The International Journal of Biochemistry & Cell Biology 39 (2007) 227237

Emodin negatively affects the phosphoinositide 3-kinase/AKT

signalling pathway: A study on its mechanism of action
Birgitte B. Olsen, Marina Bjrling-Poulsen, Barbara Guerra

University of Southern Denmark, Institute of Biochemistry and Molecular Biology, Campusvej 55, 5230 Odense M, Denmark
Received 12 June 2006; received in revised form 31 July 2006; accepted 13 August 2006
Available online 30 August 2006
Abstract
The development of selective cell-permeable inhibitors of protein kinases whose aberrant activation contributes to cell transfor-
mation is a promising approach in cancer treatment. Emodin is a natural anthraquinone derivative that exhibits anti-proliferative
effects in various cancer cell lines by efcient induction of apoptosis. The phosphoinositide 3-kinase (PI3K)/AKT pathway has
been shown to be central in the promotion of cell survival since the alteration of this signalling cascade is a frequent event in human
malignancies. Previous published results indicated that treatment of cells with inhibitors of protein kinase CK2, such as emodin,
induces apoptosis and that the anti-apoptotic effect of CK2 is partially mediated by target phosphorylation and up-regulation of
AKT by CK2. In the present study, a screening with selected CK2 inhibitors induced a variable response with respect to AKT
down-regulation, emodin being the most effective, suggesting that other mechanisms other than the inhibition of CK2 were respon-
sible for the emodin-mediated modulation of AKT. We found that emodin does not directly affect AKT kinase. Furthermore, we
show that the down-regulation of AKT is due to the emodin-mediated target inhibition of components of the PI3K pathway, which
directly or indirectly affect AKT activity, i.e. the mammalian target of rapamycin and the phosphatase and tensin homolog deleted
on chromosome 10, but not the phosphoinositide-dependent kinase 1. Taken together, our results highlight a new mechanism by
which emodin exerts anti-cancer activity and suggest the further investigation of plant polyphenols, such as emodin, as therapeutic
and preventive agents for cancer therapy.
2006 Elsevier Ltd. All rights reserved.
Keywords: CK2; AKT; mTOR; PI3K pathway; Emodin
1. Introduction
Emodin is a biologically active natural compound
extracted fromthe rhizomes of Rheumpalmatumthat can
be chemically classied as an anthraquinone derivative
(1,3,8-trihydroxy-6-methylanthraquinone). Several sci-
entic studies have been performed that indicate the vast
variety of effects mediated by this compound. Emodin
is known to have anti-microbial, immunosuppressive

Corresponding author. Tel.: +45 6550 2388; fax: +45 6550 2467.
E-mail address: bag@bmb.sdu.dk (B. Guerra).
and anti-inammatory activities (Chang et al., 1996;
Huang et al., 1992; Wang & Chung, 1997), it exerts
anti-proliferative effects in a vast array of cancer cell
lines, often enhancing the sensitivity of cancer cells
to chemotherapeutic drugs. The efcacy of emodin in
inhibiting tumorigenesis is due, at least in part, to its
ability to induce apoptosis.
Although the exact mechanism(s) of apoptosis induc-
tion by emodin remain unclear, several studies have
indicated that this compound is an effective inhibitor of
protein kinases that are known to regulate a wide range
of cellular processes, including apoptosis. Emodin is an
inhibitor of protein kinase CK2 (Yim et al., 1999), a
1357-2725/$ see front matter 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biocel.2006.08.006
228 B.B. Olsen et al. / The International Journal of Biochemistry & Cell Biology 39 (2007) 227237
constitutively active Ser/Thr kinase that is highly con-
served and ubiquitously expressed in eukaryotic cells.
CK2 is typically viewed as a tetrameric complex con-
sisting of two catalytic -subunits (and/or

