In the present study, we focussed on the colorectal carcinoma cell line SW480, in which STAT3 is consti-tutively activated [26] and found that SW480 cells were efficiently killed by the h
Trang 1signal transducer and activator of transcription (STAT) 3 hairpin decoy oligodeoxynucleotide – interference with interferon-c-STAT1-mediated killing
Ali Tadlaoui Hbibi1,2, Christelle Laguillier1,2, Ine`s Souissi1,2, Denis Lesage1,2, Ste´phanie Le Coquil1,2,
An Cao3, Valeri Metelev4, Fanny Baran-Marszak1,2,5and Remi Fagard1,2,6
1 Institut National de la Sante´ et de la Recherche Me´dicale, U978, Bobigny, France
2 Universite´ Paris 13, UFR SMBH Bobigny, France
3 Centre National de la Recherche Scientifique, UMR 7033, Bobigny, France
4 Department of Chemistry, Moscow State University, Russia
5 AP-HP, hoˆpital Avicenne, service d’he´matologie, Bobigny, France
6 AP-HP, hoˆpital Avicenne, service de biochimie, Bobigny, France
Signal transducer and activators of transcription
(STATs) are a family of transcription factors that are
activated in response to cytokines regulating cell
proli-feration, differentiation, inflammation, the immune
response, apoptosis and fetal development [1]
Sche-matically, the inactive STATs are cytoplasmic; once activated, they dimerize and enter the nucleus where they induce the expression of target genes [2]
Several studies have demonstrated that STAT3 is a key 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, hoˆpital
Avicenne 125 rue de Stalingrad, 93009
Bobigny Cedex, France
Fax: +33 014 895 5627
Tel: +33 014 895 5928
E-mail: remi.fagard@avc.aphp.fr
(Received 17 November 2008, revised 25
January 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 growth factors STAT3 is activated in response to cytokines involved mostly in cell proliferation; 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 valuable target with respect to achieving inhibition of tumour cell proliferation Indeed, its inhibition results in cell death We previously observed that inhibition of the transcription factor nuclear factor-jB, 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 cell death as efficiently as the STAT3 inhibitor stattic The hairpin STAT3 oligodeoxynucleotide co-localized with STAT3 within the cytoplasm, prevented STAT3 localization to the nucleus, blocked a cyclin D1 reporter promoter and associated with STAT3 in pull-down assays However, the same cells were efficiently killed by interferon-c This effect was counter-acted by the STAT3 oligodeoxynucleotide, which was found to efficiently inhibit STAT1 Thus, although it can inhibit STAT3, the hairpin STAT3 oligodeoxynucleotide 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, nuclear factor; ODN, oligodeoxynucleotide; PARP, poly(ADP-ribose) polymerase; STAT, signal transducer and activator of transcription; TEAPC-chol, 3b-[N-(N ¢,N ¢,N ¢-triethylaminopropane)-carbamoyl] cholesterol iodide.
