Embelin inhibits growth and induces apoptosis through the suppression of Akt/mTOR/S6K1 signaling cascades.

Abstract

BACKGROUND: Akt/mTOR/S6K1 signaling cascades play an important role both in the survival and proliferation of tumor cells. METHODS: In the present study, we investigated the effects of embelin (EB), identified primarily from the Embelia ribes plant, on the Akt/mTOR/S6K1 activation, associated gene products, cellular proliferation, and apoptosis in human prostate cancer cells. RESULTS: EB exerted significant cytotoxic and suppressive effects on Akt and mTOR activation against androgen-independent PC-3 cells as compared to androgen-dependent LNCaP cells. Moreover, EB suppressed the constitutive activation of Akt/mTOR/S6K1 signaling cascade, which correlated with the induction of apoptosis as characterized by accumulation of cells in subG1 phase, positive Annexin V binding, down-regulation of anti-apoptotic (Bcl-2, Bcl-xL, survivin, IAP-1, and IAP-2) and proliferative (cyclin D1) proteins, activation of caspase-3, and cleavage of PARP. We also observed that EB can significantly enhance the apoptotic effects of a specific pharmacological Akt inhibitor when used in combination and also caused broad inhibition of all the three kinases in Akt/mTOR/S6K1 signaling axis in PC-3 cells. CONCLUSIONS: EB inhibits multiple signaling cascades involved in tumorigenesis and can be used as a potential therapeutic candidate for both the prevention and treatment of prostate cancer. Prostate © 2012 Wiley Periodicals, Inc.

Full text

Page 1

TheProstate
EmbelinInhibitsGrowthandInducesApoptosisThroughthe
SuppressionofAkt/m TOR/S6K1SignalingCascades
Seong Won Kim,1Sung-Moo Kim,1Hang Bae,1Dongwoo Nam,1Jun-Hee Lee,1
Seok-Geun Lee,1Bum Sang Shim,1Sung-Hoon Kim,1Kyoo Seok Ahn,1Seung-Hoon Choi,1
Gautam Sethi,2** and Kwang Seok Ahn1*
1CollegeofOrientalMedicineandInstituteofOrientalMedicine,KyungHeeUniversity ,Seoul,RepublicofKorea
2DepartmentofPharmacology ,YongLooLinSchoolofMedicine,andCancerScienceInstituteofSingapore,National
UniversityofSingapore,Singapore,Singapore
BACKGROUND. Akt/mTOR/S6K1 signaling cascades play an important role both in the
survival and proliferation of tumor cells.
METHODS. In the present study, we investigated the effects of embelin (EB), identified
primarily from the Embelia ribes plant, on the Akt/mTOR/S6K1 activation, associated gene
products, cellular proliferation, and apoptosis in human prostate cancer cells.
RESULTS. EB exerted significant cytotoxic and suppressive effects on Akt and mTOR
activation against androgen-independent PC-3 cells as compared to androgen-dependent
LNCaP cells. Moreover, EB suppressed the constitutive activation of Akt/mTOR/S6K1 sig-
naling cascade, which correlated with the induction of apoptosis as characterized by accumu-
lation of cells in subG1 phase, positive Annexin V binding, down-regulation of anti-apoptotic
(Bcl-2, Bcl-xL, survivin, IAP-1, and IAP-2) and proliferative (cyclin D1) proteins, activation of
caspase-3, and cleavage of PARP. We also observed that EB can significantly enhance the
apoptotic effects of a specific pharmacological Akt inhibitor when used in combination and
also caused broad inhibition of all the three kinases in Akt/mTOR/S6K1 signaling axis in
PC-3 cells.
CONCLUSIONS. EB inhibits multiple signaling cascades involved in tumorigenesis and
can be used as a potential therapeutic candidate for both the prevention and treatment of
prostate cancer. Prostate
# 2012 Wiley Periodicals, Inc.
KEY WORDS:Akt/mTOR/S6K1; embelin; prostate cancer; apoptosis
INTRODUCTION
Embelia ribes Burm (Myrsinaceae), an herbaceous
medicinal plant of Pakistan and India, has been tradi-
tionally used to treat fever, dyspepsia, various gastro-
intestinal aliments, and skin disease [1]. The active
compound from this plant, an antagonist of XIAP, and
embelin (EB) has shown significant anti-proliferative
and pro-apoptotic effects against fibrosarcoma [2],
leukemia [3–5], pancreatic cancer [6,7], breast cancer
[8], colon cancer [9], multiple myeloma [10], and pros-
tate cancer cells [11]. Our previous reports have
clearly demonstrated that EB is a potent inhibitor of
activation of pro-inflammatory transcription factors
such as nuclear factor-kappaB (NF-kB) [4] and signal
transducer and activator of transcription 3 (STAT3)
[10]. Additionally, EB has also been reported to
Grant sponsor: Korean Ministry of Education, Science and Technology;
Grant number: 2011-0006220; Grant sponsor: National Medical
Research Council of Singapore; Grant number: R-184-000-211-213.
The authors have no conflicts of interest to disclosure.
Seong Won Kim and Sung-Moo Kim contributed equally to this
study.
*Correspondence to: Dr. Kwang Seok Ahn, Department of Oriental
Pathology, College of Oriental Medicine, Kyung Hee University, 1
Hoegi-Dong Dongdaemun-Gu, Seoul 130-701, Republic of Korea.
E-mail: ksahn@khu.ac.kr
**Correspondence to: Dr. Gautam Sethi, Department of Pharmacology,
Yong Loo Lin School of Medicine, Cancer Science Institute, National
University of Singapore, Singapore 117597, Singapore.
E-mail: phcgs@nus.edu.sg
Received 9 May 2012; Accepted 5 July 2012
DOI 10.1002/pros.22574
Published online in Wiley Online Library
(wileyonlinelibrary.com).
?2012WileyPeriodicals,Inc.

