig. 5A and B, cell apoptotic response was observed in
Cl41 (vector) cells treated with Chel A at 4 mmol/L, whereas
such apoptotic induction was impaired by overexpression
of either PHLPP1 or PHLPP2 in Cl41 cells, as demonstrated
www.aacrjournals.org
in either cell morphology alteration or western blot results
(caspase-3 cleavage, as well as PARP cleavage). These results
demonstrated that PHLPPs degradation by Chel A played a
crucial role in its apoptotic induction. Consistent with the
alteration of cell apoptosis, PHLPP overexpression also
attenuated p53 protein expression and phosphorylation at
Ser15, due to Chel A treatment as compared with those in
Cl41 vector cells (Fig. 5B). Very importantly, the inhibition
of cell transformation by Chel A was partially reversed by
ectopic expression of HA-PHLPP1 and HA-PHLPP2 as
shown in Fig. 5C and D. Those results demonstrated that
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Zhu et al.
PHLPP degradation caused by Chel A treatment was essential for c-Jun phosphorylation, p53 protein expression, and
apoptosis, which in turn led to the inhibition of EGFinduced cell transformation in Cl41 cells.
PHLPPs interacted with c-Jun and mediated its
phosphorylation
PHLPP1 and PHLPP2 act as the phosphatases, and have
been reported to exert the function through binding with
their substrates (such as AKT and PKC), and subsequently
dephosphorylating those targeted proteins. Our abovementioned results revealed that the PHLPPs and c-Jun protein
phosphorylation at Ser63/73 was inversely corelated in
Cl41 cells after Chel A treatment. Moreover, ectopic expression of either PHLPP1 or PHLPP2 could abolish the c-Jun
protein phosphorylation at Ser63/73. Those results
prompted us to test whether PHLPP proteins could interact
with c-Jun protein. To test this possibility, 293T cells were
transfected with GFP-c-Jun, GFP-c-Jun plus HA-PHLPP1, or
GFP-c-Jun plus HA-PHLPP2, respectively, and coimmunoprecipitation was performed to pull down GFP-c-Jun using
specific anti-GFP antibodies. The stable 293T GFP-c-Jun/
PHLPP1 transfectants were selected and identified as shown
in Fig. 6A. The HA-PHLPP1 was observed in the immunocomplex pull down with specific anti-GFP antibodies, suggesting that PHLPP1 protein did interact with c-Jun protein
in the intact cells. Similarly, the binding of GFP-c-Jun with
PHLPP2 protein was also observed in transfectants of GFPc-Jun and HA-PHLPP2 (Fig. 6C). To determine whether
Chel A could regulate the interaction of c-Jun with PHLPP,
the stable Cl41 GFP-c-Jun/PHLPP1 transfectants and Cl41
GFP-c-Jun/PHLPP2 were treated with Chel A and the cell
Cl41
B
293T
GFPc-Jun
HA
HAPHLPP1
Input
c-Jun
p-c-Jun
S73
p-c-Jun
S63
p-AKT
S473
p-AKT
T308
AKT
GFP
IP
HAPHLPP1
GFP
GFP-c-Jun
GAPDH
PHLPP
p-c-Jun
Chel A
p-Chk1
p53
Apoptosis
EGF-induced cell
transformation
293T
GFPc-Jun
p-GFPc-Jun S73
p-GFPc-Jun S63
HAPHLPP2
GFPc-Jun
c-Jun
GFP
GFPc-Jun
HA
HAPHLPP1
c-Jun
p-GFPc-Jun S73
p-c-Jun
S73
p-GFPc-Jun S63
p-c-Jun
S63
HA
HAPHLPP2
P2
LP un
H
-P -c-J
HA FP
G
+
– Chel A
–
+ IgG
+
– anti-GFP
GFP
GFPc-Jun
HA
HA
GFP
Input
E
P1
LP un
H
-P -c-J
HA FP
G
–
+
–
–
–
–
+
+
+
P2
.0
LP un
A3-Jun
H
N
D -c
-P -c-J
pc FP
HA FP
G
G
–
–
+ IgG
+
+
–
anti-GFP
GFP
0
1
3. un
PPJun
NAc-J
L
D
H c
pc FP
-P PG
HA GF
–
–
+ IgG
+
+
– anti-GFP
IP
P1
0
3. Jun LP -Jun
A
H
c
DN -c -P Ppc GFP HA GF
HA
D
IP
293T
C
HAPHLPP1
HAPHLPP2
p-c-Jun
S73
p-c-Jun
S63
GFP
GFPc-Jun
c-Jun
Input
A
HA
HAPHLPP1
HAPHLPP2
p-c-Jun
S73
p-c-Jun
S63
Figure 6. PHLPPs were responsible for c-Jun phosphorylation via interaction with c-Jun. A, 293T cells were cotransfected with GFP-c-Jun, along with
HA-PHLPP1, and the cell extracts were applied to Western blotting for determination of the protein expressions using specific antibodies. B and C,
coimmunoprecipitation was performed with anti-GFP antibody–conjugated agarose beads. Immunoprecipitates were then subjected to immunoblotting
for detection of PHLPP1 and PHLPP2 using HA antibody. D, Cl41 cells cotransfected with GFP-c-Jun, along with HA-PHLPP1 or HA-PHLPP2, were
treated with or without Chel A (4 mmol/L) for 6 hours, the cell extracts were used for coimmunoprecipitation by using anti-GFP antibody–conjugated agarose
beads. Immunoprecipitates were then subjected to immunoblotting for detection of various protein expressions as indicated. E, a model for Chel A–inhibited
EGF-induced cell transformation by inducing apoptosis in JB6 Cl41 cells: Chel A treatment reduced the protein level of PHLPPs which results in upregulating
the activation of c-Jun and promoting p53 activation and accumulation, apoptosis, and inhibiting EGF-induced cell transformation.