Trang 2major 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 tumour
progression [7] and its activation, as detected in breast,
head and neck, lung and colon cancers [8], is considered
to be a marker of poor prognosis The role played by
STAT3 in malignant cell growth is mediated in part by
the up-regulation of the expression of genes involved
in cell survival and proliferation, including those for
Bcl-xl, Bcl-2, c-Myc, cyclin D1, survivin, Mcl-1,
vascu-lar endothelial growth factor, IL-10 and transforming
growth factor b [9–13] The constitutive activation of
STAT3 observed in many tumours and tumour cell lines
suggests that it may be a good target for the induction
of cell death Several therapeutic approaches have been
developed to inhibit STAT3, including inhibition of its
expression [14,15], inhibition of its dimerization [16,17]
and inhibition of its binding to the DNA promoter
sequence 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 affecting
other nontranscriptional functions They have been
successfully used in the treatment of some diseases,
including rheumatoid arthritis [20] or atopic dermatitis
[21] In cancer cell lines, the STAT3 ODNs were shown
to inhibit cell proliferation [18,22]
How the STAT3 decoy ODNs interact with STAT3
within cells, including how they affect its function, has
not been thoroughly investigated One potential
diffi-culty regarding specific targeting of STAT3 is that it
shares 72% sequence homology with STAT1 STAT3
and STAT1 are generally recognized to be
antagonis-tic, with STAT3 functioning as a proliferation
acti-vator and STAT1 as an inhibitor [23–25], this
antagonism is further illustrated by the fact that
cyto-kines, such as IL-6, which favour cell proliferation,
activate principally STAT3, whereas cytokines, such as
interferon (IFN)-a⁄ b or IFN-c, which favour cell
death, activate principally STAT1 However, despite
their different functions in cells, STAT3 and STAT1
recognize very similar sequences on the gene promoters
and share common targets; they can also form
hetero-dimers, whose function has not been clearly elucidated
In the present study, we focussed on the colorectal
carcinoma cell line SW480, in which STAT3 is
consti-tutively activated [26] and found that SW480 cells were
efficiently killed by the hpST3dODN SW480 cells
were also efficiently killed by IFN-c treatment, and
this action was counteracted by hpST3dODN, which
reduced transcriptional activity and nuclear
localiza-tion of STAT1 after IFN-c treatment Thus, although
IFN-c treatment did not impair hpST3dODN-induced
cell killing, IFN-c-induced cell killing was impaired by hpST3dODN, most likely as a result of its interaction with activated STAT1
Results
The hairpin STAT3 decoy ODN induces cell death
of the colon carcinoma SW480 cells
To examine the transfection efficiency of hpST3dODN into cells, we applied different concentrations of the fluorescein isothiocyanate (FITC)-labelled hpST3d ODN combined with cationic lipid and analysed the intensity of FITC fluorescence by flow cytometry Transfection efficiency increased with increasing ODN amounts (0.5, 1 and 2 lgÆmL)1) but not linearly, sug-gesting the possibility of a saturable mechanism of entry (Fig 1A); identical results were obtained with a control ODN (not shown) Examination of the cells by light microscopy showed that untreated cells, cells treated with empty liposomes and cells treated with control ODN were identical and had a normal appear-ance, whereas cells treated with hpST3dODN became rounded and were detached from the culture dish (not shown) To further analyse cell death induced by hpST3dODN, different concentrations of ODN were added to cells (0.5, 1 and 2 lg) or, alternatively, a con-trol ODN was used (1 and 2 lg) After 48 h of culture, cell death was determined by measuring trypan blue uptake; the number of dead cells increased with hpST3dODN concentration (0.5, 1 and 2 lg), whereas control ODN (1 and 2 lg) or the liposomes alone had little effect (Fig 1C) Kinetic analysis showed that cell death was undetectable after 12 h, and became detect-able after 16, 24 and 48 h (Fig 1B); after 72 h, the amount of dead cells and debris made it difficult to count dead cells Analysis by flow cytometry clearly showed the cells that had incorporated hpST3dODN (FITC positive) were those that were dying (PI posi-tive) (Fig 1D) hpST3dODN was also applied to the 2C4 fibroblastic cell line in which STAT3 is not consti-tutively activated There was no effect on cell viability, despite the fact that hpST3dODN could efficiently enter 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 further explore the sensitivity of SW480 cells to STAT3 inhibi-tion, the inhibitor stattic, which is considered to be specific to STAT3 [29], was used and trypan blue-positive cells counted Increased cell death was observed (Fig 1F), thus strengthening the notion that specific inhibition of STAT3 is sufficient to induce the death of these cells Interestingly, in stattic-treated 2C4
