Efficient killing of SW480 colon carcinoma cells by a signal transducer and activator of transcription (STAT) 3 hairpin decoy oligodeoxynucleotide - interference with interferon-γ-STAT1-mediated killing

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The signal transducers and activators of transcription (STATs) convey signals from the membrane to the nucleus in response to cytokines or growth factors. STAT3 is activated in response to cytokines involved mostly in cell proliferation; STAT1 is
  Efficient killing of SW480 colon carcinoma cells by asignal transducer and activator of transcription (STAT) 3hairpin decoy oligodeoxynucleotide – interference withinterferon- c -STAT1-mediated killing Ali Tadlaoui Hbibi 1,2 , Christelle Laguillier 1,2 , Ine `s Souissi 1,2 , Denis Lesage 1,2 , Ste´ phanie Le Coquil 1,2 ,An Cao 3 , Valeri Metelev 4 , Fanny Baran-Marszak 1,2,5 and Remi Fagard 1,2,6 1 Institut National de la Sante´ et de la Recherche Me´dicale, U978, Bobigny, France2 Universite´ Paris 13, UFR SMBH Bobigny, France3 Centre National de la Recherche Scientifique, UMR 7033, Bobigny, France4 Department of Chemistry, Moscow State University, Russia5 AP-HP, hoˆpital Avicenne, service d’he´matologie, Bobigny, France6 AP-HP, hoˆpital Avicenne, service de biochimie, Bobigny, France Signal transducer and activators of transcription(STATs) are a family of transcription factors that areactivated in response to cytokines regulating cell proli-feration, differentiation, inflammation, the immuneresponse, apoptosis and fetal development [1]. Sche-matically, the inactive STATs are cytoplasmic; onceactivated, they dimerize and enter the nucleus wherethey induce the expression of target genes [2].Several studies have demonstrated that STAT3 is akey regulator of cell proliferation. It was shown to be a Keywords cell death; hairpin decoy oligonucleotide;interferon- c ; STAT1; STAT3 Correspondence R. Fagard, service de biochimie, hoˆpitalAvicenne 125 rue de Stalingrad, 93009Bobigny Cedex, FranceFax: +33 014 895 5627Tel: +33 014 895 5928E-mail: remi.fagard@avc.aphp.fr(Received 17 November 2008, revised 25January 2009, accepted 19 February 2009)doi:10.1111/j.1742-4658.2009.06975.x The signal transducers and activators of transcription (STATs) convey sig-nals from the membrane to the nucleus in response to cytokines or growthfactors. STAT3 is activated in response to cytokines involved mostly in cellproliferation; STAT1 is activated by cytokines, including interferon- c ,involved in defence against pathogens and the inhibition of cell prolifera-tion. STAT3, which is frequently activated in tumour cells, is a valuabletarget with respect to achieving inhibition of tumour cell proliferation.Indeed, its inhibition results in cell death. We previously observed thatinhibition of the transcription factor nuclear factor- j B, a key regulator of cell proliferation, with decoy oligodeoxynucleotides results in cell death.We used a similar approach for STAT3. A hairpin STAT3 oligodeoxy-nucleotide was added to a colon carcinoma cell line in which it induced celldeath as efficiently as the STAT3 inhibitor stattic. The hairpin STAT3oligodeoxynucleotide co-localized with STAT3 within the cytoplasm,prevented STAT3 localization to the nucleus, blocked a cyclin D1 reporterpromoter and associated with STAT3 in pull-down assays. However, thesame cells were efficiently killed by interferon- c . This effect was counter-acted by the STAT3 oligodeoxynucleotide, which was found to efficientlyinhibit STAT1. Thus, although it can inhibit STAT3, the hairpin STAT3oligodeoxynucleotide appears also to inhibit STAT1-mediated interferon- c cell killing, highlighting the need to optimize STAT3-targeting oligodeoxy-nucleotides. Abbreviations FITC, fluorescein isothiocyanate; GAS,  c -activated sequence; IFN, interferon; IL, interleukin; IRF, interferon regulatory factor; NF, nuclearfactor; ODN, oligodeoxynucleotide; PARP, poly(ADP-ribose) polymerase; STAT, signal transducer and activator of