Chemico-Biological Interactions 214 (2014) 69–76

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Chemico-Biological Interactions

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The fungal metabolite galiellalactone interferes with the nuclear importof NF-jB and inhibits HIV-1 replication

http://dx.doi.org/10.1016/j.cbi.2014.02.0120009-2797/� 2014 Elsevier Ireland Ltd. All rights reserved.

⇑ Corresponding author. Address: Instituto Maimónides de InvestigaciónBiomédica de Córdoba (IMIBIC), Departamento de Biología Celular, Fisiología eInmunología, Universidad de Córdoba, Avenida Menéndez Pidal s/n, 14004Córdoba, Spain. Tel.: +34 957 218267; fax: +34 957 218266.

E-mail address: fi1muble@uco.es (E. Muñoz).

Moisés Pérez a, Rafael Soler-Torronteras a, Juan A. Collado a, Carmen G. Limones a, Rebecka Hellsten b,Martin Johansson c, Olov Sterner c, Anders Bjartell b, Marco A. Calzado a, Eduardo Muñoz a,⇑a Maimónides Institute for Research in Biomedicine of Córdoba, Reina Sofía University Hospital, University of Córdoba, Spainb Division of Urological Cancers, Department of Clinical Sciences, Lund University, Swedenc Division of Organic Chemistry, Lund University, Sweden

a r t i c l e i n f o

Article history:Received 23 October 2013Received in revised form 27 January 2014Accepted 26 February 2014Available online 11 March 2014

Keywords:GaliellalactoneHIV-1LTRNF-jBImportins

a b s t r a c t

Galiellalactone (GL) is a metabolite produced by the fungus Galiella rufa that presents antitumor andimmunomodulatory activities. GL interferes with the binding to DNA of signal transducer and activatorof transcription (STAT)-3 and also inhibits other signal pathways such as NF-jB, but the mechanism ofaction in this pathway remains unknown. In this study we report that GL inhibits vesicular stomatitisvirus-recombinant HIV-1 infection and the NF-jB-dependent transcriptional activity of the HIV-LTR pro-moter. We found that GL prevents the binding of NF-jB to DNA but neither affects the phosphorylationand degradation of NF-jB inhibitory protein, IjBa, nor the phosphorylation and acetylation of the NF-jBp65 subunit. However, GL prevents the association of p65 with the importin a3 impairing the nucleartranslocation of this transcription factor. Using a biotinylated probe we found that GL binds to p65 butnot to importin a3. Therefore, GL is a dual NF-jB/STAT3 inhibitor that could serve as a lead compoundfor the development of novel drugs against HIV-1, cancer and inflammatory diseases.

� 2014 Elsevier Ireland Ltd. All rights reserved.

1. Introduction

Galiellalactone (GL) is a hexaketide metabolite from the asco-mycete Galiella rufa that was isolated during screening of mycelialcultures in a search for inhibitors of the IL-6-mediated signallingpathway. GL did not prevent Stat3 phosphorylation in IL-6-stimu-lated HepG2 but blocked the binding of Stat3 to DNA transcriptionelements [34]. More recently it has been shown that GL inducedapoptosis of prostate cancer cells expressing constitutively acti-vated STAT3 and inhibited prostate cancer tumour growth in vivo[18]. In addition, GL induced apoptosis of prostate cancer stemcell-like cells and reduced the expression of the putative cancerstem cell marker aldehyde dehydrogenase in vivo [19]. GL has alsoimmunomodulatory activities in a murine model of allergic asthma[14] and inhibits both TGF-b signalling and vasculogenic mimicry[27].

Despite the enormous progress made in the treatment of the dis-ease, AIDS remains one of the most important causes of mortality

produced by infectious agents. The detailed characterization ofthe HIV-1 genome and precise knowledge about the role of viralproteins and cellular factors involved in viral replication have beencritical to understand the viral cycle and to develop antiviral drugs.The HIV-1-LTR promoter contains binding sites for different cellulartranscription factors and a stem-loop RNA structure called transac-tivating response element (TAR) that binds to the HIV-1 Tat protein[26]. HIV-1 Tat/TAR interaction facilitates the recruitment of thepositive transcriptional elongation factor that phosphorylates theC-terminal domain of the RNA polymerase II [33] that is requiredfor activation of transcription elongation from the HIV-1-LTR pro-moter [40]. In addition to the TAR element, the HIV-1 LTR promotercontains three additional functional regions including the coreenhancer that has two jB elements that are critical in the regula-tion of HIV-1 transcription [3,20].

NF-jB comprises a family of transcription factors that play akey role in the control of a variety of cellular processes, such as im-mune response, cellular growth, apoptosis and HIV-1 gene expres-sion [10,16]. NF-jB is made up of homo- and heterodimers of p50,p65 (relA), p52, relB and c-rel subunits that interact with a familyof inhibitory IjB proteins, of which IjBa is the best characterized.IjB proteins sequester NF-jB in the cytoplasm by masking itsnuclear localization sequence (NLS). Stimulation of cells with a

70 M. Pérez et al. / Chemico-Biological Interactions 214 (2014) 69–76

variety of physiological or pathogenic stimuli activates IjB kinasesleading to phosphorylation and subsequent ubiquitination and thedegradation of IjBa proteins by the proteasome [12,21]. As aconsequence, the NLSs of NF-jB proteins are unmasked and trans-ported by importins to the nucleus where it binds to specific cis-acting elements present in the promoter of a large number ofinducible genes. In addition to the control of NF-jB activity exertedat the nuclear translocation level, there is increasing evidence foranother complex level of regulation that is mediated by posttrans-lational modifications, including phosphorylation, acetylation andsulfhydration of the NF-jB p65 subunit [15,29].