) and two
regulatory -subunits. Abnormally high levels of CK2
have been observed in various types of cancer and in
transformed cells as compared to normal tissues (Unger
et al., 2004). Moreover, a direct link between tumori-
genesis and CK2 has been established employing trans-
genic mice, demonstrating that targeted overexpression
of CK2 leads to lymphocyte transformation and mam-
mary tumours (Landesman-Bollag et al., 2001; Seldin
& Leder, 1995). The depletion of CK2 subunits by
the application of antisense oligodeoxynucleotides and
siRNAtechniques, respectively, incells exposedtogeno-
toxic stress has provided additional evidence that CK2
plays a prominent, positive role in cell survival (Seeber et
al., 2005; Wang et al., 2005). These ndings have further
emphasized the validity of CK2 as potential therapeutic
target with respect to its anti-apoptotic role. Treatment
of cells with emodin causes a decrease in intracellular
protein-tyrosine phosphorylation because of target inhi-
bition of p56
lck
protein tyrosine kinase (Jayasuriya et al.,
1992). Recently, it has been shown that emodin inhibits
HER-2/neu (also known as c-erbB-2) tyrosine kinase
activity and represses the transformation of HER-2/neu-
overexpressing breast cancer cells in athymic nude mice
through repression of p185
neu
tyrosine kinase (Zhang et
al., 1999). Su et al. (2005) reported that the treatment
of human lung adenocarcinoma A549 cells with emodin
leads to apoptotic cell death associated with ERK pro-
tein kinase inactivation, conrming earlier observations
obtained with cultured human breast cancer MDA-MB-
231 cells and human skin squamous carcinoma HSC5
cells.
Earlier reports have indicated that the treatment of
cells with emodin negatively affects the phosphoinosi-
tide 3-kinase (PI3K)/AKT signalling cascade (Kim et
al., 2004; Lai et al., 2003). The PI3K signal transduc-
tion pathway has been investigated extensively for its
role in oncogenic transformation and in the preven-
tion of apoptosis (reviewed in Osaki et al., 2004). The
activation of the PI3K pathway is relatively well under-
stood and is known to be a multi-step process involving
the PI3K-dependent phosphorylation of phospholipids
localized at the plasma membrane, and the subsequent
membrane localization of phosphoinositide-dependent
kinase 1 (PDK1) and Ser/Thr kinase AKT (also known
as protein kinase B) via their pleckstrin homology (PH)
domains. The activation of PI3K ultimately leads to
AKT phosphorylation at Thr308 and Ser473. Activated
AKT controls fundamental cellular processes such as
cell survival by phosphorylating and inactivating several
downstream pro-apoptotic target molecules. PI3K was
rst implicated in the suppression of apoptosis in a study
by Yao and Cooper (1995) which demonstrated that the
inhibition of PI3K activity impairs the ability of nerve
growth factor (NGF) to prevent apoptosis. The nding
that PTEN (phosphatase and tensin homolog deleted on
chromosome 10), a lipid phosphatase considered to be
a tumour suppressor gene product is able to negatively
affect the PI3Kpathway in vivo, provided additional evi-
dence of a role of this kinase in promoting cell survival.
Mutation of PTEN, which dephosphorylates PI(3,4,5)P
3
and down-regulates the PI3Kpathway, has been reported
in various primary human tumours and in human cancer
cell lines as well (reviewed in Datta et al., 1999). The
fact that AKT overexpression is found in many human
cancers, that active AKT promotes resistance to chemo-
and radiotherapy, and that AKT activity is sufcient to
block apoptosis induced by a number of death stimuli
has resulted in intensive studies on the role of AKT as a
mediator of the PI3Ksurvival signal. These observations
suggest that the inhibition of the PI3K/AKT pathway
might be therapeutically important for cancer patients.
As mentioned above, the treatment of cells with
emodin alone or in combination with other chemother-
apeutic agents has been shown to effectively counter-
act tumour progression, although the emodin-mediated
molecular mechanismresponsible for this effect remains
to be fully elucidated. Given the importance of the afore-
mentionedpathwayinthe modulationof tumour progres-
sion, the aimof the present study was to examine in detail
how emodin affects the PI3K/AKT signalling pathway,
leading to a cell death that biochemically resembles
the typical features of apoptosis. We propose a model
that supports the therapeutic validity of emodin in the
treatment of human malignancies and other pathologi-
cal conditions, since the emodin-mediated regulation of
components of the PI3Kpathway upstreamof AKTleads
to an effective down-regulation of AKT kinase activity.
2. Materials and methods
2.1. Cell culture and treatments
HeLa cell line was grown in DMEM (Gibco) supple-
mented with 10% (v/v) fetal bovine serum (FBS) and
1 mM l-glutamine. Cells were cultured at 37

C under
a 5% CO
2
atmosphere. For the in vivo activation of
AKT and mTOR kinases, cells were seeded and 24 h
thereafter they were subjected to serum starvation for
24 h, followed by treatment with 100 ng/ml IGF-1 (Cal-
biochem) for 10 and 30 min, respectively. The transient
B.B. Olsen et al. / The International Journal of Biochemistry & Cell Biology 39 (2007) 227237 229
overexpression of AKT was achieved, where indicated,
by transfecting cells with FuGene 6 reagent (Roche) with
a total of 2 g plasmid DNA in 60 mm Petri dishes fol-
lowing the manufacturers recommendations. In the case
of AKT activation, cells were transfected 24 h prior to
serum starvation. After 24 h, cells were incubated with
100 ng/ml IGF-1 for 10 min at 37

C. The incubation
of cells with apigenin, emodin and DMAT (purchased
from Calbiochem) and LY294002 (obtained from Cell
Signalling Technology), respectively, was as indicated
in the gure legends.
2.2. Antibodies
Proteins were detected by Western blotting using
the following primary antibodies: monoclonal anti-
AKT, monoclonal anti-mTOR and monoclonal anti-
PDK1 (all from BD Biosciences), polyclonal anti-AKT,
and polyclonal anti-PDK1 (both from Upstate). Poly-
clonal anti-phospho-AKT(Thr308), monoclonal anti-
phospho-AKT(Ser473), monoclonal anti-phospho-p70
S6 kinase (Thr389), polyclonal anti-phospho-PTEN
(Ser380/Thr382/383), polyclonal anti-PTEN, polyclonal
anti-p44/42 MAP kinase, monoclonal anti-phospho-
p44/42 MAP kinase (Thr202/Tyr204) and polyclonal
anti-phospho-p38 MAP kinase (Thr180/Tyr182) anti-
bodies were all from Cell Signaling Technology.
Polyclonal anti-p70 S6 kinase, polyclonal anti-mTOR,
polyclonal anti-p38 (H-14) and monoclonal anti-JNK
antibodies were purchased from Santa Cruz Biotech-
nology. Polyclonal anti-phospho-JNK (Thr183/Tyr185)
antibody was from Biosource while monoclonal
anti--actin antibody was obtained from Sigma.
Protein-antibody complexes were visualized by a
chemiluminescence Western blotting detection system
according to the manufacturers instructions (CDP-Star,
Applied Biosystems).
2.3. Preparation of cell extracts and
immunoprecipitation
Prior to harvesting, cells were rinsed with ice-cold
PBSand lysed with lysis buffer (50 mMTris/HCl pH7.5,
150 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM
DTT, 1 mM Na
3
VO
4
, 30 mM -glycerophosphate,
10 mM NaF, 100 nM okadaic acid and a protease
inhibitor cocktail, Roche). Lysates were cleared by
centrifugation at 4

