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The International Journal of Biochemistry & Cell Biology 39 (2007) 227–237

Emodin negatively affects the phosphoinositide 3-kinase/AKTsignalling pathway: A study on its mechanism of action

Birgitte B. Olsen, Marina Bjørling-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 2006Available online 30 August 2006

bstract

The development of selective cell-permeable inhibitors of protein kinases whose aberrant activation contributes to cell transfor-ation is a promising approach in cancer treatment. Emodin is a natural anthraquinone derivative that exhibits anti-proliferative

ffects in various cancer cell lines by efficient induction of apoptosis. The phosphoinositide 3-kinase (PI3K)/AKT pathway haseen shown to be central in the promotion of cell survival since the alteration of this signalling cascade is a frequent event in humanalignancies. Previous published results indicated that treatment of cells with inhibitors of protein kinase CK2, such as emodin,

nduces apoptosis and that the anti-apoptotic effect of CK2 is partially mediated by target phosphorylation and up-regulation ofKT by CK2. In the present study, a screening with selected CK2 inhibitors induced a variable response with respect to AKTown-regulation, emodin being the most effective, suggesting that other mechanisms other than the inhibition of CK2 were respon-ible for the emodin-mediated modulation of AKT. We found that emodin does not directly affect AKT kinase. Furthermore, wehow that the down-regulation of AKT is due to the emodin-mediated target inhibition of components of the PI3K pathway, which
irectly or indirectly affect AKT activity, i.e. the mammalian target of rapamycin and the phosphatase and tensin homolog deletedn chromosome 10, but not the phosphoinositide-dependent kinase 1. Taken together, our results highlight a new mechanism byhich emodin exerts anti-cancer activity and suggest the further investigation of plant polyphenols, such as emodin, as therapeutic
nd preventive agents for cancer therapy.2006 Elsevier Ltd. All rights reserved.

eywords: CK2; AKT; mTOR; PI3K pathway; Emodin

. Introduction

Emodin is a biologically active natural compoundxtracted from the rhizomes of Rheum palmatum that cane chemically classified as an anthraquinone derivative1,3,8-trihydroxy-6-methylanthraquinone). Several sci-
ntific studies have been performed that indicate the vastariety of effects mediated by this compound. Emodins known to have anti-microbial, immunosuppressive
∗ Corresponding author. Tel.: +45 6550 2388; fax: +45 6550 2467.E-mail address: [email protected] (B. Guerra).

357-2725/$ – see front matter © 2006 Elsevier Ltd. All rights reserved.doi:10.1016/j.biocel.2006.08.006

and anti-inflammatory activities (Chang et al., 1996;Huang et al., 1992; Wang & Chung, 1997), it exertsanti-proliferative effects in a vast array of cancer celllines, often enhancing the sensitivity of cancer cellsto chemotherapeutic drugs. The efficacy of emodin ininhibiting tumorigenesis is due, at least in part, to itsability 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 ofprotein kinases that are known to regulate a wide rangeof cellular processes, including apoptosis. Emodin is aninhibitor of protein kinase CK2 (Yim et al., 1999), a
mailto:[email protected]

dx.doi.org/10.1016/j.biocel.2006.08.006

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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 tworegulatory �-subunits. Abnormally high levels of CK2have been observed in various types of cancer and intransformed cells as compared to normal tissues (Ungeret al., 2004). Moreover, a direct link between tumori-genesis and CK2 has been established employing trans-genic mice, demonstrating that targeted overexpressionof CK2 leads to lymphocyte transformation and mam-mary tumours (Landesman-Bollag et al., 2001; Seldin& Leder, 1995). The depletion of CK2 subunits bythe application of antisense oligodeoxynucleotides andsiRNA techniques, respectively, in cells exposed to geno-toxic stress has provided additional evidence that CK2plays a prominent, positive role in cell survival (Seeber etal., 2005; Wang et al., 2005). These findings have furtheremphasized the validity of CK2 as potential therapeutictarget with respect to its anti-apoptotic role. Treatmentof cells with emodin causes a decrease in intracellularprotein-tyrosine phosphorylation because of target inhi-bition of p56lck protein tyrosine kinase (Jayasuriya et al.,1992). Recently, it has been shown that emodin inhibitsHER-2/neu (also known as c-erbB-2) tyrosine kinaseactivity and represses the transformation of HER-2/neu-overexpressing breast cancer cells in athymic nude micethrough repression of p185neu tyrosine kinase (Zhang etal., 1999). Su et al. (2005) reported that the treatmentof human lung adenocarcinoma A549 cells with emodinleads to apoptotic cell death associated with ERK pro-tein kinase inactivation, confirming earlier observationsobtained with cultured human breast cancer MDA-MB-231 cells and human skin squamous carcinoma HSC5cells.