Proteins that contain classical NLSs are recognized by a hetero-dimeric receptor composed of importin a, an adaptor protein be-tween the NLS (importin-cargo) and the importin b protein thatis responsible for the docking of the importin-cargo complex tothe cytoplasmic side of the nuclear pore complexes followed bytranslocation of the complex through the nuclear pore [36]. Sevenimportin a family members have been identified in humans (a1,a2, a3, a4, a5, a6 and a7). The role of importins in HIV-1 viral cy-cle is being understood [39] and it has been shown that importina3 binds to HIV-1 integrase (IN) and favour the shuttle of the viralpre-integration complex to the nucleus [1,22]. In addition, Impor-tins a3 and a4 are responsible for the nuclear translocation ofNF-jB [8,9]. Our results show that GL is a potent inhibitor ofHIV-1 replication and we present evidence that GL inhibits thetranscriptional activity of the HIV-1-LTR promoter through a sig-naling pathway involving the nuclear import of NF-jB.

2. Materials and methods

2.1. Cell lines and reagents

HeLa, Hela-Tat-Luc, HEK 293T, U2OS and A549 cells were main-tained in DMEM supplemented with 10% FCS, 2 mM L-glutamineand 1% (v/v) penicillin/streptomycin (complete medium) at 37 �Cin a humidified atmosphere containing 5% CO2. DU145, Jurkat,and 5.1 cells were maintained in complete RPMPI medium. The5.1 clone line is a Jurkat derived clone stably transfected with aplasmid containing the luciferase gene driven by the HIV-LTR pro-moter. The HeLa-Tat-Luc is a stably transfected cell line with theplasmids pLTR-Luc and pcDNA3-Tat [25]. Galiellalactone was pur-chased from Tebu-Bio (Barcelona, Spain). TNFa was obtained fromImmunoTools GmbH (Friesoythe, Germany). [c32P]-ATP (3000 Ci/mmol) was from MP Biomedicals (Irvine, CA, USA). All other re-agents were from Sigma Co (St Louis, MO, USA). The synthesis ofbiotinylated galiellalactone will be described elsewhere.

2.2. Plasmids and transient tranfections

The KBF-Luc contains three copies of the MHC enhancer jB siteupstream of the conalbumin promoter, followed by the luciferasegene. The vector pNL4-3.Luc.R-E- (AIDS Research and ReferenceReagent Program, National Institute of Allergy and Infectious Dis-ease, National Institutes of Health) was from N. Landau and con-tains the firefly luciferase gene inserted into the pNL4-3 nef gene[7]. HA-p65, Flag-IKKa, Flag-IKKb, Flag-IKKe, GFP-CBP and Flag-p300 constructions were a gift from Dr. Lienhard Schmitz (Giessen,Germany). Flag-STAT3 was a gift from Dr. David A. Frank (HarvardMedical School, Boston) and Flag-Importin a1, a3, a4, a5, and a7plasmids were from Dr. Gülsah Gabriel (Hamburg, Germany). Tran-sient transfections were performed with Rotifect (Carl Roth GmbH,Karlsruhe, Germany) and harvested 48 h after transfection. DNAamounts in each transfection were kept constant upon additionof empty expression vector.

2.3. VSV-pseudotyped HIV-1 infection assays and luciferase reporterassays

HeLa and A549 cells were infected with the VSV-pseudotypedrecombinant virus as previously described [28]. The results arerepresented as the % of activation (considering the infected andstimulated cells in the absence of GL as the 100% activation). Forluciferase assays 5.1 or transiently transfected cells were stimu-lated as indicated. Cells were collected and washed twice in PBS,lysed and luciferase assay was performed using Luciferase AssayReagent (Promega Corp., Madison, WI, USA) according to the man-ufacturer’s instructions and measured in a luminometer AutolumatLB9510 (Berthold Technologies GmbH, Bad Wildbad, Germany).Luciferase activity in different samples was normalized with pro-tein concentration. Results are represented as RLU or fold induc-tion over untreated control.

2.4. Isolation of nuclear extracts and mobility shift assays

Jurkat cells (106/mL) were pre-treated with GL and 30 min latercells were stimulated with PMA (50 ng/ml) or TNFa (30 ng/ml) asindicated. Cells were collected and washed twice with PBS. Pro-teins from nuclear extracts were isolated by standard proceduresand the concentration was determined by the Bradford method.The consensus NF-jB oligonucleotide probe (Promega Corp) wasend-labelled with [c-32P] ATP. The binding reaction mixture con-tained 3 lg of nuclear extract, 0.5 lg poly (dI-dC), 20 mM HepespH 7, 70 mM NaCl, 2 mM DTT, 0,01% NP-40, 100 lg/mL BSA, 4% Fi-coll and 100,000 cpm of end labelled DNA fragments in a total vol-ume of 20 ll. After 30 min incubation at 4 �C, the mixture was runthrough a native polyacrylamide gel containing 89 mM Tris–bo-rate, 89 mM boric acid and 1 mM EDTA. Gels were pre-electropho-resed for 30 min at 225 V and then for 2 h after loading thesamples. These gels were dried and exposed to X-ray film at�80 �C.