C for 30 min at 10,000 g. The

protein concentration of supernatants was determined
by the Bradford assay (BioRad). Whole cell extracts
were subjected to SDS-PAGE, Western blotting anal-
ysis, or protein kinase assays. For immunoprecipitation
experiments, protein A-agarose (Roche) was incubated
overnight at 4

C with the antibodies indicated in the

gure legends. Thereafter, cell lysates were added and
incubated for 3 h at 4

C with gentle rocking. Immuno-

complexes were extensively washed with NET-modied
buffer (50 mM Tris/HCl pH 8, 150 mM NaCl, 5 mM
EDTA, 0.05% NP40, 0.2% casein, 0.02% NaN
3
) con-
taining a protease inhibitor cocktail and subsequently
eluted either by adding SDS-PAGE sample buffer or
by washing twice with assay buffer prior to the pro-
tein kinase assay. In the case of mTOR immunopre-
cipitates, the procedure followed was essentially as
described above except that cells were lysed on ice with
lysis buffer containing 0.3% CHAPS instead of Triton
X-100 (CHAPS lysis buffer) and immunoprecipitation
experiments were performed with CHAPS lysis buffer.
Immunocomplexes were analyzed by immunoblot-
ting with the antibodies indicated in the gure
legends.
2.4. Protein kinase assays
To monitor the activity of AKT transiently expressed
in HeLa cells, 1 mg cell lysate was subjected to immuno-
precipitation as described above with rabbit polyclonal
anti-AKT antibody. Prior to the kinase assay, immuno-
precipitates were washed with kinase assay buffer
(25 mM Tris/HCl pH 7.5, 30 mM -glycerophosphate,
10 mM MgCl
2
, 1 mM Na
3
VO
4
, 20 mM NaF, 1 mM
DTT). Kinase assays were performed in kinase assay
buffer supplemented with 50 M ATP, 10 Ci of [-
32
P]ATP (3000 Ci/mmol, Hartmann Analytic) and 5 g
histone 2B (H2B, Roche) substrate in a total volume
of 60 l. Reaction mixtures were incubated at 30

C
for 30 min and subsequently stopped by adding SDS-
PAGE sample buffer. Samples were subjected to SDS-
PAGE. The activity of endogenous mTOR was eval-
uated after immunoprecipitation with goat polyclonal
anti-mTOR antibody, essentially as described above.
Immunoprecipitates were washed twice in mTORkinase
buffer (25 mMHepes pH7.5, 100 mMpotassiumacetate,
1 mM MgCl
2
). The kinase reaction was performed at
37

C for 20 min in a nal volume of 40 l in the

mTOR kinase buffer containing 500 M ATP, 10 Ci
of [-
32
P]ATP and 1 g recombinant inactive AKT1
(Upstate). The activity of endogenous PDK1 was tested
after immunoprecipitation with polyclonal anti-PDK1
antibody. Immunoprecipitates were washed twice in
PDK1 kinase buffer (50 mM Tris/HCl pH 7.5, 100 M
EGTA, 100 M EDTA, 1 mM DTT, 100 nM okadaic
acid, 10 mM MgAc). The kinase reaction was per-
formed at 30

C for 30 min in PDK1 kinase buffer (nal

230 B.B. Olsen et al. / The International Journal of Biochemistry & Cell Biology 39 (2007) 227237
volume 40 l) containing 100 M ATP, 10 Ci of [-
32
P]ATP and 1 g recombinant inactive AKT1. PDK1
kinase assay performed with active recombinant PDK1
(Upstate) was performed using recombinant inactive
AKT1 as a substrate according to the manufacturers
recommendations. All reactions were stopped by the
addition of SDS-PAGEsample buffer. In all experiments,
radiolabeled proteins were visualized by autoradiogra-
phy and the radioactivity incorporated was quantied by
scintillation counting of the excised radioactive bands.
AKT kinase assays in the presence of human recom-
binant AKT1, AKT2 and AKT3 (all purchased from
KinaseDetect Aps), respectively, were performed essen-
tially as in the case of immunoprecipitated AKT1 from
cell lysate using as a substrate 100 M synthetic pep-
tide (RPRAATF, Upstate) and by incubating the reaction
mixtures at 30

C for 15 min. Protein kinase CK2 activ-

ity assay was performed in a reaction mixture containing
50 mM Tris/HCl pH 7.5, 150 mM NaCl, 10 mM MgCl
2
,
50 M [-
32
P]ATP and 150 M synthetic peptide
(RRRDDDSDDD) substrate in a total volume of 30 l.
The reactions were initiated by adding 20 g of pro-
tein extracts from cells and incubated at 37