Page 2

induce apoptosis through up-regulation of PPARg
[9], targeting microtubular proteins [12], and cleavage
of receptor-interacting protein (RIP) [7]. Although
various molecular mechanisms as discussed above
have been described to account for the potent antitu-
mor activities of EB, its potential effect on Akt/
mTOR/S6K1 signaling cascades in tumor cells has
never been investigated before.
Since constitutive activation of Akt/mTOR/S6K1
activation pathways leads to tumorigenesis, metastasis,
and angiogenesis in tumor cells [13–16], we hypothe-
sized that EB exerts its effects at least partially through
the suppression of the Akt/mTOR/S6K1 signaling. Akt
promotes cell survival by phosphorylating substrates
that decrease the activity of pro-apoptotic proteins or
increase the activity of anti-apoptotic proteins [17–19].
Activation of Akt signaling results in a disturbance of
control of cell proliferation and apoptosis, resulting in
competitive growth advantage for tumor cells. Once
Akt stimulated, it can be propagate to a diverse array
of substrates, including the mammalian target of
rapamycin (mTOR), a key modulator of protein trans-
lation [20]. Also, ribosomal S6 kinase 1 (S6K1) is a
downstream component of mTOR signaling pathway
as a key mTOR effector of cell growth and prolifera-
tion in tumor cells [21]. The phosphorylation of S6K1
is often used as a indicator for mTOR activity in labo-
ratory and clinical research studies [22,23], and the
S6K1 gene amplification or overexpression of S6K1
has been associated with a poor prognosis in patients
with cancer [24,25].
Here, we aimed to investigate whether EB has any
anti-cancer effects in human prostate cancer, and also
to elucidate the molecular mechanisms involved in its
action. We particularly aimed to determine whether
the modulation of Akt/mTOR/S6K1 signaling cas-
cade by EB can play a critical role in its antitumor
effects in prostate cancer cells. The results indicate
that EB can indeed suppress Akt/mTOR/S6K1 sig-
naling axis and down-regulate the expression of cell
survival and proliferative gene products, leading to
suppression of proliferation and induction of apopto-
sis in PC-3 cells.
MATERIALSANDMETHODS
Reagents
Embelin
diphenyltetrazolium bromide (MTT), and propidium
iodide (PI) were purchased from Sigma (St. Louis,
MO). Anti-p-PI3K (Tyr458), anti-PI3K, anti-p-Akt
(Ser473), anti-Akt, anti-p-mTOR (Ser2448), anti-mTOR,
anti-p-S6K1 (Thr421/Ser424), anti-S6K1, anti-procaspase-
3, and anti-cleaved caspase-3 antibodies were purchased
from Cell Signaling Technology (Beverly, MA). Anti-
(EB),3-(4,5-dimethylthiazol-2-yl)-2,5-
Bcl-2, anti-Bcl-xL, anti-Survivin, anti-IAP-1, anti-IAP-
2, anti-Cyclin D1, anti-PARP, b-actin antibodies, and
HRP-conjugated secondary antibodies were from Santa
Cruz Biotechnology (Santa Cruz, CA). Annexin V was
from BD Biosciences (Palo Alto, CA). Akt inhibitor IV
was from Calbiochem (Nottingham, UK). Rapamycin
and NVP-BEZ235 were from Cayman Chemical Com-
pany (Ann Arbor, MI).
CellCulture
PC-3, LNCaP, and DU145 cells were grown in
Rosewell Park Memorial Institute (RPMI) 1640 medium
supplemented with 10% fetal bovine serum (FBS),
penicillin (100 units/ml), and streptomycin (100 mg/ml)
at 378C in a humidified atmosphere of 5% CO2and
95% air. All experiments were performed 1 day after
seeding the cells.
MTTAssay
Cell viability was measured by an MTT assay to
detect NADH-dependent dehydrogenase activity. 50 ml
MTT solution (5 mg/ml) in 1? phosphate-buffered sa-
line (PBS) was directly added to the cells, which were
then incubated for 4 hr to allow MTT to metabolize to
formazan. Absorbance was measured with an automat-
ed spectrophotometric plate reader at a wavelength of
570 nm. Cell viability was normalized as relative per-
centages in comparison with untreated controls.
WesternBlotAnalysis
After PC-3 and LNCaP cells were treated with the
indicated concentrations of EB, the cells were lysed
and the total protein concentration was determined
by Bradford reagent (Bio-Rad, Hercules, CA). Equal
amounts of lysates resolved on sodium dodecyl–
polyacrylamide gel electrophoresis (SDS–PAGE) were
transferred to a nitrocellulose membrane (Bio-Rad),
and the membrane was blocked with 1? TBS contain-
ing 0.1% Tween 20 and 5% skim milk or 2% BSA for
1 hr at room temperature. After blocking, the mem-
branes were incubated overnight at 48C with the re-
spective primary antibodies. The membranes were
washed thrice and incubated with diluted horserad-
ish peroxidase (HRP)-conjugated secondary antibod-
ies (1:5,000) for 1 hr at room temperature. After three
washes, the membranes were detected using the
enhanced chemiluminescence (ECL) kit (Millipore,
Bedford, MA).
CellCycleAnalysis
PC-3 cells were seeded onto 6-well plates at a den-
sity of 1 ? 106cells per well and incubated for 1 day.
After treatment with 50 mM of EB for 24 hr, the cells
were collected and washed with 1? PBS. Cell pellets
2Kimetal.
TheProstate