Earlier reports have indicated that the treatment ofcells with emodin negatively affects the phosphoinosi-tide 3-kinase (PI3K)/AKT signalling cascade (Kim etal., 2004; Lai et al., 2003). The PI3K signal transduc-tion pathway has been investigated extensively for itsrole in oncogenic transformation and in the preven-tion of apoptosis (reviewed in Osaki et al., 2004). Theactivation of the PI3K pathway is relatively well under-stood and is known to be a multi-step process involvingthe PI3K-dependent phosphorylation of phospholipidslocalized at the plasma membrane, and the subsequentmembrane localization of phosphoinositide-dependentkinase 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 toAKT phosphorylation at Thr308 and Ser473. ActivatedAKT controls fundamental cellular processes such as
hemistry & Cell Biology 39 (2007) 227–237

cell survival by phosphorylating and inactivating severaldownstream pro-apoptotic target molecules. PI3K wasfirst implicated in the suppression of apoptosis in a studyby Yao and Cooper (1995) which demonstrated that theinhibition of PI3K activity impairs the ability of nervegrowth factor (NGF) to prevent apoptosis. The findingthat PTEN (phosphatase and tensin homolog deleted onchromosome 10), a lipid phosphatase considered to bea tumour suppressor gene product is able to negativelyaffect the PI3K pathway 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)P3and down-regulates the PI3K pathway, has been reportedin various primary human tumours and in human cancercell lines as well (reviewed in Datta et al., 1999). Thefact that AKT overexpression is found in many humancancers, that active AKT promotes resistance to chemo-and radiotherapy, and that AKT activity is sufficient toblock apoptosis induced by a number of death stimulihas resulted in intensive studies on the role of AKT as amediator of the PI3K survival signal. These observationssuggest that the inhibition of the PI3K/AKT pathwaymight be therapeutically important for cancer patients.

As mentioned above, the treatment of cells withemodin alone or in combination with other chemother-apeutic agents has been shown to effectively counter-act tumour progression, although the emodin-mediatedmolecular mechanism responsible for this effect remainsto be fully elucidated. Given the importance of the afore-mentioned pathway in the modulation of tumour progres-sion, the aim of the present study was to examine in detailhow emodin affects the PI3K/AKT signalling pathway,leading to a cell death that biochemically resemblesthe typical features of apoptosis. We propose a modelthat supports the therapeutic validity of emodin in thetreatment of human malignancies and other pathologi-cal conditions, since the emodin-mediated regulation ofcomponents of the PI3K pathway upstream of AKT leadsto 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) and1 mM l-glutamine. Cells were cultured at 37 ◦C undera 5% CO atmosphere. For the in vivo activation of
2AKT and mTOR kinases, cells were seeded and 24 hthereafter they were subjected to serum starvation for24 h, followed by treatment with 100 ng/ml IGF-1 (Cal-biochem) for 10 and 30 min, respectively. The transient

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verexpression of AKT was achieved, where indicated,y transfecting cells with FuGene 6 reagent (Roche) withtotal of 2 �g plasmid DNA in 60 mm Petri dishes fol-

owing the manufacturer’s recommendations. In the casef AKT activation, cells were transfected 24 h prior toerum starvation. After 24 h, cells were incubated with00 ng/ml IGF-1 for 10 min at 37 ◦C. The incubationf cells with apigenin, emodin and DMAT (purchasedrom Calbiochem) and LY294002 (obtained from Cellignalling Technology), respectively, was as indicated

n the figure legends.

.2. Antibodies

Proteins were detected by Western blotting usinghe following primary antibodies: monoclonal anti-KT, monoclonal anti-mTOR and monoclonal anti-DK1 (all from BD Biosciences), polyclonal anti-AKT,nd polyclonal anti-PDK1 (both from Upstate). Poly-lonal anti-phospho-AKT(Thr308), monoclonal anti-hospho-AKT(Ser473), monoclonal anti-phospho-p706 kinase (Thr389), polyclonal anti-phospho-PTENSer380/Thr382/383), polyclonal anti-PTEN, polyclonalnti-p44/42 MAP kinase, monoclonal anti-phospho-44/42 MAP kinase (Thr202/Tyr204) and polyclonalnti-phospho-p38 MAP kinase (Thr180/Tyr182) anti-odies were all from Cell Signaling Technology.olyclonal anti-p70 S6 kinase, polyclonal anti-mTOR,olyclonal anti-p38 (H-14) and monoclonal anti-JNKntibodies were purchased from Santa Cruz Biotech-ology. Polyclonal anti-phospho-JNK (Thr183/Tyr185)ntibody was from Biosource while monoclonalnti-�-actin antibody was obtained from Sigma.rotein-antibody complexes were visualized by ahemiluminescence Western blotting detection systemccording to the manufacturer’s instructions (CDP-Star,pplied Biosystems).