C for 5 min.
Assays were stopped on ice and immediately after-
wards were spotted onto P81 phosphocellulose paper
lters (Whatmann). Filters were washedextensivelywith
0.75% phosphoric acid and then transferred into vials
containing a scintillation cocktail. Radioactivity was
quantied by scintillation counting (Canberra-Packard).
3. Results
3.1. Treatment of cells with emodin leads to
down-regulation of AKT kinase
Previous results have indicated the ability of
anthraquinone-derivative compounds to efciently
induce cell death in a number of different cell lines
including HeLa human cervical carcinoma cells. One
of the most studied intracellular cascade which con-
trols cell survival by preventing cells from undergo-
ing apoptosis is the PI3K/AKT-mediated signal trans-
duction pathway which has been shown to be nega-
tively affected by emodin as mentioned above (Kim
et al., 2004; Lai et al., 2003). Recent data reported by
Di Maira et al. (2005) led to the nding that down-
regulation of protein kinase CK2 activity or protein
level in cells correlates with decreased AKT kinase
activity, revealing a novel mode of regulation of AKT
by CK2-mediated constitutive phosphorylation. Because
emodin is an inhibitor of protein kinase CK2, we con-
sidered the possibility that the in vivo emodin-mediated
inhibition of CK2 might affect cell survival through
the down-regulation of AKT. In order to verify this
hypothesis, cells transiently overexpressing AKT1 were
incubated with emodin and, for comparison, two addi-
tional inhibitors of CK2, apigenin and 2-dimethylamino-
4,5,6,7-tetrabromo-1H-benzimidazole (DMAT), respec-
tively, as indicated in Fig. 1. We performed an immuno-
precipitation assay where the activity of AKT1 was
measured in a kinase assay using histone 2B (H2B)
as substrate according to the cell treatment indicated
in Fig. 1A. Surprisingly, the AKT1 kinase assay and
the subsequent densitometric analysis of the phospho-
rylated histone 2B bands demonstrated that the cellu-
lar treatment with emodin was the most effective in
inhibiting AKT in vivo. The intensity of the
32
P-H2B
protein band was reduced up to 51% (Fig. 1A, lane 2)
with respect to the control experiment (Fig. 1A, lane
1). These results suggested that the emodin-mediated
down-regulation of AKT might have been dependent on
other mechanisms not linked to CK2 inhibition. Since
a CK2 kinase test performed on total lysate from cells
treated with DMSO (Fig. 1B, Control-bar) or incubated
with the indicated compounds revealed that emodin, api-
genin and DMAT markedly inhibited CK2 activity to the
same extent (Fig. 1B), we speculated that the observed
decrease in AKT activity in cells treated with emodin
might have been due to the inhibition of either AKT
and/or protein kinases that modulate AKT activity by
target phosphorylation. To shed light on this point, we
performed an in vitro kinase assay where the activity of
1 pmol of active recombinant puried AKT1, AKT2 and
AKT3, respectively, were tested in the presence of AKT
peptide substrate and increasing amounts of emodin. As
shown in Fig. 2, 40 M emodin led to a 27% decrease
in AKT1 activity (black bar). This value was not con-
sistent with the degree of inhibition observed in the
experiment where AKT was immunoprecipitated from
the total lysates of cells treated as indicated in Fig. 1A
and subsequently subjected to kinase assays using H2B
as substrate. In the in vitro assay, a higher amount of
emodin (i.e. 8090 M) was required in order to induce
a 50% inhibition of AKT1 activity (results not shown).
In the case of AKT2 (grey bar), 40 M emodin led to a
9.6%decrease in AKTactivity with respect to the control
experiment performed in the presence of DMSO. AKT3
activity was not inhibited by the presence of emodin
(white bar). Instead, it is apparent that AKT3 kinase was
affected by the presence of DMSOas compared with the
control assay (Control). The results reported in Fig. 2
support the notion that emodin does not affect consis-
tently the activity of AKT but rather inhibits upstream
proteins that target and up-regulate AKT.
B.B. Olsen et al. / The International Journal of Biochemistry & Cell Biology 39 (2007) 227237 231
Fig. 1. Modulation of AKT kinase activity by protein kinase CK2
inhibitors. (A) Cells transiently expressing AKT1 were treated for
4 h with 40 M emodin, 50 M apigenin and 25 M DMAT, respec-
tively, as indicated in the gure. Activation of AKT was induced by the
incubation of cells with 100 ng/ml IGF-1 for 10 min prior to harvest-
ing. Total lysates were subjected to immunoprecipitation with rabbit
polyclonal anti-AKT antibody and subsequently, to kinase activity as
described in Section 2. The samples were analyzed by SDS-PAGE
and transferred afterwards to PVDF membrane. Proteins were visu-
alized by probing the membrane with the indicated antibody while
phosphorylated proteins were revealed by autoradiography. The values
reported below each lane number represent the densitometric analy-
sis (expressed in %) of the
32
P-H2B bands. The quantication was
performed with Gelworks 1D Intermediate Software assigning 100 to
the protein in lane 1. The experiment was done in triplicate. (B) CK2
kinase assays were performed as described in Section 2 with a specic
CK2 peptide substrate and 20 g total lysate from HeLa cells incu-
bated as described in (A). The averages and standard deviation of the
mean of three independent experiments are shown.