2.5. NF-jB nuclear translocation assay and confocal microscopy

HeLa cells seeded on 96 well black imaging plates (BD Falcon™Microplates) were incubated with different concentrations of GL for30 min before final stimulation with 10 ng/ml TNFa for 30 min.Cells were fixed with 3,7% of pre-warmed paraphormaldehyde/PBS for 10 min and permeabilized with 0,1% Triton X-100/PBS for15 min. Cells were then blocked with 3% BSA/PBS and incubatedovernight with 1:50 (4 lg/ml) dilution of anti-p65 primary anti-body. Then, the cells were washed with PBS and incubated 1 h withAlexa Fluor 647 goat anti-mouse IgG1 antibody diluted 1:1000 (LifeTechnologies, Carlsbad, CA, USA) and 1 lM of Hoechst 33342.NF-jB p65 localization was visualized on a BD Pathway™ 855 Bio-imager system in a non-confocal mode using BP360/10 nm excita-tion and 435 (LP) emission filters for Hoechst 33342. The filtersused for AlexaFluor647 were: BP635/20 nm for excitation, and84000bs and 84101 m (Chroma 84000 Set) for emission. For theseexperiments, a 20X U-Apo 340 Objective (Olympus, NA 0.75) wasused. Images were binned 2 � 2 and montaged at 2 � 2. Sampleswere then quantified using BD Attovision™ v1.7 software, and ana-lyzed using BDTM Image Data Explorer. For confocal microscopyexperiments, HeLa cells were seeded on coverslips and transientlytransfected as previously described with FLAG-tagged Importina3 and GFP-tagged p65. Forty-eight hours later cells were fixedwith 3,7% of pre-warmed paraformaldehyde/PBS for 10 min, per-meabilized with 0,1% Triton X-100/PBS for 15 min, blocked with3% BSA/PBS and incubated overnight with anti-FLAG (M2, SIGMA)antibody diluted 1:1000. Cells were then washed with PBS andincubated for 45 min with secondary antibody Alexa Fluor 647 goatanti-mouse IgG1 antibody diluted 1:1000. The nuclei were stained

M. Pérez et al. / Chemico-Biological Interactions 214 (2014) 69–76 71

with DAPI before mounting cells on glass slides. A confocal laser-scanning microscope LSM 5 EXCITER (Carl Zeiss MicroImagingGmbH, Jena, Germany) with 40X/1.30 oil objective (EC Plan-Neofl-uar) and ZEN 2008 software (Carl Zeiss MicroImaging GmbH) wasused to capture the fluorescence images. Images were analysedusing the ImageJ v 1.45 software (http://rsbweb.nih.gov/ij/). TheCostes’ approach was employed for co-localization analysis be-tween green (GFP) and red (Alexa 647) images to determine thePearson’s correlation coefficient (R).

2.6. Western blotting

Cells were collected, washed in PBS and centrifuged. The cellpellet was lysed in NP-40 buffer [20 mM Tris–HCl pH 7.5,150 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 10 mM NaF,0.5 mM sodium orthovanadate, leupeptine (10 lg/ml), pepstatin(10 lg/ml), aprotinin (10 lg/ml), 1% (v/v) NP-40 and 10% (v/v)glycerol]. After centrifugation the supernatants were mixed withSDS sample buffer. Proteins were resolved on SDS–PAGE gels andblotted to polyvinylidene difluoride membranes. After blockingwith non-fat milk or BSA in TBST buffer, primary antibodies wereadded. The washed membranes were incubated with appropriatesecondary antibodies coupled to horseradish peroxidase that weredetected by ECL system. The antibodies anti-IjBa (C-21, sc-371)and anti-NF-jB-p65 (F-6, sc-8008) were purchased from SantaCruz Biotechnology (San Diego, CA, USA). The antibodies againstanti-phospho-p65 (Ser536 and Ser468) and anti-phospho-IjBa(Ser32/36) were from Cell Signalling (Danvers, MA, USA). ThemAb anti-tubulin was purchased from Sigma.

2.7. Immunoprecipitation

Transfected cells were collected and washed in PBS. The cellpellet was lysed in IP buffer [50 mM Hepes pH 7.5, 50 mM NaCl,1% (v/v) Triton X-100, 2 mM EDTA, 10 mM sodium fluoride,0.5 mM sodium orthovanadate, leupeptine (10 lg/ml), aprotinin(10 lg/ml), pepstatin (10 lg/ml), leupeptin (10 lg/ml), and 1 mMPMSF]. After removing cell debris, the immunoprecipitation (6 hat 4 �C) was carried out using 1 lg of the indicated antibodies.After that, antibodies were isolated with 25 ll of protein A/G Se-pharose for 6 h at 4 �C. The immunoprecipitated proteins of inter-est were washed five times in IP buffer and eluted in 1.5� SDSsample buffer, followed by Western blotting. The anti-Acety-lated-Lysine mAb (9441) was from Cell Signalling. The anti-HAand anti-Flag mAb was from Roche (Roche Molecular Biochemicals,Indianapolis, IN, USA). The anti-Importin a3 mAb was from Abcam(Abcam, Cambridge, UK). For GL protein interaction DU145 cellswere treated with biotinylated galiellalactone for 2 h. Cells werelysed in mild lysis buffer containing 50 mM Tris–HCl, 150 mMNaCl, 1 mM EDTA, 0.1% NP-40 and complete mini protease inhibi-tor cocktail tablet (Roche Applied Sciences, Penzberg, Germany).Cell lysates were incubated with streptavidin mag Sepharose beads(GE Healthcare Life Sciences, Buckinghamshire, UK) and analyzedby Western blotting using goat polyclonal anti-KPNA3 antibody(Abcam) or anti-NF-jB p65 rabbit polyclonal antibody (Santa Cruz,CA). Anti-goat (Sigma) or anti-rabbit (GE Healthcare Life Sciences)antibodies conjugated with horseradish peroxidase were used assecondary antibodies.

2.8. Statistical analysis

Data are expressed as mean ± SD. Differences were analyzed byStudent t test. P < 0.05 was considered significant. ⁄ = P < 0,05;⁄⁄ = P < 0,01; ⁄⁄⁄ = P < 0,001. Images were analyzed and quantifiedusing the ImageJ v1.45 software. Statistical analyses were

performed using GraphPad Prism version 6.00 (GraphPad, San Die-go, CA, USA).