Trang 3cells (in which STAT3 is not activated), there were 5%
dead cells with 10 lm stattic, (28% in SW480), 10%
with 15 lm (35% in SW480), 25% with 30 lm (45% in
SW480) and 35% with 40 lm (60% in SW480)
The hairpin STAT3 decoy ODN inhibits the
transcriptional activity of STAT3 and colocalizes
with STAT3 to the cytoplasm of SW480 cells
The transcriptional activity of STAT3 after treatment
of the cells with hpST3dODN was analysed in SW480
cells transfected with a cyclin D1-promoter luciferase
reporter The luminescence of cell extracts, measured
24 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 inhibit the nuclear factor (NF)-jB-luciferase reporter in these cells and that the NF-jB inhibitory ODN [30] did not inhibit the cyclin D1-luciferase reporter (not shown)
To determine whether the subcellular localization of STAT3 had been modified by ODN, fluorescence microscopy was employed In untreated SW480 cells, phospho-STAT3 was detectable in the cytoplasm and nucleus (Fig 2B) In FITC-labelled hpST3dODN-transfected cells, phospho-STAT3 was detected in the cytoplasm, but not in the nucleus, and ODN was detected only in the cytoplasm (Fig 2C), suggesting that hpST3dODN somehow prevented the nuclear localization of phospho-STAT3 Indeed, in cells that
A
B
C
D
E
F
60 80 100
0 20 40
ODN (µg)
15 20 25
0 5 10
ODN (µg)
0.5 1 2 1 e
Cont (µg) n
20 30
16 24 48 16 24 48 16 24 48
ODN (2 µg)
15
30 2C4
10
Contr n
10 Time (h)
15 30
10
ODN (µg) Cont (µg)
40
80 60
20 10
Fig 1 Cell death induced by treatment of the SW480 colon carcinoma cell line with the STAT3 decoy ODN (A) Efficient incorporation of FITC-STAT3 decoy ODN into SW480 cells using decoy⁄ lipid complexes After 6 h of incubation, cells were placed in fresh culture medium containing 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 lg (B) SW480 cells were treated with empty lipids (n), hairpin decoy ODN (2 lg) or control ODN (con) and the dead cells were counted after 16, 24 and 48 h of culture using trypan blue staining (C) Cells were treated with 0.5, 1 and 2 lg of hpST3dODN and 1 and 2 lg of control ODN for 6 h or with lipids only; after 48 h of culture, they were stained with trypan blue and counted (n, untreated cells; e, empty liposomes) (D) Cells were treated as described in (C) and then analysed
by flow cytometry for propidium iodide (PI) and FITC uptake, the results shown are for the cells that are positive for both PI and FITC uptake (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 of Stattic 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.
Trang 4were either not treated (Fig 2B) or treated with
con-trol ODN (Fig 2D), phospho-STAT3 was found
within the nucleus
The hairpin STAT3 decoy ODN also disrupts
IFN-c-induced STAT1 signalling
Because STAT3 and STAT1 share a high degree of
homology and bind to similar promoter sequences, they
are likely to interact with the same ODN Although
hpST3dODN induced the death of SW480 cells, and
blocked the transcriptional activity of STAT3, it was
important to verify whether, within cells, this ODN was
STAT3-specific or could also interact with STAT1 and
disrupt 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 determine
whether this was also observed in SW480 cells IFN-c,
at 200 ngÆmL)1for 48 h, efficiently killed the cells;
how-ever, lower concentrations (10 ngÆmL)1) and shorter
exposures (4 h) had no effect on cell death (Fig 3A) In
addition, treatment of the cells with 100–200 ngÆmL)1
IFN-c for 24–48 h induced poly(ADP-ribose)
polymer-200 400 600 800
STAT3
0
Merge DAPI
Merge STAT3 DAPI
Merge Cont ODN STAT3 DAPI
A
B
C
Fig 2 Transcriptional activity and subcellu-lar localization of STAT3 are altered in STAT3 decoy ODN-treated SW480 cells (A) Inhibition of the transcriptional activity of STAT3 by hpST3dODN SW480 cells were cotransfected with a cyclin D1-luc plasmid, treated with either hpST3dODN or a control ODN and the luciferase activity measured after 24 h of incubation The relative STAT3 transcriptional activity in transfected cells is shown Each transfection experiment was performed in triplicate Subcellular location
of phospho-STAT3 analysed by fluorescence microscopy: (B) in nontreated cells, phopho-STAT3 was cytoplasmic and nuclear; (C) in hpST3dODN-treated cells, STAT3 was almost exclusively cytoplasmic and not detected in the nuclei (arrow); the FITC-labelled hpST3dODN was also cytoplasmic; (D) in control ODN-treated cells, phospho-STAT3 was mostly nuclear, as in control cells; the ODN was mostly cytoplasmic (scale bar = 10 lm).