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Chel A Inhibits Cell Transformation via Degradation of PHLPP
extracts were used to carry out pull-down GFP-c-Jun assay
using specific anti-GFP antibodies. The results showed that
although co-immunoprecipitated HA-PHLPP1 or HAPHLPP2 protein by pull-down assay with anti-GFP antibodies was decreased in Chel A–treated cells as compared
with vehicle-treated cells, the decreased levels were consistent with the downregulated protein levels of HA-PHLPP1
and HA-PHLPP2 upon Chel A treatment (Fig. 6D). This
result revealed that Chel A only inhibited HA-PHLPP protein expression, while it did not affect the binding of GFP-cJun with either HA-PHLPP1 or HA-PHLPP2. Thus, our
studies demonstrated that Chel A treatment resulted in
PHLPP protein degradation, which reduced PHLPP protein
interaction with c-Jun, and in turn led to the upregulation of
c-Jun protein phosphorylation, p53 protein induction,
apoptotic responses, as well as the inhibition of EGFinduced cell transformation.
Discussion
Defects in apoptosis underpin both tumorigenesis and
drug resistance, and most anticancer drugs exert their chemotherapeutic effect by inducing tumor cell apoptosis (31).
Chel A exhibits a potent cytotoxicity in HL-60 cells, and is
capable of inducing apoptosis of leukemia cells by downregulation of Bcl-2 expression (2). In addition, our recent
studies have found that Chel A exerts an inhibitory effect on
EGF-induced cell transformation with the induction of
apoptosis in Cl41 cells through stabilization and activation
of p53 (4). In the current study, we showed that Chel A
significantly inhibited EGF-induced cell transformation
accompanied with the induction of c-Jun protein phosphorylation at Ser63/Ser73 and PHLPP protein downregulation. Further studies showed that ectopic expression of
dominant-negative c-Jun–mutant TAM67 attenuated Chel
A–induced p53 protein expression, apoptotic induction,
and the inhibition of cell transformation; these indicated
that c-Jun phosphorylation and activation was the upstream
mediator for p53 protein expression. Moreover, we found
that PHLPP protein could bind to and interact with c-Jun
protein and attenuated c-Jun protein phosphorylation. In
addition, we revealed that Chel A treatment could induce
PHLPP1 and PHLPP2 protein degradation, which further
reduced the PHLPP protein interaction with c-Jun protein,
resulting in the upregulation of c-Jun protein phosphorylation. Therefore, we identify a novel molecular mechanism
underlying Chel A as a chemopreventive agent by activating
PHLPPs/c-Jun/p53 apoptotic axis as shown in Fig. 6E.
p53 is a tumor suppressor that has a crucial role in the
inhibition of cancer development (32). One of biologic
functions of p53 is to trigger cell apoptosis (33–35). Our
previous studies have strongly indicated that Chel A treatment leads to p53 protein accumulation and activation by
preventing p53 proteins from degradation (4). c-Jun is a key
member of the AP-1 family of transcription factors which
bind to AP-1 elements in their target genes (36), and c-Jun/
AP-1 activation is involved in the regulation of numerous
cell biologic activities, such as proliferation, survival, tumor-
www.aacrjournals.org
igenesis, and apoptosis (24, 25). c-Jun appears to be both a
positive and a negative regulator of apoptosis (36). The
exact function of c-Jun is likely to be cell type- and stimulusspecific. Eferl and colleagues analyzed the antiapoptotic
function of c-Jun using a cell culture system and found that
c-Jun–deficient hepatocytes are more sensitive to TNFainduced apoptosis and that this sensitization was rescued
upon p53 deficiency, showing that c-Jun–deficient liver
tumors accumulate high levels of p53 protein (37). However, elevated levels of p53 could not be detected in c-Jun–
deficient hepatocytes. Our current studies found that Chel
A–initiated c-Jun phosphorylation was crucial for this p53
protein accumulation and activation. It has been reported
that phosphorylated c-Jun has the ability to bind to p53
promoter for the induction of p53 transcriptional activity
(38). Our recent studies, however, have shown that Chel A
treatment upregulates p53 expression by decreasing protein
degradation level rather than transcription or mRNA stability (4). Although the detailed mechanism involved in cJun regulation of p53 protein expression is undergoing
investigation in our group, the phosphorylation of c-Jun is
a crucial factor for activation of p53 by Chel A.