Page 3

were fixed in 70% cold ethanol overnight at ?208C.
The fixed cells were resuspended in 1? PBS contain-
ing 1 mg/ml RNase A, incubated for 1 hr at 378C,
and the cells were stained by adding 50 mg/ml PI for
30 min at room temperature in the dark. The DNA
contents of the stained cells were analyzed using Cell
Quest Software with a FACS Vantage SE (Becton
Dickinson, Heidelberg, Germany) flow cytometry
(Becton Dickinson).
AnnexinVAnalysis
1 ? 106cells were treated with EB for 24 hr and
stained by Annexin V conjugated to FITC or with
1 mg/ml DAPI solution. The cells were washed and
observed accordingly with a flow cytometry (Becton
Dickinson).
CombinationTherapyStudies
The combination index (CI) was determined by
Chou-Talalay method and calcusyn software (Biosoft,
Ferhuson, MO) and was expressed as the average of
the CI values obtained at three different combinations
(EB30/Aki2.5, EB50/AKi5, EB75/AKi7.5). A CI of less
than one was considered synergistic; a CI of one was
additive, and a CI of greater than one antagonistic.
StatisticalAnalysis
Allnumericvalues arerepresentedas the
mean ? SD. Statistical significance of the data com-
pared with the untreated control was determined
using the Student unpaired t-test. Significance was set
at P < 0.05.
RESULTS
We have previously shown that EB is a potent
inhibitor of activation of NF-kB [4] and STAT3 [10],
pro-inflammatory transcription factors that play a
critical role in cell proliferation, survival, angiogenesis,
and metastasis of tumor cells. The present study was
undertaken to determine the effect of EB on Akt/
mTOR/S6K1 signaling cascades in human prostate
cancer cells. We also evaluated the effect of EB on var-
ious mediators of cellular proliferation, cell survival,
and apoptosis. The structure of EB is shown in
Figure 1A. The concentration and duration of EB used
for Akt/mTOR/S6K1 experiments had no effect on
cell viability (data not shown).
EBExertsMoreCytotoxicEffectAgainst
Androgen-IndependentPC-3CellsVersus
Androgen-DependentLNCaPCells
The cytotoxic effect of EB against human prostate
cancer PC-3 and LNCaP cells was evaluated by MTT
assay. As shown in Figure 1B, EB inhibited cell viabil-
ity in both PC-3 and LNCaP cells up to the 75 mM
concentration. However, EB at 100 mM dose showed
more significant cytotoxic effect against PC-3 cells
(20% of cell viability) as compared to LNCaP cells
(60% of cell viability) thereby indicating that andro-
gen-independent prostate cancer PC-3 cells are more
sensitive to the cytotoxic effects of EB.
EBBlocksAktandm TORActivationinPC-3Cells,But
NotinLNCaPandDU145Cells
We next investigated whether EB can modulate
constitutive Akt and mTOR activation in tumor cells.
Fig. 1.
in androgen-independentPC-3 cells.A:The chemical structure of
embelin, EB. B: After PC-3 and LNCaP cells (1 ?104cells/well)
wereseededonto96-wellplates,theyweretreatedwithEBat0,10,
30,5075,or100 mMconcentrationsfor24 hr.Thecellviability was
measuredusing the MTTassay.C: After PC-3,LNCaP, and DU145
cells(1 ?106cells/well)wereseededonto6-wellplates,they were
treated with 75 mM of EB for 4 hr.Then equal amounts of lysates
were analyzed by Western blot analysis using antibodies against
p-Akt(Ser473),Akt,p-mTOR(Ser2448),andmTOR.
EBreducedcellviabilityandinhibitedAkt/mTORpathway
EmbelinInhibitsAkt/m TOR/S6K1Pathways3
TheProstate

Page 4

Because PC-3 and LNCaP cells have been shown to
express constitutive Akt and mTOR activation [26,27],
we set out to determine whether EB could inhibit
Akt and mTOR activation in PC-3 and LNCaP cells.
Indeed, these two prostate tumor cells clearly exhib-
ited constitutive Akt and mTOR activation and the
activation was suppressed by EB in PC-3 cells, but not
in LNCaP cells. Also, the androgen independent
DU145 cells have been known to possess a wild-type
PTEN and lower basal Akt activity compared to PC-3
and LNCaP cells [28,29]. However, EB did not sup-
press the phosphorylation of Akt and mTOR in
DU145 cells. The data suggest that inhibition of Akt
and mTOR activation by EB was cell-type specific and
EB had no effect on the expression of total Akt and
mTOR proteins (Fig. 1C). Thus, we focused on PC-3
cells to investigate the detailed mechanism by which
EB exhibits cytotoxicity and suppresses Akt and
mTOR activation.
EBSuppressesAkt/m TOR/S6K1SignalingCascadesin
aConcentration- andTime-DependentManner
Akt/mTOR/S6K1 pathways have been recognized
to play an important role in the anti-apoptotic signal-
ing machinery that confers the survival advantage
and resistance of cancer cells against various chemo-
therapeutic agents [14]. We investigated whether EB
down-regulates constitutive Akt/mTOR/S6K1 activa-
tion in PC-3 cells. Constitutive activation of the
serine protein kinase Akt was suppressed by EB in a
concentration- (Fig. 2A) and time-dependent manner
(Fig. 2B). In addition, constitutive mTOR and S6K1
activation was also suppressed upon EB treatment
in the cells (Fig. 2A and B). We also performed appro-
priate time course studies in LNCaP cells and our
results indicate that EB did not cause substantial
inhibition of Akt, mTOR, and S6K1 phosphorylation
in LNCaP cells (Fig. 2C). As shown in Figure 2D, EB
significantly suppressed cell proliferation in the
tumor cells in a concentration- and time-dependent
manner. Our data clearly indicates that the inhibition
of Akt/mTOR/S6K1 signaling cascade by EB leads to
the suppression of cell proliferation in human pros-
tate carcinoma PC-3 cells.
EBInducestheAccumulationofSubG1PhaseofCell
Cycle
To examine the apoptotic activity of EB, cell cycle
analysis was performed in EB-treated PC-3 cells. After
PI staining, DNA contents were analyzed by flow
cytometry. PI staining revealed the peaks that repre-
sent the increased percentage in apoptotic cell popu-
lation (subG1): from 1% to 15% on treatment (Fig. 3A,
left panels), moreover EB also produced morphological
changes characteristic of cell undergoing apoptosis
with cell shrinkage and fragmentation (Fig. 3A, right
panels). These data suggest that EB treatment leads to
the suppression of cell proliferation and can induce
apoptosis as evident by the increased accumulation of
the cells in the SubG1 phase of the cell cycle.
EBSuppressesCyclinD1Expression
Because D-type cyclins are necessary for the pro-
gression of cells from the G1 phase of the cell cycle to
the S phase, we set out to determine whether EB
Fig. 2.
cells.A:PC-3cellswere treatedwithvariousindicatedconcentra-
tions of EB for 4 hr.B: PC-3 cells were treated with EB at 75 mM
concentration for the various indicated time intervals.C: LNCaP
cellswere treatedwithEB at75 mMconcentrationfor thevarious
indicated time intervals.Then equal amounts of lysates were ana-
lyzed by Western blot analysis using antibodies against p-Akt
(Ser473),Akt,p-mTOR(Ser2448),mTOR,p-S6K1(Thr421/Ser424),
and S6K1. The results shown here are representative of three
independent experiments. D: PC-3 cells were plated in triplicate
and they wereleftnon-treated (NT) or treatedwith EB at 25, 50,
and75 mMconcentrationsfor theindicatedtimeintervals.Thecell
proliferationwasmeasuredusingtheMTTassay.
EB inhibited Akt/mTOR/S6K1signaling pathways in PC-3
4 Kimetal.
TheProstate