60 70
Dead cells (%) 20
30 40 50
IFN-γ (ng·mL–1) 0 10 100 200
0 10
cPARP
cPARP
48 h IFN-γ 0 5 20 100 200 cPARP
(ng·mL –1 ) actin
A
B
Fig 3 Treatment with IFN-c induces cell death of SW480 cells (A) SW480 cells were incubated in the absence of IFN-c or with
10, 100 and 200 ngÆmL)1 for 4, 24 and 48 h of incubation and cell death was measured by trypan blue exclusion Each experiment was performed in triplicate The results are expressed
as a percentage of dead cells (B) Cleavage of PARP, induced by
24 or 48 h of treatment with IFN-c at 5, 20, 100 and
200 ngÆmL)1was analysed by western blotting using anti-cleaved-PARP serum.
Trang 5ase (PARP) cleavage (Fig 3B) The transcriptional
activity of STAT1, as measured with an interferon
regu-latory factor (IRF) 1-promoter luciferase reporter after
treatment of IFN-c-treated cells with hpST3dODN
(1 lgÆmL)1), was considerably reduced compared to the
effect of control ODN (Fig 4A) The subcellular
locali-zation of STAT1 was also modified by treatment with
hpST3dODN In IFN-c-treated cells, STAT1 was
detected in the nucleus (Fig 4B); in cells treated with
hpST3dODN, STAT1 remained in the cytoplasm and
was found to colocalize with ODN (Fig 4C); and, in
cells treated with control ODN, the nuclear
transloca-tion of STAT1 occurred normally (Fig 4D) These
observations suggest that hpST3dODN could interfere
with 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 In
cells 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 cells
with 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 is
acting on both STAT3 and STAT1 and that it has
the potential to interfere with the biological activity
of IFN-c In the SW480 cell line, IFN-c treatment resulted in the inhibition of the STAT3-dependent cyclin D1 promoter, and activation of the
STAT1-2000
Luciferase activity (Rlu per µg prot) No
0 500 1000 1500
IFN
Contr-ODN STAT1 DAPI Merge
A
B
C
D
Fig 4 Transcriptional activity and
subcellu-lar localization of STAT1 are altered in
STAT3 decoy ODN-treated SW480 cells.
(A) SW480 cells were transfected with an
IRF-1-luc plasmid, treated with IFN-c at
20 ngÆmL)1, and either not treated (no add.),
treated with hpST3dODN (ODN) or treated
with control ODN (contr.); after 24 h of
incu-bation, luciferase activity was measured.
Each transfection experiment was
per-formed in triplicate Subcellular location of
STAT1 determined by fluorescence
micros-copy: (B) cytoplasmic location of STAT1 in
untreated cells and nuclear location in IFN-c
treated cells (20 ngÆmL)1); (C) cytoplasmic
location of phospho-STAT1 (red) in
hpST3dODN-treated (1 lg) SW480 cells that
had been treated with IFN-c (20 ngÆmL)1);
the decoy ODN (green) was also
cytoplas-mic; (D) nuclear location of STAT1 (red) in
cells treated with control ODN (green) (scale
bar = 10 lm).
40
20 10
IFN- γ (ng·mL –1 ) 0 0 0 100 200 100 200 100 200
30 40 50 60 70
0 10 20
Stattic (µ M ) IFN- γ (ng·mL –1 )
A
B
Fig 5 Inhibition of IFN-c-induced cell death by the STAT3 decoy ODN in SW480 cells (A) Cells were either treated with 1 lg of hpST3dODN or control ODN for 6 h, with IFN-c alone or with ODN and IFN-c; after 48 h of culture, they were stained with trypan blue and counted (B) Cells were either not treated, or treated with stattic alone, IFN-c alone or both combined together After 48 h of incubation, dead cells were counted using trypan blue exclusion.