.3. Preparation of cell extracts andmmunoprecipitation

Prior to harvesting, cells were rinsed with ice-coldBS and lysed with lysis buffer (50 mM Tris/HCl pH 7.5,50 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mMTT, 1 mM Na3VO4, 30 mM �-glycerophosphate,0 mM NaF, 100 nM okadaic acid and a proteasenhibitor cocktail, Roche). Lysates were cleared byentrifugation at 4 ◦C for 30 min at 10,000 × g. The
rotein concentration of supernatants was determinedy the Bradford assay (BioRad). Whole cell extractsere subjected to SDS-PAGE, Western blotting anal-sis, or protein kinase assays. For immunoprecipitation
hemistry & Cell Biology 39 (2007) 227–237 229

experiments, protein A-agarose (Roche) was incubatedovernight at 4 ◦C with the antibodies indicated in thefigure legends. Thereafter, cell lysates were added andincubated for 3 h at 4 ◦C with gentle rocking. Immuno-complexes were extensively washed with NET-modifiedbuffer (50 mM Tris/HCl pH 8, 150 mM NaCl, 5 mMEDTA, 0.05% NP40, 0.2% casein, 0.02% NaN3) con-taining a protease inhibitor cocktail and subsequentlyeluted either by adding SDS-PAGE sample buffer orby washing twice with assay buffer prior to the pro-tein kinase assay. In the case of mTOR immunopre-cipitates, the procedure followed was essentially asdescribed above except that cells were lysed on ice withlysis buffer containing 0.3% CHAPS instead of TritonX-100 (CHAPS lysis buffer) and immunoprecipitationexperiments were performed with CHAPS lysis buffer.Immunocomplexes were analyzed by immunoblot-ting with the antibodies indicated in the figurelegends.

2.4. Protein kinase assays

To monitor the activity of AKT transiently expressedin HeLa cells, 1 mg cell lysate was subjected to immuno-precipitation as described above with rabbit polyclonalanti-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 MgCl2, 1 mM Na3VO4, 20 mM NaF, 1 mMDTT). Kinase assays were performed in kinase assaybuffer supplemented with 50 �M ATP, 10 �Ci of [�-32P]ATP (3000 Ci/mmol, Hartmann Analytic) and 5 �ghistone 2B (H2B, Roche) substrate in a total volumeof 60 �l. Reaction mixtures were incubated at 30 ◦Cfor 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 polyclonalanti-mTOR antibody, essentially as described above.Immunoprecipitates were washed twice in mTOR kinasebuffer (25 mM Hepes pH 7.5, 100 mM potassium acetate,1 mM MgCl2). The kinase reaction was performed at37 ◦C for 20 min in a final volume of 40 �l in themTOR kinase buffer containing 500 �M ATP, 10 �Ciof [�-32P]ATP and 1 �g recombinant inactive AKT1(Upstate). The activity of endogenous PDK1 was testedafter immunoprecipitation with polyclonal anti-PDK1antibody. Immunoprecipitates were washed twice in
PDK1 kinase buffer (50 mM Tris/HCl pH 7.5, 100 �MEGTA, 100 �M EDTA, 1 mM DTT, 100 nM okadaicacid, 10 mM MgAc). The kinase reaction was per-formed at 30 ◦C for 30 min in PDK1 kinase buffer (final

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volume 40 �l) containing 100 �M ATP, 10 �Ci of [�-32P]ATP and 1 �g recombinant inactive AKT1. PDK1kinase assay performed with active recombinant PDK1(Upstate) was performed using recombinant inactiveAKT1 as a substrate according to the manufacturer’srecommendations. All reactions were stopped by theaddition of SDS-PAGE sample buffer. In all experiments,radiolabeled proteins were visualized by autoradiogra-phy and the radioactivity incorporated was quantified byscintillation counting of the excised radioactive bands.AKT kinase assays in the presence of human recom-binant AKT1, AKT2 and AKT3 (all purchased fromKinaseDetect Aps), respectively, were performed essen-tially as in the case of immunoprecipitated AKT1 fromcell lysate using as a substrate 100 �M synthetic pep-tide (RPRAATF, Upstate) and by incubating the reactionmixtures at 30 ◦C for 15 min. Protein kinase CK2 activ-ity assay was performed in a reaction mixture containing50 mM Tris/HCl pH 7.5, 150 mM NaCl, 10 mM MgCl2,50 �M [�-32P]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 paperfilters (Whatmann). Filters were washed extensively with0.75% phosphoric acid and then transferred into vialscontaining a scintillation cocktail. Radioactivity wasquantified by scintillation counting (Canberra-Packard).