transcription; TEAPC-chol,3 b -[ N  -( N   ¢ , N   ¢ , N   ¢ -triethylaminopropane)-carbamoyl] cholesterol iodide. FEBS Journal  276  (2009) 2505–2515  ª  2009 The Authors Journal compilation  ª  2009 FEBS  2505  major effector of epidermal growth factor receptor sig-nalling [3–5] and of cytokines such as interleukin (IL)-6[6]. It is also involved in transformation and tumourprogression [7] and its activation, as detected in breast,head and neck, lung and colon cancers [8], is consideredto be a marker of poor prognosis. The role played bySTAT3 in malignant cell growth is mediated in part bythe up-regulation of the expression of genes involvedin cell survival and proliferation, including those forBcl-xl, Bcl-2, c-Myc, cyclin D1, survivin, Mcl-1, vascu-lar endothelial growth factor, IL-10 and transforminggrowth factor  b  [9–13]. The constitutive activation of STAT3 observed in many tumours and tumour cell linessuggests that it may be a good target for the inductionof cell death. Several therapeutic approaches have beendeveloped to inhibit STAT3, including inhibition of itsexpression [14,15], inhibition of its dimerization [16,17]and inhibition of its binding to the DNA promotersequence using decoy oligodeoxynucleotides (ODN)[18,19]. ODNs comprise a valuable approach for inhi-biting transcription factors because they have the poten-tial to inhibit transcriptional function without affectingother nontranscriptional functions. They have beensuccessfully used in the treatment of some diseases,including rheumatoid arthritis [20] or atopic dermatitis[21]. In cancer cell lines, the STAT3 ODNs were shownto inhibit cell proliferation [18,22].How the STAT3 decoy ODNs interact with STAT3within cells, including how they affect its function, hasnot been thoroughly investigated. One potential diffi-culty regarding specific targeting of STAT3 is that itshares 72% sequence homology with STAT1. STAT3and STAT1 are generally recognized to be antagonis-tic, with STAT3 functioning as a proliferation acti-vator and STAT1 as an inhibitor [23–25], thisantagonism is further illustrated by the fact that cyto-kines, such as IL-6, which favour cell proliferation,activate principally STAT3, whereas cytokines, such asinterferon (IFN)- a   ⁄   b  or IFN- c , which favour celldeath, activate principally STAT1. However, despitetheir different functions in cells, STAT3 and STAT1recognize very similar sequences on the gene promotersand share common targets; they can also form hetero-dimers, whose function has not been clearly elucidated.In the present study, we focussed on the colorectalcarcinoma cell line SW480, in which STAT3 is consti-tutively activated [26] and found that SW480 cells wereefficiently killed by the hpST3dODN. SW480 cellswere also efficiently killed by IFN- c  treatment, andthis action was counteracted by hpST3dODN, whichreduced transcriptional activity and nuclear localiza-tion of STAT1 after IFN- c  treatment. Thus, althoughIFN- c  treatment did not impair hpST3dODN-inducedcell killing, IFN- c -induced cell killing was impaired byhpST3dODN, most likely as a result of its interactionwith activated STAT1. Results The hairpin STAT3 decoy ODN induces cell deathof the colon carcinoma SW480 cells To examine the transfection efficiency of hpST3dODNinto cells, we applied different concentrations of thefluorescein isothiocyanate (FITC)-labelled hpST3dODN combined with cationic lipid and analysed theintensity of FITC fluorescence by flow cytometry.Transfection efficiency increased with increasing ODNamounts (0.5, 1 and 2  l g Æ mL ) 1 ) but not linearly, sug-gesting the possibility of a saturable mechanism of entry (Fig. 1A); identical results were obtained with acontrol ODN (not shown). Examination of the cells bylight microscopy showed that untreated cells, cellstreated with empty liposomes and cells treated withcontrol ODN were identical and had a normal appear-ance, whereas cells treated with hpST3dODN becamerounded and were detached from the culture dish (notshown). To further