Trang 6dependent IRF1 promoter (Fig 6A) To determine
whether this correlated with phosphorylation levels,
the phosphorylation of STAT1 on tyrosine 701, and of
STAT3 on tyrosine 705, was examined in
IFN-c-trea-ted SW480 cells STAT1 phosphorylation increased
dramatically, even at the lowest concentration used,
whereas STAT3 phosphorylation never increased by
more than twofold (Fig 6B) In the absence of IFN-c
treatment, there was a low but clearly detectable
phosphorylation of STAT3 Finally, to analyse the
interaction of both STAT1 and STAT3 with hpST3dODN, pull-down experiments were performed using a biotinylated version of this ODN This was fol-lowed by gel separation and western blotting with anti-phospho-STAT1 or anti-phospho-STAT3 The results obtained show that, in SW480 cells that have not been stimulated, there is a basal level of binding of phospho-STAT3 to hpST3dODN This binding is increased in cells treated with IL-6 and, to a lesser extent, in cells treated with IFN-c (Fig 6C, lanes 1 and 2) On the other hand, binding of phospho-STAT1
is detected only in cells that have been treated with IFN-c (Fig 6C, lane 3)
Discussion
In the present study, we observed that a hairpin decoy ODN targeting STAT3 (hpST3dODN) induces cell death of the carcinoma cell line SW480, apparently by trapping STAT3 within the cytoplasm
The hairpin decoy, but not control ODN, inhibited cell proliferation, eliminating any possible effects as a result of the introduction of DNA within cells, and indicating that, in itself, the interaction of ODN with STAT3 induced these effects (i.e inhibition of the cyclin-D1-dependent promoter, colocalization with STAT3 and STAT3 binding in pull-down assays) Our data indicate a correlation between inhibition of STAT3 by hpST3dODN and induction of cell death
In addition, they confirm previous observations made
in head and neck nonsquamous carcinoma cell lines, with a nonhairpin ODN containing the c-activated sequence (GAS) sequence [18] Taken together with our observation that ODN does not kill the fibrosar-coma cell line 2C4, in which STAT3 is not activated, these results suggest that the effect of ODN may be restricted to cells in which STAT3 is activated These results are also in agreement with a previous study showing that inhibition of the constitutively activated Janus kinase⁄ STAT3 pathway with AG490 resulted in the diminished viability of SW480 cells [26] The mech-anism by which hpST3dODN inhibits STAT3 is not clearly understood Our immunofluorescence micros-copy data suggest that activated STAT3 may be trapped by ODN within the cytoplasm This view is supported by our pull-down assays, which indicate a direct interaction of hpST3dODN with activated STAT3 Cytoplasmic trapping of a transcription factor was previously observed in the laboratory with a NF-jB decoy ODN [30], indicating that the mecha-nism involved is probably not specific to one transcrip-tion factor Nevertheless, the mechanism by which binding of a hairpin to STAT3 prevents nuclear entry
Cyclin D1
0
100
200
(100 ng·mL–1) (100 ng·mL –1 )
0 1000 2000
IRF1
A
Western blotting
IFN-γ (ng·mL–1) 0 5 20 100 200
P-STAT1
P-STAT3
STAT1
STAT3
Pull-down
P-STAT1
IL6
P-STAT3
B
C
Fig 6 Binding of the STAT3 decoy ODN to STAT3 and STAT1 (A)
SW480 cells were transfected with a cyclin D1-luc plasmid (left
panel) or an IRF1-luc plasmid (right panel) and either treated or not
with IFN-c at 100 ngÆmL)1 After 24 h of incubation, luciferase
activity was measured Relative STAT1 and STAT3 transcriptional
activities in transfected cells are shown (B) Phosphorylation levels
of STAT3 and STAT1 in SW480 cells after treatment with different
concentrations of IFN-c analysed by western blotting using
anti-P-STAT1 and anti-P-STAT3 sera (C) Interaction of the STAT3 decoy
ODN with STAT3 and STAT1 as determined by pull-down assays.