3. Results

3.1. Treatment of cells with emodin leads todown-regulation of AKT kinase

Previous results have indicated the ability ofanthraquinone-derivative compounds to efficientlyinduce cell death in a number of different cell linesincluding HeLa human cervical carcinoma cells. Oneof 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 (Kimet al., 2004; Lai et al., 2003). Recent data reported byDi Maira et al. (2005) led to the finding that down-regulation of protein kinase CK2 activity or proteinlevel in cells correlates with decreased AKT kinase
activity, revealing a novel mode of regulation of AKTby CK2-mediated constitutive phosphorylation. Becauseemodin 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 throughthe down-regulation of AKT. In order to verify thishypothesis, cells transiently overexpressing AKT1 wereincubated 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 wasmeasured in a kinase assay using histone 2B (H2B)as substrate according to the cell treatment indicatedin Fig. 1A. Surprisingly, the AKT1 kinase assay andthe subsequent densitometric analysis of the phospho-rylated histone 2B bands demonstrated that the cellu-lar treatment with emodin was the most effective ininhibiting AKT in vivo. The intensity of the 32P-H2Bprotein band was reduced up to 51% (Fig. 1A, lane 2)with respect to the control experiment (Fig. 1A, lane1). These results suggested that the emodin-mediateddown-regulation of AKT might have been dependent onother mechanisms not linked to CK2 inhibition. Sincea CK2 kinase test performed on total lysate from cellstreated with DMSO (Fig. 1B, Control-bar) or incubatedwith the indicated compounds revealed that emodin, api-genin and DMAT markedly inhibited CK2 activity to thesame extent (Fig. 1B), we speculated that the observeddecrease in AKT activity in cells treated with emodinmight have been due to the inhibition of either AKTand/or protein kinases that modulate AKT activity bytarget phosphorylation. To shed light on this point, weperformed an in vitro kinase assay where the activity of1 pmol of active recombinant purified AKT1, AKT2 andAKT3, respectively, were tested in the presence of AKTpeptide substrate and increasing amounts of emodin. Asshown in Fig. 2, 40 �M emodin led to a 27% decreasein AKT1 activity (black bar). This value was not con-sistent with the degree of inhibition observed in theexperiment where AKT was immunoprecipitated fromthe total lysates of cells treated as indicated in Fig. 1Aand subsequently subjected to kinase assays using H2Bas substrate. In the in vitro assay, a higher amount ofemodin (i.e. 80–90 �M) was required in order to inducea 50% inhibition of AKT1 activity (results not shown).In the case of AKT2 (grey bar), 40 �M emodin led to a9.6% decrease in AKT activity with respect to the controlexperiment performed in the presence of DMSO. AKT3activity was not inhibited by the presence of emodin(white bar). Instead, it is apparent that AKT3 kinase wasaffected by the presence of DMSO as compared with the
control assay (Control). The results reported in Fig. 2support the notion that emodin does not affect consis-tently the activity of AKT but rather inhibits upstreamproteins that target and up-regulate AKT.

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Fig. 1. Modulation of AKT kinase activity by protein kinase CK2inhibitors. (A) Cells transiently expressing AKT1 were treated for4 h with 40 �M emodin, 50 �M apigenin and 25 �M DMAT, respec-tively, as indicated in the figure. Activation of AKT was induced by theincubation of cells with 100 ng/ml IGF-1 for 10 min prior to harvest-ing. Total lysates were subjected to immunoprecipitation with rabbitpolyclonal anti-AKT antibody and subsequently, to kinase activity asdescribed in Section 2. The samples were analyzed by SDS-PAGEand transferred afterwards to PVDF membrane. Proteins were visu-alized by probing the membrane with the indicated antibody whilephosphorylated proteins were revealed by autoradiography. The valuesreported below each lane number represent the densitometric analy-sis (expressed in %) of the 32P-H2B bands. The quantification wasperformed with Gelworks 1D Intermediate Software assigning 100 tothe protein in lane 1. The experiment was done in triplicate. (B) CK2kinase assays were performed as described in Section 2 with a specificCbm

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Fig. 2. Emodin is not an inhibitor of AKT. AKT kinase activity wasperformed in the presence of 60 ng active recombinant AKT1, AKT2and AKT3 kinases with increasing amounts of emodin as indicated in

K2 peptide substrate and 20 �g total lysate from HeLa cells incu-ated as described in (A). The averages and standard deviation of theean of three independent experiments are shown.