3.2. Emodin is an inhibitor of mTOR kinase
In order to gain insight into the mechanism by which
emodin modulates AKT kinase activity in vivo, we then
Fig. 2. Emodin is not an inhibitor of AKT. AKT kinase activity was
performed in the presence of 60 ng active recombinant AKT1, AKT2
and AKT3 kinases with increasing amounts of emodin as indicated in
the gure. Kinase activity, performed as described in Section 2, is pre-
sented as percentage of control activity and represents the mean S.D.
of three independent experiments.
analysed the phosphorylation status of AKT by Western
blotting using phospho-specic antibodies. The analysis
was carried out with cells left untreated or stimulated
with the insulin-like growth factor (IGF-1) allowing the
investigationof the effect of emodinonactivatedproteins
that play a role in the regulation of AKT kinase activity.
As shown in Fig. 3, the phosphorylation level of AKT at
Ser473, one of the two target amino acids whose phos-
phorylation up-regulates AKT kinase activity, was sig-
nicantly inhibited in cells treated with 30 M emodin
for 12 h (Fig. 3, lane 4) as compared to the control experi-
ment (Fig. 3, lane 3). As a positive control, cells were also
incubated with 100 M LY294002 for 1 h, a avonoid
derivative which has been reported to efciently inhibit
different members of the PI3K family as well as mTOR
kinase (Fig. 3, lane 5). In all experiments, activation
of AKT was induced by brief incubation of cells with
IGF-1 prior to harvesting as described in Section 2. A
recent study by Sarbassov et al. (2005) demonstrated
that the mammalian target of rapamycin (mTOR) pro-
tein kinase in complex with Rictor:GL targets AKT
for phosphorylation at Ser473. In cells, mTOR can be
part of two distinct complexes dened by Rictor and
Raptor proteins and characterized by distinct substrate
targets. In order to test whether emodin targets directly
mTOR or exclusively the complex RictormTOR in
vivo, we included the analysis of the phosphorylation
level of RaptormTOR-downstream protein target (i.e.
p70 S6K), before and after cell treatment with the indi-
cated compounds (i.e. emodin and LY294002). Cell
232 B.B. Olsen et al. / The International Journal of Biochemistry & Cell Biology 39 (2007) 227237
Fig. 3. Treatment of cells with emodin affects the phosphorylation sta-
tus of AKT at Ser473. HeLa cells were incubated with 30 M emodin
for 12 h (lanes 2 and 4) and with 100 M LY294002 for 1 h (lane 5),
respectively. Where indicated, cells were stimulated with IGF-1 before
harvesting. Whole-cell lysates were analysed by Western blotting prob-
ing the membranes with the indicated antibodies. Note that monoclonal
anti-phospho-p70 S6K (Thr389) antibody detects endogenous levels
of p70 S6 kinase that has been phosphorylated at Thr389. Endogenous
p85 S6 kinase is detected when phosphorylated at the analogous site,
i.e. Thr412. Lane 1 represents a control experiment (DMSO) where
the endogenous level of the indicated proteins in whole lysate fromthe
DMSO-treated cells was analysed.
incubation with emodin markedly reduced the level of
RaptormTOR-catalyzed phosphorylation of p70 S6K
at Thr389 (Fig. 3, lane 4). As expected, LY294002 treat-
ment signicantly attenuated the level of phosphoryla-
tion of p70 S6K as a consequence of target inhibition
mTOR (Fig. 3, lane 5). Moreover, the sole treatment
of cells with emodin (i.e. without IGF-1-mediated AKT
activation) apparently did not affect the phosphorylation
of AKTas comparedtoDMSO-treatedcells (Fig. 3, lanes
1 and 2). Next, we veried whether emodin is indeed
a direct inhibitor of mTOR kinase (Fig. 4). HeLa cells
were starved for 24 h in the absence of serum and subse-
quentlystimulatedwith100 ng/ml IGF-1for 30 min. Cell
extracts were subjected to immunoprecipitation assays.
Immunoprecipitated endogenous mTOR was subjected
tokinase assayusingrecombinant inactive AKT1protein
as a substrate in the absence (Fig. 4, lanes 1 and 2) and in
the presence of 60 M emodin (Fig. 4, lane 3), respec-
tively. The observed reduced level of phosphorylation of
Fig. 4. Emodin inhibits mTOR kinase activity. Immunoprecipitates
prepared from HeLa cell lysates (stimulated with IGF-1 prior to har-
vesting) with either polyclonal anti-mTOR antibody (lanes 2 and 3)
or a rabbit control serum (C, lane 1), were subjected to kinase assays
with inactive recombinant AKT1 as substrate. As indicated, the phos-
phorylation assay was performed in the presence of DMSO (lane 2)
and 60 M emodin (lane 3), respectively. Samples were analysed by
immunoblotting for the indicated protein levels. The phosphorylation
of AKT1 was revealed by autoradiograpy.
AKT protein indicates that the presence of emodin in the
kinase assay signicantly inhibits the catalytic activity
of mTOR kinase.
3.3. Emodin modulates the phosphorylation of
PTEN protein phosphatase but does not inuence
PDK1 activity in vivo
The catalytic activity of AKT is regulated by the
level of phosphorylation of another important amino acid
residue, Thr308, which is targeted by PDK1 (Alessi et
al., 1997). In order to obtain a complete overviewof how
emodin affects the PI3K pathway, protein extracts from
HeLa cells treated as indicated in Fig. 5 were subjected
to Western blotting analysis and the level of phospho-
rylation of AKT at Thr308 was determined by employ-
ing a specic anti-phospho-AKT antibody. As shown
in Fig. 5A, the treatment of cells with emodin markedly