3. Results

3.1. Effect of Galiellalactone on HIV-1 replication

To study the inhibitory effects of GL on HIV-1-LTR transcrip-tional activity we used two stably HIV-LTR transfected cell lines,namely 5.1, which is responsive to TNFa through the NF-jB path-way, and HeLa-Tat-Luc cells where the HIV-1 LTR is directly acti-vated by the HIV-1 Tat protein. We found that GL clearlyinhibited the LTR transcriptional activity induced by TNFa(Fig. 1A) but did not affect HIV-Tat-induced LTR transactivation(Fig. 1B). As expected, the high levels of luciferase activity inducedby Tat were inhibited by the pTEFb inhibitor 5,6-dichloro-1-b-D-ribofuranosyl benzimidazole (DRB) (Fig. 1B). These results ruleout the possibility that the inhibitory effects of GL were due tointerference with the transcriptional machinery or with thein vitro activity of the luciferase enzyme. Next, we investigatedthe effect of GL on p65-induced HIV-LTR activation and we foundthat GL also inhibited the transcriptional activity of p65 in a con-centration dependent manner (Fig. 1C). To further confirm theanti-HIV activity of GL, we infected HeLa and A549 cells with thepNL4-3 HIV-1 clone pseudotyped with the VSV envelope, whichbypasses the natural mode of HIV-1 entry into these cells that sup-port robust HIV-1 replication [5]. Upon integration, this recombi-nant virus expresses the luciferase gene and consequentlyluciferase activity in infected cells correlates with the rate of viralreplication. Thus, high luciferase activity levels were detected 12 hafter cellular infection with the VSV-pseudotyped HIV-1 clone, andpretreatment of the cells 60 min prior infection with increasingdoses of GL inhibited significantly the luciferase activity (Fig. 1D).

3.2. Effects of GL in the NF-jB signalling pathway

We were interested in studying if GL interferes with the bindingof NF-jB to DNA and, therefore, we stimulated Jurkat cells withTNFa in the presence of GL and the binding of NF-jB to DNAwas analyzed by EMSA. The DNA-binding specificity was studiedby supershift experiments with specific anti-p50 and anti-p65(RelA) antibodies and by cold competition experiments with unla-belled competitors, and the heterodimer p50/p65 was identified asthe main complex (data not shown and [28]). We found that GLinhibited the binding of NF-jB in stimulated cells (Fig. 2A). It hasbeen suggested that GL interacts directly with STAT3 thus prevent-ing the binding to DNA and to analyze if this mechanism also ac-counts for the binding of NF-jB to DNA, we included GL in theEMSA binding buffer that contains nuclear extracts isolated fromPMA stimulated cells. Under these conditions we found that GLwas unable to prevent the binding of NF-jB to DNA (Fig. 2B).

To investigate the level at which GL exerted its inhibitory effecton NF-jB activation, we co-transfected HeLa cells with the up-stream IjBa kinases IKKa, IKKb and IKKe and the reporter plas-mids KBF-Luc. Twenty-four hours after transfection the cellswere stimulated with GL for six hours and the luciferase activitywas measured in the cell lysates. In Fig. 3A we show that GL inhib-ited the activity of NF-jB induced by these IKKs. Since the effects ofGL were mediated downstream IKKs we studied the phosphoryla-tion status of IjBa and p65, which are two key targets for IKKs inthe NF-jB pathway. GL did not inhibit either IjBa (Ser32/36) orp65 (ser536 and 468) in TNFa-stimulated cells. In addition, GLdid not prevent IjBa degradation but it clearly prevented therecovery of steady state IjBa protein that is clearly detectable after60 min of TNFa stimulation in the absence of GL (Fig. 3B). Next, we

A B

C D

Fig. 1. GL inhibits HIV-1-LTR transcription and HIV-1 replication. (A) 5.1 cells were stimulated with increasing concentrations of GL. Thirty min later cells were stimulatedwith 30 ng/ml TNFa for 6 h and luciferase activity was analyzed. Results represent mean ± SD of at least four different experiments. ⁄⁄P = 0.0075; ⁄⁄⁄P < 0.001. (B) HeLa-Tat-Luc cells were stimulated with DRB or with increasing concentrations of GL for 12 h and luciferase activity was analyzed. Data represent the mean ± SD of three independentexperiments considering 100% activation the values obtained in activated cells in the absence of GL. (C) HeLa cells transiently co-transfected with p65 and HIV-LTR-Luc weretreated with increasing concentrations of GL for 6 h and luciferase activity was analyzed. Data are expressed as fold induction over the basal level and represent themean ± SD of three independent experiments. ⁄P = 0.0487; ⁄⁄P = 0.0055; ⁄⁄⁄P < 0.001. (D) HeLa and A549 cells were treated with GL at the indicated doses and then infectedwith VSV-pseudotyped pNL4-3.Luc.R-E- (200 ng of p24) for 24 h. Luciferase activity in the cell extracts was then measured. Results represent mean ± SD of five differentexperiments. ⁄⁄⁄P < 0.001.

0.5-GL (μM)

+ +++TNF -

NF- B

+ + +

1 5 10 25 50- - ++-++ +++PMA -+ --- vehicle -

+--

Cells N.E.

NF- B

A B

Fig. 2. GL inhibits the binding of NF-jB to DNA. (A) Jurkat cells were incubated with increasing concentrations of GL and 30 min later stimulated with TNFa (30 ng/ml). After60 min the nuclear extracts were obtained and analyzed for NF-jB binding by EMSA. (B) Effect of GL on the binding of NF-jB to DNA. Nuclear extracts prepared from PMAactivated Jurkat cells were incubated at 37 �C with GL (20 M) for 30 min and then analyzed for NF-jB activation. We show a representative result of three independentexperiments.