.2. Emodin is an inhibitor of mTOR kinase

In order to gain insight into the mechanism by whichmodin modulates AKT kinase activity in vivo, we then

the figure. 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 Westernblotting using phospho-specific antibodies. The analysiswas carried out with cells left untreated or stimulatedwith the insulin-like growth factor (IGF-1) allowing theinvestigation of the effect of emodin on activated proteinsthat play a role in the regulation of AKT kinase activity.As shown in Fig. 3, the phosphorylation level of AKT atSer473, one of the two target amino acids whose phos-phorylation up-regulates AKT kinase activity, was sig-nificantly inhibited in cells treated with 30 �M emodinfor 12 h (Fig. 3, lane 4) as compared to the control experi-ment (Fig. 3, lane 3). As a positive control, cells were alsoincubated with 100 �M LY294002 for 1 h, a flavonoidderivative which has been reported to efficiently inhibitdifferent members of the PI3K family as well as mTORkinase (Fig. 3, lane 5). In all experiments, activationof AKT was induced by brief incubation of cells withIGF-1 prior to harvesting as described in Section 2. Arecent study by Sarbassov et al. (2005) demonstratedthat the mammalian target of rapamycin (mTOR) pro-tein kinase in complex with Rictor:G�L targets AKTfor phosphorylation at Ser473. In cells, mTOR can bepart of two distinct complexes defined by Rictor andRaptor proteins and characterized by distinct substratetargets. In order to test whether emodin targets directlymTOR or exclusively the complex Rictor–mTOR in
vivo, we included the analysis of the phosphorylationlevel of Raptor–mTOR-downstream protein target (i.e.p70 S6K), before and after cell treatment with the indi-cated compounds (i.e. emodin and LY294002). Cell

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Fig. 3. Treatment of cells with emodin affects the phosphorylation sta-tus of AKT at Ser473. HeLa cells were incubated with 30 �M emodinfor 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 beforeharvesting. Whole-cell lysates were analysed by Western blotting prob-ing the membranes with the indicated antibodies. Note that monoclonalanti-phospho-p70 S6K (Thr389) antibody detects endogenous levelsof p70 S6 kinase that has been phosphorylated at Thr389. Endogenousp85 S6 kinase is detected when phosphorylated at the analogous site,

Fig. 4. Emodin inhibits mTOR kinase activity. Immunoprecipitatesprepared 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 assayswith inactive recombinant AKT1 as substrate. As indicated, the phos-phorylation assay was performed in the presence of DMSO (lane 2)

i.e. Thr412. Lane 1 represents a control experiment (DMSO) wherethe endogenous level of the indicated proteins in whole lysate from theDMSO-treated cells was analysed.

incubation with emodin markedly reduced the level ofRaptor–mTOR-catalyzed phosphorylation of p70 S6Kat Thr389 (Fig. 3, lane 4). As expected, LY294002 treat-ment significantly attenuated the level of phosphoryla-tion of p70 S6K as a consequence of target inhibitionmTOR (Fig. 3, lane 5). Moreover, the sole treatmentof cells with emodin (i.e. without IGF-1-mediated AKTactivation) apparently did not affect the phosphorylationof AKT as compared to DMSO-treated cells (Fig. 3, lanes1 and 2). Next, we verified whether emodin is indeeda direct inhibitor of mTOR kinase (Fig. 4). HeLa cellswere starved for 24 h in the absence of serum and subse-quently stimulated with 100 ng/ml IGF-1 for 30 min. Cellextracts were subjected to immunoprecipitation assays.Immunoprecipitated endogenous mTOR was subjected
to kinase assay using recombinant inactive AKT1 proteinas a substrate in the absence (Fig. 4, lanes 1 and 2) and inthe presence of 60 �M emodin (Fig. 4, lane 3), respec-tively. The observed reduced level of phosphorylation of
and 60 �M emodin (lane 3), respectively. Samples were analysed byimmunoblotting for the indicated protein levels. The phosphorylationof AKT1 was revealed by autoradiograpy.

AKT protein indicates that the presence of emodin in thekinase assay significantly inhibits the catalytic activityof mTOR kinase.