reduced the phosphorylation of AKTat Thr308 (Fig. 5A,
lane 4), as we observed with cells treated with LY294002
(Fig. 5A, lane 5). The activation of AKT indicated by
enhanced phosphorylation at Thr308 (Fig. 5A, lane 3)
was induced by treatment with IGF-1. As expected,
DMSO-treated cells (Fig. 5A, lane 1) or those solely
incubated with emodin (Fig. 5A, lane 2) did not dis-
play Thr308 phosphorylation. Since the incubation of
cells with emodin lowers the phosphorylation of AKT at
Thr308 leaving the expression level of AKT intact, we
analysed whether emodin directly inhibits PDK1 kinase.
B.B. Olsen et al. / The International Journal of Biochemistry & Cell Biology 39 (2007) 227237 233
Fig. 5. Emodin affects the phosphorylation of AKTat Thr308 and PTENat Ser380/Thr382/383. (A) HeLa cells were treated as described in Fig. 3 and
as indicated in the gure. Total cell lysate was subjected to SDS-PAGE, transferred to PVDF membrane and probed with the indicated antibodies. (B)
The kinase activity of recombinant PDK1 was tested in the presence of inactive AKT1 as substrate. The bar graph shows the inuence of increasing
amounts of emodin (i.e. bar 2: 5 M; bar 3: 10 M; bar 4: 20 M; bar 5: 40 M; bar 6: 100 M) on PDK1 activity. Bar 1 shows the phosphorylation of
AKT1 by PDK1 in the absence of emodin, bars 7 and 8 are control experiments where PDK1 was incubated with the reaction mixture in the absence
of substrate (bar 7) and inactive recombinant AKT1 was incubated with the reaction mixture in the absence of enzyme (i.e. PDK1, bar 8), respectively.
The radioactivity incorporated into AKT1 substrate was quantied by scintillation counting of the excised radioactive bands. The values represent
the average S.D. of three independent experiments. (C) Total lysate from HeLa cells incubated with DMSO (lanes 1 and 2) or treated with 40 M
emodin for 12 h was subjected to immunoprecipitation with either control serum (lane 1) or polyclonal anti-PDK1 antibody (lanes 2 and 3). The
activity of endogenous PDK1 was tested with inactive AKT1 as a substrate. (D) Western blotting analysis was performed to analyse the total amount
and/or phosphorylation status of the indicated proteins. HeLa cells were treated as indicated in the gure. Lane 1 represents a control experiment
(DMSO) where the cell extract was from DMSO-treated cells. The values expressed in % below lane numbers refer to the densitometric analysis
of the protein bands revealed probing the Western blotting membrane with anti-phospho-PTEN (Ser380/Thr382/383) antibody. The quantication
was performed assigning value 100 to the band in lane 3. Results shown in the gure are representative of three independent experiments.
234 B.B. Olsen et al. / The International Journal of Biochemistry & Cell Biology 39 (2007) 227237
We performed a kinase assay where active recombi-
nant PDK1 was incubated with increasing amounts of
emodin, as indicated in the gure legend in the pres-
ence of recombinant inactive AKT1 substrate. Results
shown in Fig. 5Bindicate that emodin does not affect the
activity of recombinant PDK1. We also veried whether
emodin affect the activity of the aforementioned kinase
in vivo. Cells were treated as indicated in the gure leg-
end. Endogenous PDK1 was immunoprecipitated from
cell extract and subjected to kinase assay. As shown in
Fig. 5C, the incubation of cells with emodin did not affect
the activity of PDK1 in vivo. These results suggested
that the decreased phosphorylation of AKT at Thr308
observed upon incubation of HeLa cells with emodin,
might have been due to the emodin-dependent inhibi-
tion of enzymes upstream of PDK1, rather than by a
direct inhibition of the latter. After examining current
models that describe the mode of regulation of AKT
by receptor tyrosine kinases-activation, we focused on
two important upstream modulators of AKT, PI3K and
PTEN. A literature search revealed that emodin targets
PI3K in vivo by inhibiting human cancer cell migra-
tion via suppression of the PI3K-Cdc42/Rac1 signalling
pathway (Huang et al., 2005). Therefore, for determin-
ing whether emodin affects PTEN in vivo, we checked
the phosphorylation level of PTENin crude extracts from
HeLa cells (which express wild-type PTEN) treated with
emodin. As shown in Fig. 5D, the incubation with IGF-
1 and emodin (lane 4) decreased Ser380/Thr382/383
phosphorylationof PTENby50%, as determinedbyden-
sitometric analysis, in comparison with cells stimulated
only with IGF-1 (lane 3). Interestingly, Western blotting
analysis of protein extracts using anti PTEN antibody
revealed that the incubation with emodin is accompa-
nied by a concomitant slight reduction in the expression
level of the PTEN protein (Fig. 5D, lanes 2 and 4).
3.4. Emodin inuences the MAP kinase signalling
pathways
Because of the central role of the MAP kinase sig-
nalling pathways in the regulation not only of cell growth
and differentiation but also cell survival, we examined
the effect of cell treatment with emodin with respect
to the phosphorylation levels of selected members of
the mammalian MAP kinase sub-families (reviewed
in Rennefarht et al., 2005). We employed phospho-
specic antibodies against the extracellular-regulated
kinase (ERK), also known as p44/42 MAP kinase,
whose activation correlates with cancer progression and
increases the cell death threshold, p38 MAP kinase
whose activation is generally associated with enhanced