72 M. Pérez et al. / Chemico-Biological Interactions 214 (2014) 69–76

p65

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Fig. 3. Effect of GL on NF-jB signalling. (A) HeLa cells were transiently transfected with KBF-Luc alone or in combination with IKKa, IKKb and IKKc. After 24 h of transfectioncells were incubated with different doses of GL for 6 h and luciferase activity was assayed. The results show the percentage of activation and represent the mean ± SD of threeindependent experiments. ⁄⁄⁄P < 0.001. (B) HeLa cells were incubated with GL and 15 min later stimulated with TNFa (30 ng/ml) for the indicated periods of time. Cell lysateswere analyzed by Western blots with the indicated antibodies. We show a representative blot out of three independent experiments. (C) HeLa cells were incubated withincreasing concentrations of GL for 30 min and stimulated with TNFa (30 ng/ml) for 30 min. Cellular p65 localization was visualized by + immunofluorescence microscopyand the intensity difference of p65 between nucleus and cytoplasm is shown on a bar chart. Data are mean ± SD of n = 3 experiments. ⁄P = 0.0157; ⁄⁄P = 0.005; ⁄⁄⁄P < 0.001.

M. Pérez et al. / Chemico-Biological Interactions 214 (2014) 69–76 73

analyzed the effects of GL on the nuclear translocation of p65 andwe found that GL significantly impaired TNFa-induced p65 nucleartranslocation in HeLa cells (Fig. 3C).

It is well known that acetylation is a key post-transcriptionalmechanism that modulates the transcriptional activity of bothp65 and STAT3 [32,38]. To further study whether p65 and STAT3acetylation is affected by GL, HeLa cells were co-transfected witha plasmid encoding the histone acetylase p300 and either Flag-STAT3 or HA-p65 plasmid. Twenty-four hours after transfectionSTAT3 and p65 were immunoprecipitated and the acetylation sta-tus of both proteins determined by Western blot. We found that GLdid not affect the p300-induced acetylation of p65 (Fig. 4A) orSTAT3 (Fig. 4B). As expected, STAT3 acetylation was inhibited byanacardic acid, a histone acetyltransferase p300 inhibitor [2]. In

addition, GL only at the higher concentration (20 lM) slightlyinhibited the p65 transcriptional activity induced by p300 (Fig. 4C).

3.3. Galiellalactone interferes with the interaction between importina3 and p65 NF-jB

Previous studies have shown that p65 interacts with importinsa3, a4 and a5 [8,9]. Since GL inhibits the nuclear translocation ofp65 (Fig. 3C) we investigated if this compound could interfere withp65-importins interaction. Thus, 293T cells were co-transfectedwith the HA-p65 plasmid and Flag-importins a3, a4 and a5 plas-mids and twenty-four hours later the cells were treated with GLfor six hours. As depicted in Fig. 5A, we found that GL was ableto disrupt the interaction of p65 with importin a3. However, the

Flag-STAT3

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Fig. 4. Acetylation of p65 and STAT3 is not affected by GL. (A) HeLa cells co-transfected with HA-p65 and Flag-p300 plasmids were treated with increasingamounts of GL during 6 h before lysis. A fraction was subjected to immunoprecip-itation (IP) using anti-HA antibody. After elution acetylated p65 protein wasdetected by western blotting with antibody specific for anti-Ac-Lys. The remainingextract fraction was tested for the occurrence of the p65 by immunoblotting(Inputs). We show a representative blot out of three independent experiments. (B)HeLa cells were cotransfected with the indicated plasmids and treated withincreasing amounts of GL during 6 h before lysis. A fraction was subjected toimmunoprecipitation (IP) using anti-Flag antibody and STAT3 detected by immu-noblotting with the indicated antibody. We show a representative blot out of threeindependent experiments. (C) HeLa cells transiently co-transfected with p65, p300and KBF-Luc plasmids were incubated for 12 h with the indicated amounts of GL.Cell lysates were analyzed for luciferase activity. Data are expressed as foldinduction over the basal level and represent the mean ± SD of three independentexperiments. ⁄P = 0.0422.

74 M. Pérez et al. / Chemico-Biological Interactions 214 (2014) 69–76

interaction of p65 with importin a4 and a5 was not affected signif-icantly by GL treatment. To analyze the effect of GL on endogenousp65 and importin a3 interactions, A549 cells were preincubatedwith GL (10 lM) for 15 min and then stimulated with TNFa during30 min. We show in Fig. 5B that TNFa induced an interaction be-tween p65 and importin a3 that was almost completely abolishedby the treatment with GL (Fig. 5B).

To further confirm the effect of GL on p65/importin a3 interac-tion, we over-expressed p65 (GFP-tagged) and importin a3(Flag-tagged) in U2OS cells. In untreated cells the p65 localization

was mainly cytoplasmic while importin a3 localized in perinuclearregion. Following TNFa stimulation p65 clearly translocated to thenucleus and a perinuclear co-localization with importin a3 wasalso detectable. Interestingly, GL prevented not only p65 nucleartranslocation but also the perinuclear co-localization with impor-tin a3 (Fig. 5C). To get an insight into the mechanism by whichGL could prevent p65/importin a3 interactions, we incubatedDU145 cells with biotinylated GL and interacting proteins wereimmunoprecipitated by streptavidin magnetics beads. In Fig. 5Dit is shown that GL binds to p65 but not to importin a3. Altogether,these results show that GL inhibits the NF-jB pathway at the levelof p65 nuclear import by interacting with p65 and possibly pre-venting its interaction with importin a3.