Page 5

Fig. 3.
A:AfterPC-3(1 ?106cells/well)cellswereseededonto6-wellplates,theyweretreatedwithEBat75and100 mMconcentrationsfor24 hr.
Then,thecellswerefixedandanalyzedusingaflowcytometer(leftpanel),orobservedusingamicroscope(rightpanel).(B)PC-3cellswere
treatedwithEBat75 mMconcentrationfor thevariousindicatedtimeintervals.ThenequalamountsoflysateswereanalyzedbyWesternblot
analysisusingantibodyagainstcyclinD1.C:PC-3cellswere treatedwithEBat75 mMconcentrationfor thevariousindicatedtimeintervals.
Thereafter,equalamountsoflysateswereanalyzedbyWesternblotanalysisusingantibodiesagainstBcl-2,Bcl-xL,Survivin,IAP-1,andIAP-2.
D:PC-3cellsweretreatedwithEBat75and100 mMconcentrationsfor24 hrandthecellswereincubatedwithanFITC-conjugatedAnnexinV
antibodyandthenanalyzedbyaflowcytometry .E:PC-3cellsweretreatedwithEBat75 mMconcentrationforthevariousindicatedtimeintervals.
Thereafter, equalamounts oflysates were analyzedby Westernblot analysisusing antibodies againstprocaspase-3, cleavedcaspase-3, and
PARP.b-actinwasusedasaloadingcontrol.Theresultsshownherearerepresentativeofthreeindependentexperiments.
EBcausedtheaccumulationofcellsinsubG1phase,downregulatedtheexpressionofvariousgeneproductsandinducedapoptosis.
EmbelinInhibitsAkt/m TOR/S6K1Pathways5
TheProstate

Page 6

suppresses the expression of cyclin D1. Indeed, we
observed a clear decline in the levels of cyclin D1 in
the EB-treated cells (Fig. 3B).
EBDown-RegulatesExpressionofVariousProteins
InvolvedinAnti-Apoptosis
Because Bcl-2, Bcl-xL, survivin, IAP-1, and IAP-2
have been implicated in apoptosis and mitochondrial
dysfunction, and act downstream of Akt/mTOR/
S6K1 signaling cascade, we next examined the effects
of EB on the constitutive expression of these proteins.
We found that EB suppressed the expression of anti-
apoptotic gene products in a time-dependent manner
(Fig. 3C).
EBInducesEarlyApoptosis
To confirm the anti-cancer effects of EB, we exam-
ined its ability to induce early apoptosis using the
Annexin V antibody. The Annexin V positive cells
(regarded as early apoptotic cells) were increased as
compared to the non-treated cells (NT) as observed
by flow cytometric analysis (Fig. 3D).
EBInducesApoptosisThroughActivationof
Caspase-3
Procaspase-3 is mainly activated in the apoptotic
cell via both the extrinsic and intrinsic mitochondrial
pathways, and causes cleavage of PARP involved in
the repair of DNA damage. Therefore, we also investi-
gated the effect of EB on procaspase-3 level and PARP
cleavage in PC-3 cells. EB clearly induced the cleav-
age of procaspase-3 as observed by the disappearance
of the procaspase-3 band and appearance of its
cleaved forms. Sequentially, EB induced PARP cleav-
age in a time-dependent manner (Fig. 3E). Collectively,
these results clearly demonstrate that EB can induce
apoptosis via the down-regulation of anti-apoptotic
and proliferative proteins via caspase-3 activation in
the tumor cells.
EBEnhancestheApoptoticEffectsof
PharmacologicalAktInhibitor(Aki)inT umorCells
We next determined whether EB can synergistically
enhance Aki-induced cell death. PC-3 cells were co-
treated with Aki for 1 hr before being treated with EB
for 24 hr. We found that EB clearly synergized Aki-
induced cell death in PC-3 cells (Fig. 4A). Also, we
further examined whether EB can potentiate the apo-
ptotic effect of Akt inhibitor in PC-3 cells using cell
cycle analysis. EB enhanced the accumulation of
subG1 phase of cell cycle induced by Aki from 9% to
19% in PC-3 cells (Fig. 4B). When we examined the
cells for caspase-mediated PARP cleavage, we again
observed that EB enhanced apoptosis induced by Aki
(Fig. 4C). Together, these results support the conclu-
sion that EB can indeed potentiate the apoptotic
effects of pharmacological Akt inhibitor in the tumor
cells.
DualRegulationofAktandm TORProteinsbyEB
To better understand that how EB can modulate
complex cell signaling pathways, the potential effects
of EB on Akt/mTOR/S6K1 signaling cascade were
compared to those of an Akt inhibitor (Aki), a mTOR
inhibitor (rapamycin), and dual PI3K/mTOR inhibi-
tor (NVP-BEZ235). Western blot analysis showed that
Aki and rapamycin were the most potent in inhibiting
Akt and mTOR activation, respectively (Fig. 4D).
However, NVP-BEZ235 and EB could broadly block
all the three kinases in Akt/mTOR/S6K1 signaling
axis when compared with Aki and rapamycin, thereby
indicating that EB can regulate multiple molecular
targets and signaling pathways in tumor cells.
DISCUSSION
In light of the reported chemopreventive and che-
mosensitive effects of EB on various tumor cells and
animal models, we postulated that this benzoquinone
may mediate its effects through the suppression of
Fig. 4.
S6K1signalingaxisinPC-3cells.A:AfterPC-3cellswereseededonto96-wellplates,thecellsweretreatedwithEB(50 mM)andAktinhibitor
IV(Aki,2.5and7 .5 mM)andthensubjectedtotheMTTassay(leftpanel).TheaverageoftheCIvaluesobtainedatthreedifferentcombinations
(EB30/Aki2.5,EB50/AKi5, and EB75/AKi7 .5). ACI of less than one was considered synergistic; a CI of one was considered additive and a CI
greater thanone antagonistic.B: AfterPC-3cellswere seededonto6-wellplates,the cellswere treatedwith EB (50 mM),AktinhibitorIV
(AKi,7 .5 mM)andtheindicatedcombinationofEBwithpharmacologicalAktblockersfor24 hr.Then,thecellswerefixedandanalyzedusinga
flowcytometer.C:AfterPC-3cellswereseededonto6-wellplates,thecellsweretreatedwithEB(50 mM),AktinhibitorIV(Aki,7 .5 mM)and
theindicatedcombinationofEBwithpharmacologicalAktblockersfor24 hr.Thereafter,equalamountsoflysateswereanalyzedby Western
blot analysis using antibodies against procaspase-3,PARP, and b-actin.D: PC-3 cells were treated with the Aktinhibitor IV (Aki, 7 .5 mM),
Rapamycin(20 nM),NVP-BEZ235(50 nM),orEB(50 mM)for4 hrandthenanalyzedbyWesternblotanalysisusingantibodiesagainstp-Akt
(Ser473),Akt,p-mTOR(Ser2448),mTOR,p-S6K1(Thr421/Ser424),andS6K1.Theresultsshownherearerepresentativeofthreeindependent
experiments.E:AschematicdiagramshowingtheeffectsofEBonAkt/mTOR/S6K1andapoptosis.
EB can synergize the apoptotic effects of pharmacological Akt blocker and broadly suppress all the three kinases in Akt/mTOR/
6Kimetal.
TheProstate