Fig. 6. Effect of emodin on p44/42MAPK, p38 and JNK phospho-
rylation levels. HeLa cells were treated with emodin as described in
Fig. 3. After cell treatment, phosphorylated p44/42MAPK, p38 and
JNK kinases were assayed by Western blotting analysis probing the
membrane with the indicated antibodies. -Actin was used as loading
control.
activation of the apoptotic program and the c-jun NH2-
terminal kinase (JNK) that has also been associated with
apoptosis and survival signalling. Cells were treated as
described in the legend to Fig. 3. As shown in Fig. 6, the
phosphorylation of p44/42 MAP kinase was inhibited
by cell treatment with emodin and it was independent
from the mitogenic stimulation induced by IGF-1 (lanes
2 and 4). With respect to p38 kinase, there was no signi-
cant change in the phosphorylation level according to the
indicated cell treatments while we observed increased
phosphorylation of JNK after incubation with emodin
(lanes 2and4). Total proteinlevel of p44/42MAPkinase,
p38 and JNK did not change following IGF-1 and/or
emodin treatments.
4. Discussion
To date, there are a number of reports showing
the effectiveness of emodin and structurally similar
compounds in inducing apoptosis in different cells such
as lung carcinoma, breast cancer, cervical cancer, and
human hepatoma cell lines. However, the signalling
pathways responsible for the apoptotic feature in cells
exposed to variable concentrations of emodin remain
largely undened. Srinivas et al. (2003) reported
that in human cervical cancer cells, emodin-induced
B.B. Olsen et al. / The International Journal of Biochemistry & Cell Biology 39 (2007) 227237 235
apoptosis is caspase-dependent and presumably occurs
through the mitochondrial pathway. Shieh et al. (2004)
demonstrated that the treatment of various human
hepatoma cell lines with emodin was accompanied by
the appearance of DNA fragmentation and increased
expression of apoptosis-related proteins such as p53,
p21, Fas and caspase 3. In addition, it has also been
shown that the enhanced sensitivity towards chemother-
apeutic drugs, such as paclitaxel which is employed in
the treatment of breast cancer by pre-incubation of cells
with emodin, is due to the effective, emodin-mediated
inhibition of endogenous tyrosine kinases (Zhang et al.,
1999).
In the present study, considering that emodin is an
effective inducer of apoptosis, we aimed to investigate
in detail its mechanism(s) of action with respect to
the PI3K/AKT pathway, whose deregulation has been
linked numerous times to malignant transformation. In
this respect, inhibition of this pathway is considered a
promising and effective approach for cancer treatment.
Our investigation demonstrated that the incubation of
cells with emodin negatively affects several components
of the PI3K/AKT pathway (Fig. 7), leading to the down-
regulation of AKT kinase activity because of dephos-
phorylation of two regulatory residues (i.e. Ser473 and
Thr308), rather than through a direct inhibition of AKT
Fig. 7. Proposed regulation of the PI3K/AKT signalling pathway in cells treated with emodin. AKT phosphorylation at regulatory amino acid sites
(i.e. Ser473 and Thr308) is negatively modulated by emodin. The emodin-mediated inhibition of mTOR leads to dephosphorylation of AKT at
Ser473 while the selective inhibition of PI3K by emodin negatively inuences the PDK1-mediated phosphorylation level of AKT at Thr308. The
tumour suppressor and lipid phosphatase PTEN counteracts PI3K activity. PI3K phosphorylates PtIns(4,5)P2 to generate PtIns(3,4,5)P3 (PIP3)
which represents a membrane-docking site for the PH-domain-containing proteins AKT and PDK1, while PTEN, in the active, unphosphorylated
form, targets inositol phospholipids for dephosphorylation. Emodin inhibits the phosphorylation of PTEN at the C-terminal tail that in turn regulates
PTEN stability and may play an important role in the regulation of its biological activity. Active, unphosphorylated PTEN contributes indirectly
to the down-regulation of AKT kinase activity. The model shows also a possible inuence of emodin on members of the MAP kinase signalling
pathways. The symbol P indicates phosphorylation sites, but is not indicative of the absolute number of phosphorylated residues. Broken lines
indicate poorly dened connections. IRS: insulin receptor substrate; R: receptor.
236 B.B. Olsen et al. / The International Journal of Biochemistry & Cell Biology 39 (2007) 227237
kinase. Moreover, considering that emodin is an inhibitor
of protein kinase CK2 and that CK2 phosphorylates and
up-regulates AKT (Di Maira et al., 2005), we show that
the inhibition of AKT in cells incubated with emodin is
not specically due to impaired CK2 activity. Incubation
of cells with two additional CK2 inhibitors i.e. apigenin
and DMAT (the latter is considered one of the most spe-
cic, ATP-competitive CK2 inhibitors characterized by
an IC
50
of 150 nM) did not induce down-regulation of
AKT activity to the same extent as that seen in cells
treated with emodin, despite the fact that apigenin and
DMAT were equally effective in the inhibition of CK2
activity.
As shown in Fig. 3, incubation of cells with emodin
leads to the dephosphorylation of AKT at Ser473, a
phosphorylation site located in the hydrophobic motif
of AKT which has been recently shown to be a tar-
get residue of the RictormTOR complex (Sarbassov
et al., 2005). As mentioned above, mammalian TOR is
a protein kinase that exists in two distinct intracellular