4. Discussion

Biologically active natural products are making a significantcontribution in modern drug discovery programs. Most of thesenatural compounds are often multi-targeted in nature, which isgenerally a very desirable property for anti-cancer, anti-inflamma-tory and anti-HIV-1 agents. The use of natural or synthetic com-pounds targeting cellular proteins involved in HIV-1 replicationhas opened new research avenues in AIDS. Within these agents,biologically active small molecules for interfering with HIV-1-LTRpromoter regulatory proteins are of special interest, since thesecompounds are unlikely to induce mutations in the host genomeand drug resistances. Here we show that GL inhibits the nucleartranslocation of the transcription factor NF-jB that plays a key rolein HIV-1 replication.

It has been suggested that GL inhibits STAT3 transcriptionalactivation via a Michael addition of a cysteine residue at the sur-face of STAT3 to the a,b-unsaturated lactone moiety of GL, whichis believed to prevent the binding of STAT3 to DNA [34]. The p65NF-jB subunit contains eight cysteines from which cysteine at po-sition 38 (Cys-38) is required for interaction with the phosphatebackbone of NF-jB binding sites [6] and its oxidation inhibits theDNA binding activity [23]. More recently it has been demonstratedthat p65 Cys-38 is also subjected to hydrogen sulfide-linked sulf-hydration and this effect also modulates the binding of p65 toDNA [29].

Lactones and related compounds that contain a,b-unsaturatedcarbonyl moieties target p65 and IKKb kinase in the NF-jB signal-ling pathway by modification of Cys-38 and Cys-179 in p65 andIKKb kinase, respectively [10]. For example, it has been shown thatdehydroxymethyl-epoxyquinomicin and (11S)-2a-Bromo-3-oxoe-udesmano-12,6a-lactone inhibits the nuclear translocation of p65and its subsequent DNA binding by covalent modification of Cys-38 [30,35]. Our results indicate that GL does not interfere withthe binding to DNA of nuclear NF-jB complexes (p50/p65) butinteracts directly with p65. Therefore, it is possible that the forma-tion of a covalent bond between the sulfur atom of cysteines, oth-ers than Cys-38, in p65 and the carbon at position 3 of GL mayblock the interaction of p65 with importin a3, thus preventingits nuclear translocation.

In addition to p65 and STAT3, GL may covalently modify cyste-ines in other cellular or viral proteins impairing its function. In thissense the integrase (IN) of HIV-1, which has been implicated in dif-ferent steps during viral replication, contains cysteine residues thatare critical for dimerization and biological function [31,41]. More-over, it has been shown that IN nuclear import pathway is medi-ated in part by interaction with importin a3 [1,22], and wefound that GL inhibits HIV-1 replication in non-activated HeLaand A549 cells. Although these cells have a basal level of NF-jBactivation that can be inhibited by GL, it is tempting to speculatethat GL may also induce a covalent modification of IN cysteines

A

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Fig. 5. GL interferes the p65-Importin a3 interaction. (A) (Left panel) 293T cells co-transfected with HA-p65 and Flag-Importin-a3, -a4 and -a5 plasmids were treated withincreasing amounts of GL during 6 h before lysis. A fraction was subjected to immunoprecipitation (IP) using anti-Flag antibody. After elution p65 protein was detected bywestern blotting with an antibody specific for anti-HA. The remaining extract fraction was tested for the occurrence of the indicated proteins by immunoblotting (Inputs). Weshow a representative blot out of three independent experiments. (Right panel) Densitometric analysis of the bands obtained with Importin-a3. Values were expressed asrelative optical density immunoprecipitated p65/total p65. Data are mean ± SD of n = 3 experiments. ⁄⁄⁄P < 0.001. (B) A549 cells were stimulated with TNFa for 30 min in thepresence of GL. After lysis, a fraction was subjected to immunoprecipitation (IP) using Importin-a3 antibody. After elution p65 was detected by western blotting with aspecific antibody. The remaining extract fraction was tested for the occurrence of the indicated proteins by immunoblot (Inputs). We show a representative blot out of threeindependent experiments. (C) U2OS cells were transfected with Flag-Importin a3 and analyzed for the localization of Importin a3 and endogenous p65 byimmunofluorescence. Overlapping localization in merged pictures is shown in yellow. Nuclear DNA was stained with DAPI. (D) DU145 cells treated with biotinylated GL(GL-biotin) (50 lM) for 2 h. Proteins bound to GL-biotin were isolated with streptavidin magnetic beads (IP) and both p65 and Importin-a3 proteins detected by Western blot.We show a representative blot out of three independent experiments.

M. Pérez et al. / Chemico-Biological Interactions 214 (2014) 69–76 75

affecting it functionally and/or binding to importin a3. We are cur-rently performing experiments to investigate this possibility.

GL may also qualify as a lead compound for the development ofnovel therapies against inflammation and cancer [18]. In the pastfew years a cross talking between NF-jB and STAT3 has been dem-onstrated in different models [11]. NF-jB activation in human can-cers has been reported to be positively or negatively correlated

with STAT3 activation in the control of tumorigenesis [17], andboth factors are found constitutively activated in cancer cells andin tumour-associated myeloid cells and regulate in a cooperativemanner the expression of a plethora of genes linked to inflamma-tion and carcinogenesis [4]. For instance, in human gastric cancertumours there is a positive correlation between NF-jB and STAT3activation. In addition, both NF-jB inhibition and STAT3 silencing

76 M. Pérez et al. / Chemico-Biological Interactions 214 (2014) 69–76

decreased gastric cancer cell migration and invasion [37]. STAT3also maintains NF-jB activation and retention in the nucleus inmelanoma cells and prostate cancer cell [24]. On the other hand,a positive crosstalk between NF-jB and STAT3 was found in B celllymphoma, which prevents apoptosis and increased cell prolifera-tion [13]. Therefore, dual NF-jB and STAT3 inhibitors such as GLrepresent promising lead compounds for the development of anti-cancer therapies.