3.3. Emodin modulates the phosphorylation ofPTEN protein phosphatase but does not influencePDK1 activity in vivo

The catalytic activity of AKT is regulated by thelevel of phosphorylation of another important amino acidresidue, Thr308, which is targeted by PDK1 (Alessi etal., 1997). In order to obtain a complete overview of howemodin affects the PI3K pathway, protein extracts fromHeLa cells treated as indicated in Fig. 5 were subjectedto Western blotting analysis and the level of phospho-rylation of AKT at Thr308 was determined by employ-ing a specific anti-phospho-AKT antibody. As shownin Fig. 5A, the treatment of cells with emodin markedlyreduced the phosphorylation of AKT at Thr308 (Fig. 5A,lane 4), as we observed with cells treated with LY294002(Fig. 5A, lane 5). The activation of AKT indicated byenhanced 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 solelyincubated with emodin (Fig. 5A, lane 2) did not dis-
play Thr308 phosphorylation. Since the incubation ofcells with emodin lowers the phosphorylation of AKT atThr308 leaving the expression level of AKT intact, weanalysed whether emodin directly inhibits PDK1 kinase.

B.B. Olsen et al. / The International Journal of Biochemistry & Cell Biology 39 (2007) 227–237 233

Fig. 5. Emodin affects the phosphorylation of AKT at Thr308 and PTEN at Ser380/Thr382/383. (A) HeLa cells were treated as described in Fig. 3 andas indicated in the figure. 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 influence of increasingamounts 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 ofAKT1 by PDK1 in the absence of emodin, bars 7 and 8 are control experiments where PDK1 was incubated with the reaction mixture in the absenceof 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 quantified by scintillation counting of the excised radioactive bands. The values representthe average ± S.D. of three independent experiments. (C) Total lysate from HeLa cells incubated with DMSO (lanes 1 and 2) or treated with 40 �Memodin for 12 h was subjected to immunoprecipitation with either control serum (lane 1) or polyclonal anti-PDK1 antibody (lanes 2 and 3). Theactivity of endogenous PDK1 was tested with inactive AKT1 as a substrate. (D) Western blotting analysis was performed to analyse the total amountand/or phosphorylation status of the indicated proteins. HeLa cells were treated as indicated in the figure. 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 analysisof the protein bands revealed probing the Western blotting membrane with anti-phospho-PTEN (Ser380/Thr382/383) antibody. The quantificationwas performed assigning value 100 to the band in lane 3. Results shown in the figure are representative of three independent experiments.

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Fig. 6. Effect of emodin on p44/42MAPK, p38 and JNK phospho-rylation levels. HeLa cells were treated with emodin as described in


We performed a kinase assay where active recombi-nant PDK1 was incubated with increasing amounts ofemodin, as indicated in the figure legend in the pres-ence of recombinant inactive AKT1 substrate. Resultsshown in Fig. 5B indicate that emodin does not affect theactivity of recombinant PDK1. We also verified whetheremodin affect the activity of the aforementioned kinasein vivo. Cells were treated as indicated in the figure leg-end. Endogenous PDK1 was immunoprecipitated fromcell extract and subjected to kinase assay. As shown inFig. 5C, the incubation of cells with emodin did not affectthe activity of PDK1 in vivo. These results suggestedthat the decreased phosphorylation of AKT at Thr308observed 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 adirect inhibition of the latter. After examining currentmodels that describe the mode of regulation of AKTby receptor tyrosine kinases-activation, we focused ontwo important upstream modulators of AKT, PI3K andPTEN. A literature search revealed that emodin targetsPI3K in vivo by inhibiting human cancer cell migra-tion via suppression of the PI3K-Cdc42/Rac1 signallingpathway (Huang et al., 2005). Therefore, for determin-ing whether emodin affects PTEN in vivo, we checkedthe phosphorylation level of PTEN in crude extracts fromHeLa cells (which express wild-type PTEN) treated withemodin. As shown in Fig. 5D, the incubation with IGF-1 and emodin (lane 4) decreased Ser380/Thr382/383phosphorylation of PTEN by 50%, as determined by den-sitometric analysis, in comparison with cells stimulatedonly with IGF-1 (lane 3). Interestingly, Western blottinganalysis of protein extracts using anti PTEN antibodyrevealed that the incubation with emodin is accompa-nied by a concomitant slight reduction in the expressionlevel of the PTEN protein (Fig. 5D, lanes 2 and 4).