Page 7

the Akt/mTOR/S6K1 activation pathways. Here, we
observed that EB can indeed suppress constitutive
Akt/mTOR/S6K1 activation and exert significant
anti-proliferative and apoptotic effects in androgen-
independent
dependent LNCaP cells. Subsequently, this agent
down-regulated various gene products involved in
cell proliferation and anti-apoptosis. We also clearly
PC-3 cells, butnot inandrogen-
Fig. 4.
EmbelinInhibitsAkt/m TOR/S6K1Pathways7
TheProstate

Page 8

demonstrate that EB can target Akt/mTOR/S6K1
pathways and induce apoptosis through caspase-3
activation in human prostate cancer PC-3 cells.
Prostate cancer is very uncommon in men younger
than 45, but becomes more common with advancing
age [30]. Autopsy studies of Chinese, German, Israeli,
Jamaican, Swedish, and Ugandan men who died of
other causes have found prostate cancer in 30% of
men in their 50s, and in 80% of men in their 70s [31].
Many of the risk factors for prostate cancer are more
prevalent in the developed world, including longer
life expectancy, alcohol/tobacco intake, and diets
high in red meat [32]. Interestingly, the American
Dietetic Association and Dieticians of Canada report a
decreased the risk of developing prostate cancer
for those following a vegetarian diet [33]. However,
the specific causes of prostate cancer still remain
unknown [34].
Researchers have established a few prostate cancer
cells to investigate the mechanism involved in the
progression of prostate cancer. PC-3, LNCaP, and
DU145 cells are commonly used prostate cancer cell
lines. LNCaP cells highly express androgen receptor;
however, PC-3 and DU145 cells express very little or
no androgen receptor. Although previous studies
have shown that EB can inhibit the cell proliferation
in these human prostate cancer cells including PC-3
[3,11], DU145 [3], and LNCaP [35] cells, our study is
the first report to specifically examine Akt/mTOR/
S6K1, gene products, and the cellular responses in
human prostate cancer PC-3 cells upon EB exposure.
Recently, Hussain et al. [36] have demonstrated that
EB can overcome anti-apoptotic effects through si-
multaneous pharmacological inhibition of XIAP and
PI3K/Akt signaling in diffuse large B-cell lymphoma.
However, our findings more specifically indicate that
the constitutive Akt/mTOR/S6K1 activation was ab-
rogated by EB in human prostate cancer cells. It has
been previously reported that the level of Akt activa-
tion is drastically enhanced in androgen-independent
PC-3 cells as compared with the androgen-dependent
cells [37]. Hence, in the present report, we investigat-
ed the effects of EB on Akt/mTOR/S6K pathways
in androgen-independent prostate cancer (PC-3 cells).
We found that EB inhibited the expression of
phospho-Akt (Ser473) in a concentration-dependent
manner in PC-3 cells, which further indicates the
possibility that EB could interfere with TORC2 (the
mTOR-rictor complex that phosphrylates Akt at
Ser473). Akt is also reported to modulate the NF-kB
transcription factor through the phosphorylation of
p65 to enhance the transcriptional activity of NF-kB
[38]. NF-kB activation is also known to regulate
the expression of various cell survival, proliferative,
metastatic, and angiogenic gene products [39]. We
have previously reported that the potential effects of
EB on the modulation of NF-kB signaling cascade and
induction of apoptosis [4]. Accordingly, it is clear
from our cumulative results that the simultaneous
inhibition of NF-kB and Akt/mTOR/S6K1 signaling
can significantly contribute to the anti-cancer effects
of EB in tumor cells.
Many prior reports indicate that the aberrant acti-
vation of mTOR is closely linked with tumorigenesis
[40–42]. Recently, specific mTOR inhibitors have
shown great promise in clinical trials for the treat-
ment of various malignant tumors [43]. We for the
first time found that EB repressed constitutive mTOR
(Ser2448) activation in the cells. Thus, it is possible
that targeting the mTOR signaling pathway with a
range of agents derived from natural sources can be a
useful therapeutic strategy both for the prevention
and treatment of cancer. Because the S6K1 gene am-
plification or overexpression of S6K1 has been linked
with cell growth and proliferation in tumor cells
[24,25], it can be considered a good molecular target
in cancer. Our results also indicate, for the first time,
that EB inhibited constitutive S6K1 (Thr421/Ser424)
activation, which is a direct downstream target of
mTOR [21].
We further found that the expression of various
gene products involved in tumor initiation and pro-
motion was also down-regulated by EB and it may
explain the previously reported anti-cancer activities
of EB. These include proliferative (cyclin D1) and
anti-apoptotic (Bcl-2, Bcl-xL, survivin, IAP-1, and
IAP-2) gene products. Akt signaling through mTOR
is an important mechanism of oncogenesis that can
protect cancer cells from apoptosis and drug resis-
tance in vivo [44]. Thus, the suppression of Akt/
mTOR/S6K1 activation by EB could facilitate down-
regulation of various cell survival genes and lead to
apoptosis in tumor cells. Activated Akt can also
exert anti-apoptotic effects, positively regulate NF-kB
transcription, modulate angiogenesis, promote tumor
invasion/metastasis, and antagonize cell cycle arrest
[45]. We previously reported that EB reduced TNF-
induced COX-2 and VEGF proteins through inhibi-
tion of NF-kB activation in leukemic KBM-5 cells [4],
thereby indicating that the suppression of COX-2 and
VEGF is the potential link for the inhibition of metas-
tasis, angiogenesis, and invasion by EB. Since Bcl-2
and Bcl-xL differentially protect human prostate
cancer cells from the induction of apoptosis [46], their
down-regulation could attribute to the capability of
EB to induce apoptosis in tumor cells. Zhang et al.
[47] have indeed shown before that survivin can me-
diate resistance to anti-androgen therapy in prostate
cancer. Thus, EB may also have a potential applica-
tion to overcome resistance to anti-androgen therapy
8Kimetal.
TheProstate