Cells were treated with biotinylated hpST3dODN (1 lg) (lanes 1, 2
and 3) or control biotinylated ODN (lanes 4 and 5) for 16 h and
lysed; the complexes were bound to streptavidine and separated
on gels Cells were either not treated (lane 1), treated with IL-6
(30 ngÆmL)1, lanes 2 and 4) or treated with IFN-c (100 ngÆmL)1,
lanes 3 and 5) Western blotting was performed using
anti-phospho-STAT3 and anti-phospho-STAT1 sera Identical amounts of
cellular extracts were used, as determined by the Bradford method.
The experiment was repeated several times, with identical results
being obtained.
Trang 7is not understood Because nuclear transport is a
highly regulated process, one possibility is that binding
to hpST3dODN modifies the conformation of key
components of the STAT3 protein complex, thereby
impairing normal interaction with the nuclear
trans-port machinery Alternatively, the hairpin ODN itself
might interact with components of the nuclear
trans-port machinery through its hairpin structure Although
the combination of induced cell death, inhibited
tran-scription activity and nuclear entry strongly indicates
that hpST3dODN functions by preventing nuclear
entry, further studies are required, including cell
frac-tionation assays, to directly demonstrate this proposal
and to identify the cellular components involved
Nevertheless, these results suggest that nuclear entry of
a decoy ODN is not a prerequisite for the inhibition of
transcription factors, as previously assumed [33]
There is an intriguing similarity between STAT3 and
STAT1 Both factors share activating stimuli and a
high homology of sequence, and they have common
gene targets and recognize very similar consensus
sequences; yet, they have clearly distinct functions in
cells STAT3 is mostly involved in cell survival and
proliferation, and STAT1 is involved in anti-viral and
immune defence and cell death, in response to
interfer-ons, including IFN-c [34] Inhibition of STAT1 using
GAS-based decoy ODNs was previously found to
effi-ciently inhibit inflammation-linked processes such as
graft rejection [35,36], arthritis [37] and contact
hyper-sensitivity [38] The decoy ODN used in these studies
was considered STAT1-specific However, a recent
study, using a GAS sequence-based nonhairpin decoy
ODN [39] to inhibit STAT3 in head and neck
squa-mous carcinoma cell lines, although demonstrating
inhibition of IFN-c-activated STAT1, concluded that
there was an absence of interference with
STAT1-mediated actions The present study of the colon
carcinoma cell line SW480 demonstrates that
IFN-c-treatment induces cell death, using conditions similar
to previous studies [31,32,40] Treatments of at least
2 days with IFN-c concentrations of at least
100 ngÆmL)1 are necessary; indeed, when using lower
concentrations of IFN-c [39], we did not observe any
cell killing If the decoy ODNs used do not
discrimi-nate between STAT3 and STAT1, then a STAT3
decoy ODN may potentially inhibit the action of
IFN-c beIFN-cause STAT1 has long been reIFN-cognized as a key
component of this action [23] We therefore verified
whether hpST3dODN inhibited STAT1, and whether
this would result in an impaired action of IFN-c The
results obtained demonstrate that, in the SW480 cell
line, hpST3dODN inhibited STAT1: it inhibited its
transcriptional activity on an IRF1 reporter and its
nuclear localization, which is associated with inhibited IFN-c-induced cell death Although these results could mean that STAT3 has no effect on IFN-c-induced cell death, they show that, in our cell system, the action of hpST3dODN must be interpreted with caution because
it has the potential to inhibit IFN-c-induced cell death Alternatively, the STAT3-inhibitor stattic did not prevent cell death induced by IFN-c Because the hairpin decoy STAT3 ODN induces cell death of the nontreated SW480 cells in which there is a constitutive level of activated STAT3, which is necessary for the survival of these cells [30], and because it inhibits STAT1 in these cells when they are treated with IFN-c, we can tentatively conclude that it interacts with the activated forms of STAT3 and STAT1 The actions of STAT3 and STAT1 are highly entangled, they also have antagonistic activities, and they regulate each others activity Thus, the inhibition of both factors in vivo may have unpredictible results For example, in cardiac ischaemia, the action of STAT3 is protective and that of STAT1 increases cardiomyocyte apoptosis [41,42] Thus, the inhibition of STAT3 using decoy ODNs that are not strictly STAT3-specific may lead to unpredictable results (particularly in whole ani-mals) by impairing the action of STAT1-dependent interferon