3.4. Emodin influences the MAP kinase signallingpathways

Because of the central role of the MAP kinase sig-nalling pathways in the regulation not only of cell growthand differentiation but also cell survival, we examinedthe effect of cell treatment with emodin with respectto the phosphorylation levels of selected members ofthe mammalian MAP kinase sub-families (reviewedin Rennefarht et al., 2005). We employed phospho-specific antibodies against the extracellular-regulated
kinase (ERK), also known as p44/42 MAP kinase,whose activation correlates with cancer progression andincreases the cell death threshold, p38 MAP kinasewhose activation is generally associated with enhanced
Fig. 3. After cell treatment, phosphorylated p44/42MAPK, p38 andJNK kinases were assayed by Western blotting analysis probing themembrane with the indicated antibodies. �-Actin was used as loadingcontrol.

activation of the apoptotic program and the c-jun NH2-terminal kinase (JNK) that has also been associated withapoptosis and survival signalling. Cells were treated asdescribed in the legend to Fig. 3. As shown in Fig. 6, thephosphorylation of p44/42 MAP kinase was inhibitedby cell treatment with emodin and it was independentfrom the mitogenic stimulation induced by IGF-1 (lanes2 and 4). With respect to p38 kinase, there was no signifi-cant change in the phosphorylation level according to theindicated cell treatments while we observed increasedphosphorylation of JNK after incubation with emodin(lanes 2 and 4). Total protein level of p44/42 MAP kinase,p38 and JNK did not change following IGF-1 and/oremodin treatments.

4. Discussion

To date, there are a number of reports showingthe effectiveness of emodin and structurally similarcompounds in inducing apoptosis in different cells suchas lung carcinoma, breast cancer, cervical cancer, andhuman hepatoma cell lines. However, the signalling
pathways responsible for the apoptotic feature in cellsexposed to variable concentrations of emodin remainlargely undefined. Srinivas et al. (2003) reportedthat in human cervical cancer cells, emodin-induced

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poptosis is caspase-dependent and presumably occurshrough the mitochondrial pathway. Shieh et al. (2004)emonstrated that the treatment of various humanepatoma cell lines with emodin was accompanied byhe appearance of DNA fragmentation and increasedxpression of apoptosis-related proteins such as p53,21, Fas and caspase 3. In addition, it has also beenhown that the enhanced sensitivity towards chemother-peutic drugs, such as paclitaxel which is employed in
he treatment of breast cancer by pre-incubation of cellsith emodin, is due to the effective, emodin-mediated
nhibition of endogenous tyrosine kinases (Zhang et al.,999).

ig. 7. Proposed regulation of the PI3K/AKT signalling pathway in cells treai.e. Ser473 and Thr308) is negatively modulated by emodin. The emodin-mer473 while the selective inhibition of PI3K by emodin negatively influence

umour suppressor and lipid phosphatase PTEN counteracts PI3K activity. Phich represents a membrane-docking site for the PH-domain-containing pro

orm, targets inositol phospholipids for dephosphorylation. Emodin inhibits thTEN stability and may play an important role in the regulation of its biolo

o the down-regulation of AKT kinase activity. The model shows also a possathways. The symbol “P” indicates phosphorylation sites, but is not indicatndicate poorly defined connections. IRS: insulin receptor substrate; R: recep


In the present study, considering that emodin is aneffective inducer of apoptosis, we aimed to investigatein detail its mechanism(s) of action with respect tothe PI3K/AKT pathway, whose deregulation has beenlinked numerous times to malignant transformation. Inthis respect, inhibition of this pathway is considered apromising and effective approach for cancer treatment.Our investigation demonstrated that the incubation ofcells 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 andThr308), rather than through a direct inhibition of AKT
ted with emodin. AKT phosphorylation at regulatory amino acid sitesediated inhibition of mTOR leads to dephosphorylation of AKT ats the PDK1-mediated phosphorylation level of AKT at Thr308. TheI3K phosphorylates PtIns(4,5)P2 to generate PtIns(3,4,5)P3 (PIP3)teins AKT and PDK1, while PTEN, in the active, unphosphorylated

e phosphorylation of PTEN at the C-terminal tail that in turn regulatesgical activity. Active, unphosphorylated PTEN contributes indirectlyible influence of emodin on members of the MAP kinase signallingive of the absolute number of phosphorylated residues. Broken linestor.

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kinase. Moreover, considering that emodin is an inhibitorof protein kinase CK2 and that CK2 phosphorylates andup-regulates AKT (Di Maira et al., 2005), we show thatthe inhibition of AKT in cells incubated with emodin isnot specifically due to impaired CK2 activity. Incubationof cells with two additional CK2 inhibitors i.e. apigeninand DMAT (the latter is considered one of the most spe-cific, ATP-competitive CK2 inhibitors characterized byan IC50 of 150 nM) did not induce down-regulation ofAKT activity to the same extent as that seen in cellstreated with emodin, despite the fact that apigenin andDMAT were equally effective in the inhibition of CK2activity.