Conflict of interest statement

The authors declare no conflict of interest.

Acknowledgments

This work was supported by MICINN grants SAF2010-19292,ISCIII-RETIC RD06/006 and P09-CTS-4973 to E.M. and bySAF2010-17122 and PI-0650-2010 to M.A.C. The authors wish tothank Carmen Cabrero-Doncel for her assistance with themanuscript.

References

[1] Z. Ao, K. Danappa Jayappa, B. Wang, Y. Zheng, S. Kung, E. Rassart, R. Depping, M.Kohler, E.A. Cohen, X. Yao, Importin alpha3 interacts with HIV-1 integrase andcontributes to HIV-1 nuclear import and replication, J. Virol. 84 (2010) 8650–8663.

[2] K. Balasubramanyam, V. Swaminathan, A. Ranganathan, T.K. Kundu, Smallmolecule modulators of histone acetyltransferase p300, J. Biol. Chem. 278(2003) 19134–19140.

[3] B. Berkhout, K.T. Jeang, Functional roles for the TATA promoter and enhancersin basal and Tat-induced expression of the human immunodeficiency virustype 1 long terminal repeat, J. Virol. 66 (1992) 139–149.

[4] J. Bollrath, F.R. Greten, IKK/NF-kappaB and STAT3 pathways: central signallinghubs in inflammation-mediated tumour promotion and metastasis, EMBO Rep.10 (2009) 1314–1319.

[5] M. Canki, J.N. Thai, W. Chao, A. Ghorpade, M.J. Potash, D.J. Volsky, Highlyproductive infection with pseudotyped human immunodeficiency virus type 1(HIV-1) indicates no intracellular restrictions to HIV-1 replication in primaryhuman astrocytes, J. Virol. 75 (2001) 7925–7933.

[6] Y.Q. Chen, S. Ghosh, G. Ghosh, A novel DNA recognition mode by the NF-kappaB p65 homodimer, Nat. Struct. Biol. 5 (1998) 67–73.

[7] R.I. Connor, B.K. Chen, S. Choe, N.R. Landau, Vpr is required for efficientreplication of human immunodeficiency virus type-1 in mononuclearphagocytes, Virology 206 (1995) 935–944.

[8] R. Fagerlund, L. Kinnunen, M. Kohler, I. Julkunen, K. Melen, NF-{kappa}B istransported into the nucleus by importin {alpha}3 and importin {alpha}4, J.Biol. Chem. 280 (2005) 15942–15951.

[9] R. Fagerlund, K. Melen, X. Cao, I. Julkunen, NF-kappaB p52, RelB and c-Rel aretransported into the nucleus via a subset of importin alpha molecules, CellSignal. 20 (2008) 1442–1451.

[10] T.D. Gilmore, Introduction to NF-kappaB: players, pathways, perspectives,Oncogene 25 (2006) 6680–6684.

[11] S.I. Grivennikov, M. Karin, Dangerous liaisons: STAT3 and NF-kappaBcollaboration and crosstalk in cancer, Cytokine Growth Factor Rev. 21 (2010)11–19.

[12] H. Hacker, M. Karin, Regulation and function of IKK and IKK-related kinases,Sci. STKE 2006 (2006) re13.

[13] S.S. Han, H. Yun, D.J. Son, V.S. Tompkins, L. Peng, S.T. Chung, J.S. Kim, E.S. Park,S. Janz, NF-kappaB/STAT3/PI3K signaling crosstalk in iMyc E mu B lymphoma,Mol. Cancer 9 (2010) 97.

[14] M. Hausding, M. Tepe, C. Ubel, H.A. Lehr, B. Rohrig, Y. Hohn, A. Pautz, T.Eigenbrod, T. Anke, H. Kleinert, G. Erkel, S. Finotto, Induction of tolerogeniclung CD4+ T cells by local treatment with a pSTAT-3 and pSTAT-5 inhibitorameliorated experimental allergic asthma, Int. Immunol. 23 (2011) 1–15.

[15] M.S. Hayden, S. Ghosh, Signaling to NF-kappaB, Genes Dev. 18 (2004) 2195–2224.

[16] M.S. Hayden, S. Ghosh, Shared principles in NF-kappaB signaling, Cell 132(2008) 344–362.

[17] G. He, G.Y. Yu, V. Temkin, H. Ogata, C. Kuntzen, T. Sakurai, W. Sieghart, M.Peck-Radosavljevic, H.L. Leffert, M. Karin, Hepatocyte IKKbeta/NF-kappaB

inhibits tumor promotion and progression by preventing oxidative stress-driven STAT3 activation, Cancer Cell 17 (2010) 286–297.

[18] R. Hellsten, M. Johansson, A. Dahlman, N. Dizeyi, O. Sterner, A. Bjartell,Galiellalactone is a novel therapeutic candidate against hormone-refractoryprostate cancer expressing activated Stat3, Prostate 68 (2008) 269–280.

[19] R. Hellsten, M. Johansson, A. Dahlman, O. Sterner, A. Bjartell, Galiellalactoneinhibits stem cell-like ALDH-positive prostate cancer cells, PLoS One 6 (2011)e22118.

[20] J. Hiscott, H. Kwon, P. Genin, Hostile takeovers: viral appropriation of the NF-kappaB pathway, J. Clin. Invest. 107 (2001) 143–151.

[21] A. Hoffmann, G. Natoli, G. Ghosh, Transcriptional regulation via the NF-kappaBsignaling module, Oncogene 25 (2006) 6706–6716.