analyse cell death induced byhpST3dODN, different concentrations of ODN wereadded to cells (0.5, 1 and 2  l g) or, alternatively, a con-trol ODN was used (1 and 2  l g). After 48 h of culture,cell death was determined by measuring trypan blueuptake; the number of dead cells increased withhpST3dODN concentration (0.5, 1 and 2  l g), whereascontrol ODN (1 and 2  l g) or the liposomes alone hadlittle effect (Fig. 1C). Kinetic analysis showed that celldeath was undetectable after 12 h, and became detect-able after 16, 24 and 48 h (Fig. 1B); after 72 h, theamount of dead cells and debris made it difficult tocount dead cells. Analysis by flow cytometry clearlyshowed the cells that had incorporated hpST3dODN(FITC positive) were those that were dying (PI posi-tive) (Fig. 1D). hpST3dODN was also applied to the2C4 fibroblastic cell line in which STAT3 is not consti-tutively activated. There was no effect on cell viability,despite the fact that hpST3dODN could efficientlyenter the cells (not shown). However, curcumin, a non-specific inhibitor [27,28], could kill the cells (Fig. 1E)as efficiently as SW480 cells (not shown). To furtherexplore the sensitivity of SW480 cells to STAT3 inhibi-tion, the inhibitor stattic, which is considered to bespecific to STAT3 [29], was used and trypan blue-positive cells counted. Increased cell death wasobserved (Fig. 1F), thus strengthening the notion thatspecific inhibition of STAT3 is sufficient to induce thedeath of these cells. Interestingly, in stattic-treated 2C4 STAT3 hairpin decoy oligonucleotide cell killing  A. Tadlaoui Hbibi  et al. 2506  FEBS Journal  276  (2009) 2505–2515  ª  2009 The Authors Journal compilation  ª  2009 FEBS  cells (in which STAT3 is not activated), there were 5%dead cells with 10  l m  stattic, (28% in SW480), 10%with 15  l m  (35% in SW480), 25% with 30  l m  (45% inSW480) and 35% with 40  l m  (60% in SW480). The hairpin STAT3 decoy ODN inhibits thetranscriptional activity of STAT3 and colocalizeswith STAT3 to the cytoplasm of SW480 cells The transcriptional activity of STAT3 after treatmentof the cells with hpST3dODN was analysed in SW480cells transfected with a cyclin D1-promoter luciferasereporter. The luminescence of cell extracts, measured24 h after transfection, was found to decrease by 86%,whereas control ODN had no measurable effect(Fig. 2A). To assess the specificity of the effect of ODN, we verified that the hST3dODN did not inhibitthe nuclear factor (NF)- j B-luciferase reporter in thesecells and that the NF- j B inhibitory ODN [30] did notinhibit the cyclin D1-luciferase reporter (not shown).To determine whether the subcellular localization of STAT3 had been modified by ODN, fluorescencemicroscopy was employed. In untreated SW480 cells,phospho-STAT3 was detectable in the cytoplasm andnucleus (Fig. 2B). In FITC-labelled hpST3dODN-transfected cells, phospho-STAT3 was detected in thecytoplasm, but not in the nucleus, and ODN wasdetected only in the cytoplasm (Fig. 2C), suggestingthat hpST3dODN somehow prevented the nuclearlocalization of phospho-STAT3. Indeed, in cells that ABCDEF 608010002040    F   I   T   C   l  a   b   i  n   t  e  n  s   i   t  y 0.512ODN (µg)1520250510ODN (µg)    P   I   /   F   I   T   C  -  p  o  s  c  e   l   l  s   (   %   ) 0.5 1 2 1eCont. (µg)n203016 24 48 16 24 48 16 24 48 ODN(2 µg)    D  e  a   d  c  e   l   l  s   (   %   ) 15302C410 E. Lip ODN cont Curc    D  e  a   d  c  e   l   l  s   (   %   ) Contrn 10Time (h)1530   c  e   l   l  s   (   %   ) 10    D  e  a   d  c ODN (µg)Cont. (µg) ne0.51212408060 Stattic (µ M ) 01510153040    D  e  a   d  c  e   l   l  s   (   %   ) 2010 Fig. 1.  