PHLPP1 and PHLPP2 are the phosphatases that directly
dephosphorylate Akt, promote apoptosis, and suppress
tumor growth (5). PHLPP acts as a tumor suppressor in
several types of cancer due to its ability to block growth
factor–induced signaling in cancer cells (5, 6). However, our
most recent studies have indicated that PHLPP1 downregulation by miR-190 in arsenite responses could mediate
apoptosis via promotion of p53 translation via upregulation of Akt/p70S6K pathway (7). Our current results
showed that Chel A treatment promoted PHLPP protein
degradation, leading to an increase in c-Jun protein phosphorylation at Ser63/73, and in turn resulting in p53
protein induction and further leading to apoptosis and
inhibition of cell transformation. Given only a limited
number of Ser/Thr phosphatases to balance the actions of
more than 400 Ser/Thr kinases in human cells (39),
PHLPPs, a family of Ser/Thr phosphatases with multiple
regulatory domains, are predicted to have multiple targets
in addition to Akt and PKC that have been identified. On the
basis of previous studies, PHLPPs are critical for tumor
suppression, whereas their loss can activate p53, leading
to cellular senescence and apoptosis (40). Nevertheless, our
studies showed that PHLPPs interacted with c-Jun, which
inhibited c-Jun phosphorylation at Ser63/73, p53 protein
expression and activation, as well as apoptosis. Very importantly, we found that ectopic expression of HA-PHLPP1 or
HA-PHLPP2 could almost completely impair c-Jun phosphorylation at Ser63/73, p53 protein expression, and apoptosis, as well as reverse EGF-induced cell transformation in
Cl41 cells, which strongly demonstrated that Chel Ainduced apoptosis via PHLPPs/c-Jun/p53 was crucial for
the chemopreventive activity of Chel A. In addition, our
results revealed that PHLPPs might act as phosphatases of cJun, which is currently under investigation in our group.
In summary, our current studies elucidated a novel effect
of Chel A in regulation of PHLPP protein degradation. We
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Zhu et al.
also found that PHLPP protein degradation could reduce
the interaction of PHLPP protein with c-Jun protein and
resulting in an increase in c-Jun phosphorylation at Ser-63/
Ser-73, further promoting p53 protein expression, apoptosis, which inhibited EGF-induced cell transformation.
Those results provide a new mechanistic insight into the
understanding chemopreventive effect of Chel A as a cancer
chemopreventive agent.
Disclosure of Potential Conflicts of Interest
No potential conflicts of interest were disclosed.
Authors' Contributions
Conception and design: J. Zhang, H. Huang, J. Gao, Q. Zhao
Development of methodology: J. Zhang, X. Deng
Acquisition of data (provided animals, acquired and managed patients,
provided facilities, etc.): J. Zhu, J. Li, H. Jin, Y. Li, X. Deng, C. Huang
Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): J. Zhang, H. Huang, Y. Yu, Y. Li, X. Deng,
Q. Zhao, C. Huang
Writing, review, and/or revision of the manuscript: J. Zhang, H. Huang,
Y. Yu, Y. Li, X. Deng, J. Gao, C. Huang
Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): H. Huang, C. Huang
Study supervision: J. Gao, C. Huang
Grant Support
This study was partially supported by grants from NIH/NCI RO1
R01CA177665, CA112557, CA165980, and NIH/NIEHS ES000260 (to
C.S. Huang).
The costs of publication of this article were defrayed in part by the
payment of page charges. This article must therefore be hereby marked
advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate
this fact.
Received July 24, 2014; revised September 16, 2014; accepted September
29, 2014; published OnlineFirst October 3, 2014.
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Published OnlineFirst October 3, 2014; DOI: 10.1158/1940-6207.CAPR-14-0233
Crucial Role of c-Jun Phosphorylation at Ser63/73 Mediated by
PHLPP Protein Degradation in the Cheliensisin A Inhibition of Cell
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Junlan Zhu, Jingjie Zhang, Haishan Huang, et al.
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