Page 9

for prostate cancer. We also observed that EB inhib-
ited both the proliferation and the expression of cyclin
D1, which is consistent with previous reports that
both cyclin D1 [4], as well as S6K1 are required for
cell growth and G1 cell cycle progression in tumor
cells [48]. Additionally, we found that this benzoqui-
none can also significantly enhance the apoptotic
effects of specific Aki inhibitors, and it is very likely
that this potentiation is mediated through the accu-
mulation of cells in subG1 phase, caspase-3 activation,
and the suppression of anti-apoptotic gene products.
Our results also clearly indicate that EB exerts its
potent anti-proliferative and pro-apoptotic effects
through the modulation of Akt/mTOR/S6K1, NF-kB,
and STAT3 signal transduction pathways in tumor
cells and does not act specifically on any one cellular
signaling cascade. Interestingly, it is obvious that EB
is not active against a specific signaling cascade but it
can interfere with a multitude of targets in tumor
cells. This is quite relevant to the changing paradigm
in cancer therapy, as the increasing evidences indicate
that the mono-targeted drugs, once called smart
drugs have not had a significant impact on cancer
treatment and the use of multi-targeted drugs has be-
come increasingly accepted over the course of last
few years, as it is becoming more clearer that cancer is
caused by dysregulation of multiple pathways [49].
This is also evident by the fact that number of recent
drugs approved for cancer treatment by FDA like sora-
fenib and sunitinb modulate several tyrosine kinases/
signal transduction pathways in cancer patients.
Thus, we strongly believe that agents derived from
natural sources, like EB that can affect multiple signal
transduction cascades in tumor cells have a great po-
tential for both the prevention of cancer. Further in
vivo studies are needed to demonstrate the relevance
of these observations to prostate cancer treatment.
ACKNOWLEDGMENTS
This work was supported by the Korea Science
and Engineering Foundation (KOSEF) grant funded
by the Korean Ministry of Education, Science and
Technology (MoEST) (No. 2011-0006220). This work
was also supported in part by grants from National
Medical Research Council of Singapore [R-184-000-
211-213] to G.S.
REFERENCES
1. Gupta OP, Ali MM, Ray Ghatak BJ, Atal CK. Some pharmaco-
logical investigations of embelin and its semisynthetic deriva-
tives. Indian J Physiol Pharmacol 1977;21:31–39.
2. Chitra M, Sukumar E, Devi CS. [3H]-thymidine uptake and
lipid peroxidation by tumor cells on embelin treatment: An in
vitro study. Oncology 1995;52:66–68.
3. Nikolovska-Coleska Z, Xu L, Hu Z, Tomita Y, Li P, Roller PP,
Wang R, Fang X, Guo R, Zhang M, Lippman ME, Yang D,
Wang S. Discovery of embelin as a cell-permeable, small-
molecular weight inhibitor of XIAP through structure-based
computational screening of a traditional herbal medicine
three-dimensional structure database. J Med Chem 2004;47:
2430–2440.
4. Ahn KS, Sethi G, Aggarwal BB. Embelin, an inhibitor of X
chromosome-linkedinhibitor-of-apoptosis
nuclear factor-kappaB (NF-kappaB) signaling pathway leading
to suppression of NF-kappaB-regulated antiapoptotic and
metastatic gene products. Mol Pharmacol 2007;71:209–219.
5. Sasaki Y, Minamizawa M, Ambo A, Sugawara S, Ogawa Y,
Nitta K. Cell-penetrating peptide-conjugated XIAP-inhibitory
cyclic hexapeptides enter into Jurkat cells and inhibit cell
proliferation. Febs J 2008;275:6011–6021.
6. Mori T, Doi R, Kida A, Nagai K, Kami K, Ito D, Toyoda E,
Kawaguchi Y, Uemoto S. Effect of the XIAP inhibitor embelin
on TRAIL-induced apoptosis of pancreatic cancer cells. J Surg
Res 2007;142:281–286.
7. Mareninova OA, Sung KF, Hong P, Lugea A, Pandol SJ,
Gukovsky I, Gukovskaya AS. Cell death in pancreatitis:
Caspases protect from necrotizing pancreatitis. J Biol Chem
2006;281:3370–3381.
8. Aird KM, Ding X, Baras A, Wei J, Morse MA, Clay T, Lyerly
HK, Devi GR. Trastuzumab signaling in ErbB2-overexpressing
inflammatory breast cancer correlates with X-linked inhibitor
of apoptosis protein expression. Mol Cancer Ther 2008;7:38–47.
9. Dai Y, Qiao L, Chan KW, Yang M, Ye J, Ma J, Zou B, Gu Q,
Wang J, Pang R, Lan HY, Wong BC. Peroxisome proliferator-
activated receptor-gamma contributes to the inhibitory effects
of embelin on colon carcinogenesis. Cancer Res 2009;69:4776–
4783.
10. Heo JY, Kim HJ, Kim SM, Park KR, Park SY, Kim SW, Nam D,
Jang HJ, Lee SG, Ahn KS, Kim SH, Shim BS, Choi SH. Embelin
suppresses STAT3 signaling, proliferation, and survival of
multiple myeloma via the protein tyrosine phosphatase PTEN.
Cancer Lett 2011;308:71–80.
11. Dai Y, Desano J, Qu Y, Tang W, Meng Y, Lawrence TS, Xu L.
Natural IAP inhibitor embelin enhances therapeutic efficacy of
ionizing radiation in prostate cancer. Am J Cancer Res 2011;
1:128–143.
12. Xu M, Cui J, Fu H, Proksch P, Lin W, Li M. Embelin derivatives
and their anticancer activity through microtubule disassembly.
Planta Med 2005;71:944–948.
13. Gao N, Zhang Z, Jiang BH, Shi X. Role of PI3K/AKT/mTOR
signaling in the cell cycle progression of human prostate
cancer. Biochem Biophys Res Commun 2003;310:1124–1132.
14. Hay N. The Akt-mTOR tango and its relevance to cancer.
Cancer Cell 2005;8:179–183.
15. Bartlett JM. Biomarkers and patient selection for PI3K/Akt/
mTOR targeted therapies: Current status and future directions.
Clin Breast Cancer 2010;10(Suppl. 3):S86–S95.
16. McAuliffe PF, Meric-Bernstam F, Mills GB, Gonzalez-Angulo
AM. Deciphering the role of PI3K/Akt/mTOR pathway in
breast cancer biology and pathogenesis. Clin Breast Cancer
2010;10(Suppl. 3):S59–S65.
17. Datta SR, Dudek H, Tao X, Masters S, Fu H, Gotoh Y, Greenberg
ME. Akt phosphorylation of BAD couples survival signals to the
cell-intrinsic death machinery. Cell 1997;91:231–241.
18. Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS,
Anderson MJ, Arden KC, Blenis J, Greenberg ME. Akt
protein,blocks
EmbelinInhibitsAkt/m TOR/S6K1Pathways9
TheProstate