Cell death induced by treatment of the SW480 colon carcinoma cell line with the STAT3 decoy ODN. (A) Efficient incorporation ofFITC-STAT3 decoy ODN into SW480 cells using decoy   ⁄   lipid complexes. After 6 h of incubation, cells were placed in fresh culture mediumcontaining 10% serum for 24 h. Fluorescence intensity was measured by flow cytometry after treatment with lipids combined with increas-ing concentrations of FITC-labelled hpST3dODN in the range 0.5–2  l g. (B) SW480 cells were treated with empty lipids (n), hairpin decoyODN (2  l g) or control ODN (con) and the dead cells were counted after 16, 24 and 48 h of culture using trypan blue staining. (C) Cells weretreated with 0.5, 1 and 2  l g of hpST3dODN and 1 and 2  l g of control ODN for 6 h or with lipids only; after 48 h of culture, they werestained with trypan blue and counted (n, untreated cells; e, empty liposomes). (D) Cells were treated as described in (C) and then analysedby flow cytometry for propidium iodide (PI) and FITC uptake, the results shown are for the cells that are positive for both PI and FITCuptake. (E) Cells of the fibroblastic line 2C4 were treated with empty lipids (E. lip), hairpin decoy ODN, control ODN (con) and curcumin(40  l M ) (curc) and the dead cells were counted after 48 h of culture using trypan blue staining. (F) Cells were treated with concentrations ofStattic in the range 0–30  l M , stained with trypan blue and dead cells were counted. To facilitate the comparison of different experiments,the results are expressed as a percentage.A. Tadlaoui Hbibi  et al.  STAT3 hairpin decoy oligonucleotide cell killing FEBS Journal  276  (2009) 2505–2515  ª  2009 The Authors Journal compilation  ª  2009 FEBS  2507  were either not treated (Fig. 2B) or treated with con-trol ODN (Fig. 2D), phospho-STAT3 was foundwithin the nucleus. The hairpin STAT3 decoy ODN also disruptsIFN- c -induced STAT1 signalling Because STAT3 and STAT1 share a high degree of homology and bind to similar promoter sequences, theyare likely to interact with the same ODN. AlthoughhpST3dODN induced the death of SW480 cells, andblocked the transcriptional activity of STAT3, it wasimportant to verify whether, within cells, this ODN wasSTAT3-specific or could also interact with STAT1 anddisrupt its signalling. In colorectal carcinoma cells,treatment with IFN- c  sensitizes cells to cytotoxic com-pounds, and can also induce cell death on its own[11,25,31,32]. Experiments were performed to determinewhether this was also observed in SW480 cells. IFN- c ,at 200 ng Æ mL ) 1 for 48 h, efficiently killed the cells; how-ever, lower concentrations (10 ng Æ mL ) 1 ) and shorterexposures (4 h) had no effect on cell death (Fig. 3A). Inaddition, treatment of the cells with 100–200 ng Æ mL ) 1 IFN- c  for 24–48 h induced poly(ADP-ribose) polymer- 200400600800 STAT3 0    C  y  c   l   i  n   D   1  -   L  u  c   i   f  e  r  a  s  e  a  c   t   i  v   i   t  y   (   R   l  u  p  e  r  µ  g  p  r  o   t   ) ODNCont. ODNNo add MergeDAPIMergeSTAT3DAPIMergeCont. ODNSTAT3DAPI ABC Fig. 2.  Transcriptional activity and subcellu-lar localization of STAT3 are altered inSTAT3 decoy ODN-treated SW480 cells. (A)Inhibition of the transcriptional activity ofSTAT3 by hpST3dODN. SW480 cells werecotransfected with a cyclin D1-luc plasmid,treated with either hpST3dODN or a controlODN and the luciferase activity measuredafter 24 h of incubation. The relative STAT3transcriptional activity in transfected cells isshown. Each transfection experiment wasperformed in triplicate. Subcellular locationof phospho-STAT3 analysed by fluorescencemicroscopy: (B) in nontreated cells, phopho-STAT3 was cytoplasmic and nuclear; (C) inhpST3dODN-treated cells, STAT3 wasalmost exclusively cytoplasmic and notdetected in the nuclei (arrow); the FITC-labelled hpST3dODN was also cytoplasmic;(D) in control ODN-treated cells, phospho-STAT3 was mostly nuclear, as in controlcells; the ODN was mostly cytoplasmic(scale bar = 10  l m). 6070    D  e  a   d  c  e   l   l  s   (   %   ) 20304050 IFN- γ   (ng·mL –1 ) 010100200010 cPARPcPARPactin 24 h48 h IFN- γ  0 5 20 100 200cPARP(ng·mL –1 )actin AB Fig. 3.  Treatment with IFN- c  induces cell death of SW480 cells.