As shown in Fig. 3, incubation of cells with emodinleads to the dephosphorylation of AKT at Ser473, aphosphorylation site located in the hydrophobic motifof AKT which has been recently shown to be a tar-get residue of the Rictor–mTOR complex (Sarbassovet al., 2005). As mentioned above, mammalian TOR isa protein kinase that exists in two distinct intracellularcomplexes, one composed of mTOR, G�L and Raptorand the other one containing mTOR, G�L and Ric-tor. The Raptor-containing complex is perhaps the bestcharacterized; it is sensitive to the drug rapamycin andregulates mitogen activated signalling pathway throughthe direct phosphorylation at Thr389 and activation ofp70 S6 kinase. The Rictor-containing complex does notappear to be rapamycin-sensitive since it does not targetp70 S6 kinase and its mechanism of action remains tobe fully elucidated. The fact that cells incubated withemodin leads to dephosphorylation of p70 S6 kinase, aknown target of the Raptor–mTOR complex, supportsthe idea that emodin is a direct inhibitor of mTOR, as isLY294002. Indeed, we show that the phosphorylationof inactive recombinant AKT1, used in phosphoryla-tion assays as an mTOR substrate target, is inhibitedwhen immunoprecipitated endogenous mTOR is incu-bated with emodin. The prior phosphorylation of AKT atSer473 by Rictor–mTOR increases the subsequent phos-phorylation of Thr308 by PDK1, leading to a four- tofive-fold increase in AKT kinase activity as comparedto AKT phosphorylated only by PDK1 (Sarbassov et al.,2005). Our results indicate that the treatment of cellswith emodin leads to the dephosphorylation of AKT atThr308 and that emodin does not affect PDK1 kinase.Thus, it might be that the decreased phosphorylation ofThr308 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 upstreammodulators of AKT activity are affected by emodin.Indeed, we observed that the treatment of cells withemodin, modifies the phosphorylation level of PTEN.


PTEN exists in a predominantly phosphorylated statethat restricts the activity of the phosphatase but pre-serves its stability (Vazquez et al., 2000). According tothis model, dephosphorylation of the C-terminal domainleads to a loss of stability and a gain of PTEN func-tion. The treatment of cells with emodin results indephosphorylation of Ser380/Thr382/383 which mightindicate the up-regulation of PTEN phosphatase activ-ity; a critical event that leads to destabilization anddown-regulation of the PI3K pathway. Protein kinaseCK2 has been shown to be the major cellular PTENkinase (Torres & Pulido, 2001). Residues 369–386 ofthe 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 identified Ser370,Ser385 and Thr366 as in vivo phosphorylation sitesof PTEN (Miller et al., 2002), we cannot exclude thatthe dephosphorylation of PTEN at Ser380/Thr382/383observed in cells treated with emodin might be due to theinhibition of CK2. In this respect, in vivo 32P-labellingstudies have revealed a significant reduction in the phos-phorylation of PTEN Ser380Ala/Thr382Ala/Thr383Alamutants (Torres & Pulido, 2001).

The analysis of selected components of the MAPkinase pathways (i.e. p44/42 MAPK, p38 and JNK)with respect to cell treatment with emodin, suggeststhat the inactivation of ERK but not p38 might alsobe important determinant, beside the PI3K signallingpathway, of emodin-mediated cell death that remains tobe fully elucidated. Moreover, when cells were treatedwith emodin alone or in combination with IGF-1, weobserved increased phosphorylation of JNK, which hasbeen reported to correlate with increased kinase activ-ity and the promotion of apoptosis in a variety of celllines (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 cellswith a selective PI3K inhibitor (i.e. wortmannin) led toincreased JNK phosphorylation and enhanced cell death.

In summary, the induction of apoptosis by cell treat-ment with chemotherapeutic agents is a fundamentalmechanism in the inhibition of tumour cell growth. Inthis respect, emodin has been shown to be a natural com-pound with potent anti-cancer activity affecting severalintracellular pathways. As mentioned, one of the most
studied signalling pathways linked to uncontrolled pro-liferation and malignant transformation is the PI3K/AKTpathway. In this study, we have investigated in detailthe mode by which emodin affects the aforementioned

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ignalling cascade in vivo. We have shown that its effec-iveness in enhancing cell growth suppression might beue to its broad specificity towards components of theI3K/AKT pathway. Our data support the notion that

he PI3K/AKT pathway represents an attractive targetor anti-cancer drug discovery and that the effectivemodin-mediated inhibition of the aforementioned sig-alling cascade, in combination with other chemothera-eutic inhibitors, might prove to be an effective cancerreatment.

cknowledgments

This work was supported by the Danish Cancer Soci-ty (grant no. DP03093 to BG) and the Danish Researchouncil (grant no. 21-03-0508 to BG). We thank Dr.ary Schoenhals and Dr. O.-G. Issinger for critical read-

ng of the manuscript and Tina Holm for excellent tech-ical assistance.

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