complexes, one composed of mTOR, GL and Raptor
and the other one containing mTOR, GL and Ric-
tor. The Raptor-containing complex is perhaps the best
characterized; it is sensitive to the drug rapamycin and
regulates mitogen activated signalling pathway through
the direct phosphorylation at Thr389 and activation of
p70 S6 kinase. The Rictor-containing complex does not
appear to be rapamycin-sensitive since it does not target
p70 S6 kinase and its mechanism of action remains to
be fully elucidated. The fact that cells incubated with
emodin leads to dephosphorylation of p70 S6 kinase, a
known target of the RaptormTOR complex, supports
the idea that emodin is a direct inhibitor of mTOR, as is
LY294002. Indeed, we show that the phosphorylation
of inactive recombinant AKT1, used in phosphoryla-
tion assays as an mTOR substrate target, is inhibited
when immunoprecipitated endogenous mTOR is incu-
bated with emodin. The prior phosphorylation of AKTat
Ser473 by RictormTORincreases the subsequent phos-
phorylation of Thr308 by PDK1, leading to a four- to
ve-fold increase in AKT kinase activity as compared
to AKT phosphorylated only by PDK1 (Sarbassov et al.,
2005). Our results indicate that the treatment of cells
with emodin leads to the dephosphorylation of AKT at
Thr308 and that emodin does not affect PDK1 kinase.
Thus, it might be that the decreased phosphorylation of
Thr308 seen in cells incubated with emodin is a conse-
quence of a lack of phosphorylation at Ser473.
Our data also support the notion that other upstream
modulators of AKT activity are affected by emodin.
Indeed, we observed that the treatment of cells with
emodin, modies the phosphorylation level of PTEN.
PTEN exists in a predominantly phosphorylated state
that restricts the activity of the phosphatase but pre-
serves its stability (Vazquez et al., 2000). According to
this model, dephosphorylation of the C-terminal domain
leads to a loss of stability and a gain of PTEN func-
tion. The treatment of cells with emodin results in
dephosphorylation of Ser380/Thr382/383 which might
indicate the up-regulation of PTEN phosphatase activ-
ity; a critical event that leads to destabilization and
down-regulation of the PI3K pathway. Protein kinase
CK2 has been shown to be the major cellular PTEN
kinase (Torres & Pulido, 2001). Residues 369386 of
the PTEN amino acid sequence include several con-
sensus phosphorylation sites for protein kinase CK2
(S/TXXD/E/S(P)/T(P)) including Ser380/Thr382/383.
Although mass spectrometry analysis identied Ser370,
Ser385 and Thr366 as in vivo phosphorylation sites
of PTEN (Miller et al., 2002), we cannot exclude that
the dephosphorylation of PTEN at Ser380/Thr382/383
observed in cells treated with emodin might be due to the
inhibition of CK2. In this respect, in vivo 32P-labelling
studies have revealed a signicant reduction in the phos-
phorylation of PTEN Ser380Ala/Thr382Ala/Thr383Ala
mutants (Torres & Pulido, 2001).
The analysis of selected components of the MAP
kinase pathways (i.e. p44/42 MAPK, p38 and JNK)
with respect to cell treatment with emodin, suggests
that the inactivation of ERK but not p38 might also
be important determinant, beside the PI3K signalling
pathway, of emodin-mediated cell death that remains to
be fully elucidated. Moreover, when cells were treated
with emodin alone or in combination with IGF-1, we
observed increased phosphorylation of JNK, which has
been reported to correlate with increased kinase activ-
ity and the promotion of apoptosis in a variety of cell
lines (reviewed in Davis, 2000). The increased phospho-
rylation of JNK observed after treatment with emodin,
is consistent with the observations of Aikin et al. (2004)
who suggested a direct link between JNK and compo-
nents of the PI3K/AKT pathway as treatment of cells
with a selective PI3K inhibitor (i.e. wortmannin) led to
increased JNKphosphorylation and enhanced cell death.
In summary, the induction of apoptosis by cell treat-
ment with chemotherapeutic agents is a fundamental
mechanism in the inhibition of tumour cell growth. In
this respect, emodin has been shown to be a natural com-
pound with potent anti-cancer activity affecting several
intracellular pathways. As mentioned, one of the most
studied signalling pathways linked to uncontrolled pro-
liferationandmalignant transformationis the PI3K/AKT
pathway. In this study, we have investigated in detail
the mode by which emodin affects the aforementioned
B.B. Olsen et al. / The International Journal of Biochemistry & Cell Biology 39 (2007) 227237 237
signalling cascade in vivo. We have shown that its effec-
tiveness in enhancing cell growth suppression might be
due to its broad specicity towards components of the
PI3K/AKT pathway. Our data support the notion that
the PI3K/AKT pathway represents an attractive target
for anti-cancer drug discovery and that the effective
emodin-mediated inhibition of the aforementioned sig-
nalling cascade, in combination with other chemothera-
peutic inhibitors, might prove to be an effective cancer
treatment.
Acknowledgments
This work was supported by the Danish Cancer Soci-
ety (grant no. DP03093 to BG) and the Danish Research
Council (grant no. 21-03-0508 to BG). We thank Dr.
Gary Schoenhals and Dr. O.-G. Issinger for critical read-
ing of the manuscript and Tina Holm for excellent tech-
nical assistance.
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