[22] K.D. Jayappa, Z. Ao, M. Yang, J. Wang, X. Yao, Identification of critical motifswithin HIV-1 integrase required for importin alpha3 interaction and viralcDNA nuclear import, J. Mol. Biol. 410 (2011) 847–862.

[23] Z.T. Kelleher, A. Matsumoto, J.S. Stamler, H.E. Marshall, NOS2 regulation of NF-kappaB by S-nitrosylation of p65, J. Biol. Chem. 282 (2007) 30667–30672.

[24] H. Lee, A. Herrmann, J.H. Deng, M. Kujawski, G. Niu, Z. Li, S. Forman, R. Jove,D.M. Pardoll, H. Yu, Persistently activated Stat3 maintains constitutive NF-kappaB activity in tumors, Cancer Cell 15 (2009) 283–293.

[25] N. Marquez, R. Sancho, A. Macho, A. Moure, I. Masip, A. Messeguer, E. Munoz,Anti-Tat and anti-HIV activities of trimers of n-alkylglycines, Biochem.Pharmacol. 71 (2006) 596–604.

[26] L.A. Pereira, K. Bentley, A. Peeters, M.J. Churchill, N.J. Deacon, A compilation ofcellular transcription factor interactions with the HIV-1 LTR promoter, NucleicAcids Res. 28 (2000) 663–668.

[27] K. Rudolph, A. Serwe, G. Erkel, Inhibition of TGF-beta signaling by the fungallactones (S)-curvularin, dehydrocurvularin, oxacyclododecindione andgaliellalactone, Cytokine 61 (2013) 285–296.

[28] R. Sancho, N. Marquez, M. Gomez-Gonzalo, M.A. Calzado, G. Bettoni, M.T.Coiras, J. Alcami, M. Lopez-Cabrera, G. Appendino, E. Munoz, Imperatorininhibits HIV-1 replication through an Sp1-dependent pathway, J. Biol. Chem.279 (2004) 37349–37359.

[29] N. Sen, B.D. Paul, M.M. Gadalla, A.K. Mustafa, T. Sen, R. Xu, S. Kim, S.H. Snyder,Hydrogen sulfide-linked sulfhydration of NF-kappaB mediates itsantiapoptotic actions, Mol. Cell 45 (2012) 13–24.

[30] R. Tamura, K. Morimoto, S. Hirano, L. Wang, M. Zhao, M. Ando, T. Kataoka,Santonin-related compound 2 inhibits the nuclear translocation of NF-kappaBsubunit p65 by targeting cysteine 38 in TNF-alpha-induced NF-kappaBsignaling pathway, Biosci. Biotechnol. Biochem. 76 (2012) 2360–2363.

[31] M. Tsiang, G.S. Jones, M. Hung, D. Samuel, N. Novikov, S. Mukund, K.M.Brendza, A. Niedziela-Majka, D. Jin, X. Liu, M. Mitchell, R. Sakowicz, R.Geleziunas, Dithiothreitol causes HIV-1 integrase dimer dissociation whileagents interacting with the integrase dimer interface promote dimerformation, Biochemistry 50 (2011) 1567–1581.

[32] L. Vermeulen, G. De Wilde, S. Notebaert, W. Vanden Berghe, G. Haegeman,Regulation of the transcriptional activity of the nuclear factor-kappaB p65subunit, Biochem. Pharmacol. 64 (2002) 963–970.

[33] P. Wei, M.E. Garber, S.M. Fang, W.H. Fischer, K.A. Jones, A novel CDK9-associated C-type cyclin interacts directly with HIV-1 Tat and mediates itshigh-affinity, loop-specific binding to TAR RNA, Cell 92 (1998) 451–462.

[34] M. Weidler, J. Rether, T. Anke, G. Erkel, Inhibition of interleukin-6 signaling andStat3 activation by a new class of bioactive cyclopentenone derivatives,Biochem. Biophys. Res. Commun. 276 (2000) 447–453.

[35] M. Yamamoto, R. Horie, M. Takeiri, I. Kozawa, K. Umezawa, Inactivation of NF-kappaB components by covalent binding of (-)-dehydroxymethylepoxyquinomicinto specific cysteine residues, J. Med. Chem. 51 (2008) 5780–5788.

[36] W. Ye, W. Lin, A.M. Tartakoff, T. Tao, Karyopherins in nuclear transport ofhomeodomain proteins during development, Biochim. Biophys. Acta 2011(1813) 1654–1662.

[37] J. Yoon, S.J. Cho, Y.S. Ko, J. Park, D.H. Shin, I.C. Hwang, S.Y. Han, S.Y. Nam, M.A.Kim, M.S. Chang, H.S. Lee, W.H. Kim, B.L. Lee, A synergistic interaction betweentranscription factors nuclear factor-kappaB and signal transducers andactivators of transcription 3 promotes gastric cancer cell migration andinvasion, BMC Gastroenterol. 13 (2013) 29.

[38] Z.L. Yuan, Y.J. Guan, D. Chatterjee, Y.E. Chin, Stat3 dimerization regulated byreversible acetylation of a single lysine residue, Science 307 (2005) 269–273.

[39] P. Zhan, X. Liu, E. De Clercq, Blocking nuclear import of pre-integrationcomplex: an emerging anti-HIV-1 drug discovery paradigm, Curr. Med. Chem.17 (2010) 495–503.

[40] C. Zhou, T.M. Rana, A bimolecular mechanism of HIV-1 Tat protein interactionwith RNA polymerase II transcription elongation complexes, J. Mol. Biol. 320(2002) 925–942.

[41] K. Zhu, C. Dobard, S.A. Chow, Requirement for integrase during reversetranscription of human immunodeficiency virus type 1 and the effect ofcysteine mutations of integrase on its interactions with reverse transcriptase,J. Virol. 78 (2004) 5045–5055.