Page 10

promotes cell survival by phosphorylating and inhibiting a
Forkhead transcription factor. Cell 1999;96:857–868.
19. Collado M, Medema RH, Garcia-Cao I, Dubuisson ML,
Barradas M, Glassford J, Rivas C, Burgering BM, Serrano M,
Lam EW. Inhibition of the phosphoinositide 3-kinase pathway
induces a senescence-like arrest mediated by p27Kip1. J Biol
Chem 2000;275:21960–21968.
20. LoPiccolo J, Blumenthal GM, Bernstein WB, Dennis PA. Target-
ing the PI3K/Akt/mTOR pathway: Effective combinations and
clinical considerations. Drug Resist Updat 2008;11:32–50.
21. Hay N, Sonenberg N. Upstream and downstream of mTOR.
Genes Dev 2004;18:1926–1945.
22. Noh WC, Mondesire WH, Peng J, Jian W, Zhang H, Dong J,
Mills GB, Hung MC, Meric-Bernstam F. Determinants of
rapamycin sensitivity in breast cancer cells. Clin Cancer Res
2004;10:1013–1023.
23. Dancey JE. Therapeutic targets: MTOR and related pathways.
Cancer Biol Ther 2006;5:1065–1073.
24. Barlund M, Forozan F, Kononen J, Bubendorf L, Chen Y,
Bittner ML, Torhorst J, Haas P, Bucher C, Sauter G, Kallioniemi
OP, Kallioniemi A. Detecting activation of ribosomal protein
S6 kinase by complementary DNA and tissue microarray anal-
ysis. J Natl Cancer Inst 2000;92:1252–1259.
25. van der Hage JA, van den Broek LJ, Legrand C, Clahsen PC,
Bosch CJ, Robanus-Maandag EC, van de Velde CJ, van de
Vijver MJ. Overexpression of P70 S6 kinase protein is associat-
ed with increased risk of locoregional recurrence in node-
negativepremenopausalearly
Br J Cancer 2004;90:1543–1550.
26. Park KR, Nam D, Yun HM, Lee SG, Jang HJ, Sethi G, Cho SK,
Ahn KS. beta-Caryophyllene oxide inhibits growth and indu-
ces apoptosis through the suppression of PI3K/AKT/mTOR/
S6K1 pathways and ROS-mediated MAPKs activation. Cancer
Lett 2011;312:178–188.
27. Xu Y, Chen SY, Ross KN, Balk SP. Androgens induce prostate
cancer cell proliferation through mammalian target of rapamy-
cin activation and post-transcriptional increases in cyclin D
proteins. Cancer Res 2006;66:7783–7792.
breastcancerpatients.
28. Putz T, Culig Z, Eder IE, Nessler-Menardi C, Bartsch G,
Grunicke H, Uberall F, Klocker H. Epidermal growth factor
(EGF) receptor blockade inhibits the action of EGF, insulin-like
growth factor I, and a protein kinase A activator on the
mitogen-activated protein kinase pathway in prostate cancer
cell lines. Cancer Res 1999;59:227–233.
29. Uzgare AR, Isaacs JT. Enhanced redundancy in Akt and mito-
gen-activated protein kinase-induced survival of malignant
versus normal prostate epithelial cells. Cancer Res 2004;
64:6190–6199.
30. Hankey BF, Feuer EJ, Clegg LX, Hayes RB, Legler JM, Prorok
PC, Ries LA, Merrill RM, Kaplan RS. Cancer surveillance
series: Interpreting trends in prostate cancer–part I: Evidence
of the effects of screening in recent prostate cancer incidence,
mortality, and survival rates. J Natl Cancer Inst 1999;91:1017–
1024.
31. Breslow N, Chan CW, Dhom G, Drury RA, Franks LM, Gellei
B, Lee YS, Lundberg S, Sparke B, Sternby NH, Tulinius H.
Latent carcinoma of prostate at autopsy in seven areas: The
International Agency for Research on Cancer, Lyons, France.