(A) SW480 cells were incubated in the absence of IFN- c  or with10, 100 and 200 ng Æ mL ) 1 for 4, 24 and 48 h of incubationand cell death was measured by trypan blue exclusion. Eachexperiment was performed in triplicate. The results are expressedas a percentage of dead cells. (B) Cleavage of PARP, induced by24 or 48 h of treatment with IFN- c  at 5, 20, 100 and200 ng Æ mL ) 1 was analysed by western blotting using anti-cleaved-PARP serum. STAT3 hairpin decoy oligonucleotide cell killing  A. Tadlaoui Hbibi  et al. 2508  FEBS Journal  276  (2009) 2505–2515  ª  2009 The Authors Journal compilation  ª  2009 FEBS  ase (PARP) cleavage (Fig. 3B). The transcriptionalactivity of STAT1, as measured with an interferon regu-latory factor (IRF) 1-promoter luciferase reporter aftertreatment of IFN- c -treated cells with hpST3dODN(1  l g Æ mL ) 1 ), was considerably reduced compared to theeffect of control ODN (Fig. 4A). The subcellular locali-zation of STAT1 was also modified by treatment withhpST3dODN. In IFN- c -treated cells, STAT1 wasdetected in the nucleus (Fig. 4B); in cells treated withhpST3dODN, STAT1 remained in the cytoplasm andwas found to colocalize with ODN (Fig. 4C); and, incells treated with control ODN, the nuclear transloca-tion of STAT1 occurred normally (Fig. 4D). Theseobservations suggest that hpST3dODN could interferewith STAT1, a key signalling factor for IFN- c . Accord-ingly, cell death was analysed in SW480 cells after treat-ment with IFN- c  and the addition of hpST3dODN. Incells that were treated with IFN- c , the addition of hpST3dODN reduced cell death by more than 50%(Fig. 5A); interestingly, such a reduction of IFN- c -induced cell death was not observed when treating cellswith stattic, a compound that binds the SH2 domain of STAT3 with high affinity (Fig. 5B). hpST3dODN binds both STAT3 and STAT1 The results obtained indicate that hpST3dODN isacting on both STAT3 and STAT1 and that it hasthe potential to interfere with the biological activityof IFN- c . In the SW480 cell line, IFN- c  treatmentresulted in the inhibition of the STAT3-dependentcyclin D1 promoter, and activation of the STAT1- 20002500 IRF1    L  u  c   i   f  e  r  a  s  e  a  c   t   i  v   i   t  y   (   R   l  u  p  e  r  µ  g  p  r  o   t   ) NoaddODNContr 050010001500 IFN0IFN- γ  ODNSTAT1MergeDAPIContr-ODNMergeDAPISTAT1 ABCD Fig. 4.  Transcriptional activity and subcellu-lar localization of STAT1 are altered inSTAT3 decoy ODN-treated SW480 cells.(A) SW480 cells were transfected with anIRF-1-luc plasmid, treated with IFN- c  at20 ng Æ mL ) 1 , and either not treated (no add.),treated with hpST3dODN (ODN) or treatedwith control ODN (contr.); after 24 h of incu-bation, luciferase activity was measured.Each transfection experiment was per-formed in triplicate. Subcellular location ofSTAT1 determined by fluorescence micros-copy: (B) cytoplasmic location of STAT1 inuntreated cells and nuclear location in IFN- c treated cells (20 ng Æ mL ) 1 ); (C) cytoplasmiclocation of phospho-STAT1 (red) inhpST3dODN-treated (1  l g) SW480 cells thathad been treated with IFN- c  (20 ng Æ mL ) 1 );the decoy ODN (green) was also cytoplas-mic; (D) nuclear location of STAT1 (red) incells treated with control ODN (green) (scalebar = 10  l m).    D  e  a   d  c  e   l   l  s   (   %   ) 402010ODN0+000++00IFN- γ   (ng·mL –1 )00 0 100 200 100 200 100 200Control00+0000++304050607000015 15 150102001002000100200Stattic (µ M )IFN- γ   (ng·mL –1 )    D  e  a   d  c  e   l   l  s   (   %   ) AB Fig. 5.  Inhibition of IFN- c -induced cell death by the STAT3 decoyODN in SW480 cells. (A) Cells were either treated with 1  l g ofhpST3dODN or control ODN for 6 h, with IFN- c  alone or with ODNand IFN- c ; after 48 h of culture, they were stained with trypan blueand counted. (B) Cells were either not treated, or treated withstattic alone, IFN- c  alone or both combined together. After 48 h ofincubation, dead cells were counted using trypan blue exclusion.A. Tadlaoui Hbibi  et al.  STAT3 hairpin decoy oligonucleotide cell killing FEBS Journal  276  (2009) 2505–2515  ª  2009 The Authors Journal compilation  ª  2009 FEBS  2509
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