Int J Cancer 1977;20:680–688.
32. Ganesh B, Saoba SL, Sarade MN, Pinjari SV. Risk factors for
prostate cancer: An hospital-based case-control study from
Mumbai, India. Indian J Urol 2011;27:345–350.
33. Canada. ADADo. Position of the American Dietetic Associa-
tion and Dietitians of Canada: Vegetarian diets. J Am Diet
Assoc 2003;103:748–765.
34. Hsing AW, Chokkalingam AP. Prostate cancer epidemiology.
Front Biosci 2006;11:1388–1413.
35. Danquah M, Li F, Duke CB III, Miller DD, Mahato RI. Micellar
delivery of bicalutamide and embelin for treating prostate
cancer. Pharm Res 2009;26:2081–2092.
36. Hussain AR, Uddin S, Ahmed M, Bu R, Ahmed SO, Abubaker
J, Sultana M, Ajarim D, Al-Dayel F, Bavi PP, Al-Kuraya KS.
Prognostic significance of XIAP expression in DLBCL and
effect of its inhibition on AKT signalling. J Pathol 2010;222:180–
190.
37. Murillo H, Huang H, Schmidt LJ, Smith DI, Tindall DJ. Role of
PI3K signaling in survival and progression of LNCaP prostate
cancer cells to the androgen refractory state. Endocrinology
2001;142:4795–4805.
38. Madrid LV, Mayo MW, Reuther JY, Baldwin AS Jr. Akt stimu-
lates the transactivation potential of the RelA/p65 Subunit of
NF-kappa B through utilization of the Ikappa B kinase and
activation of the mitogen-activated protein kinase p38. J Biol
Chem 2001;276:18934–18940.
39. Ahn KS, Sethi G, Aggarwal BB. Nuclear factor-kappa B: From
clone to clinic. Curr Mol Med 2007;7:619–637.
40. Strimpakos AS, Karapanagiotou EM, Saif MW, Syrigos KN.
The role of mTOR in the management of solid tumors: An
overview. Cancer Treat Rev 2009;35:148–159.
41. Inoki K, Corradetti MN, Guan KL. Dysregulation of the TSC-
mTOR pathway in human disease. Nat Genet 2005;37:19–24.
42. DeBerardinis RJ, Lum JJ, Hatzivassiliou G, Thompson CB. The
biology of cancer: Metabolic reprogramming fuels cell growth
and proliferation. Cell Metab 2008;7:11–20.
43. Campone M, Levy V, Bourbouloux E, Berton Rigaud D, Bootle
D, Dutreix C, Zoellner U, Shand N, Calvo F, Raymond E. Safety
and pharmacokinetics of paclitaxel and the oral mTOR inhibi-
tor everolimus in advanced solid tumours. Br J Cancer 2009;
100:315–321.
44. Wendel HG, De Stanchina E, Fridman JS, Malina A, Ray S,
Kogan S, Cordon-Cardo C, Pelletier J, Lowe SW. Survival
signalling by Akt and eIF4E in oncogenesis and cancer therapy.
Nature 2004;428:332–337.
45. Testa JR, Bellacosa A. AKT plays a central role in tumorigene-
sis. Proc Natl Acad Sci USA 2001;98:10983–10985.
46. Lebedeva IV, Sarkar D, Su ZZ, Kitada S, Dent P, Stein CA,
Reed JC, Fisher PB. Bcl-2 and Bcl-x(L) differentially protect
human prostate cancer cells from induction of apoptosis by
melanoma differentiation associated gene-7, mda-7/IL-24.
Oncogene 2003;22:8758–8773.
47. Zhang M, Latham DE, Delaney MA, Chakravarti A. Survivin
mediates resistance to antiandrogen therapy in prostate cancer.
Oncogene 2005;24:2474–2482.
48. Pullen N, Thomas G. The modular phosphorylation and activa-
tion of p70s6k. FEBS Lett 1997;410:78–82.
49. Mencher SK, Wang LG. Promiscuous drugs compared to selec-
tive drugs (promiscuity can be a virtue). BMC Clin Pharmacol
2005;5:3.
10Kimetal.
TheProstate