The results obtained in the present study, including the ability of the hairpin decoy STAT3 ODN to inhibit both activated STAT3 and STAT1, also reveal that, in SW480 cells, survival may depend in part upon an equilibrium between the two STATs This equilibrium
is in favour of activated STAT3 in our untreated colo-carcinoma cells, and is tilted in favour of activated STAT1 in IFN-c-treated cells Such an equilibrium was observed in cells treated with the Janus kinase-family inhibitor AG490, where only limited potentia-tion of the pro-apoptotic effect of doxorubicin was found, whereas inhibition of STAT3 with a dominant negative or a platinum derivative increased the pro-apoptotic effect of doxorubicin [43] Thus, the efficiency of blocking STAT3 may depend on the absence of the inhibition of STAT1 Indeed, cell death induced by ODN and by IFN-c may be the result of completely different mechanisms Further-more, ODN might trap STAT1⁄ STAT3 heterodimers whose function remains to be elucidated One way to explore the complex interaction between STAT1 and STAT3 in SW480 cells is to suppress their expression Such an approach is indeed in progress in our labora-tory: using shRNA transduction, we are presently examining whether STAT3 silencing causes pro-apoptotic effects and whether STAT1 silencing causes anti-apoptotic effects
Trang 8The involvement of STAT1 may depend on the cell
line used In some cell lines, ODN bound both STAT3
and STAT1 but did not inhibit STAT1 [32] However,
the functions of STAT1 and STAT3 differ
consider-ably and this must depend at some point on the
specific recognition of a DNA-binding motif Specific
inhibition of STAT3 by DNA-binding targeting may
require an improved consensus sequence that would be
recognized principally by STAT3
As noted above, an NF-jB decoy ODN induced cell
death in the same cells [30], suggesting that the STAT3
and the NF-jB pathway are connected in these cells,
as described in other systems in which cytokines,
secreted as a result of NF-jB activation, activate
STAT3 Unphosphorylated STAT3-dependent
activa-tion of NF-jB has also been reported [35], although
our data indicate that the hairpin ODN blocks
acti-vated STAT3 and may not inhibit unphosphorylated
STAT3
Experimental procedures
Cell culture
SW480 (colon), 2C4 (fibrosarcoma) cell lines were grown in
10% FBS⁄ DMEM (Gibco BRL, Life Technologies,
Cergy-Pontoise, France), 100 UÆmL)1penicillin, 10 lgÆmL)1
strep-tomycin (Gibco BRL), 1 mm sodium pyruvate (Gibco
BRL), MEM vitamins 100· (Gibco BRL) and 5 lgÆmL)1
plasmocin (Cayla InvivoGen, Toulouse, France) Curcumin
was obtained from Acros Organics (Halluin, France)
Synthesis of the hairpin STAT3 decoy ODN
The oligodeoxynucleotides used comprised: RHN(CH2)6
-CATTTCCCGTAATCGAAGATTACGGGAAATG-(CH2)3
NHR (hpST3dODN), which was derived from the
serum-inducible element of the human c-fos promoter, and
RHN(CH2)6- CATTTCCCTTAAATCGAAGATTTAAG
GGAAATG-(CH2)3NHR (mutated hairpin control ODN)
(Sigma-Proligo; Sigma-Aldrich Corp., St Louis, MO, USA),
where R is either H, FITC or biotin To synthesize
oligo-deoxynucleotides with biotin, 7–10 nmol of the
oligodeoxy-nucleotide bearing 3¢- and 5¢-aminoalkyl linkers were
dissolved in 20 lL of 0.1 m NaHCO3 EZ-Link NHS-biotin
(Pierce, Rockford, IL, USA) (10 lL of a 65 mm solution in
dimethyl sulfoxide) was added, and the mixture was
incu-bated at room temperature for 6–16 h in the dark Next,
25 lL of water were added, and the modified
oligodeoxy-nucleotide was separated from the excess of hydrolyzed
reagent by two consecutive separations on Micro Bio-Spin 6
columns (Bio-Rad, Foster City, CA, USA) in accordance
with the manufacturer’s instructions After the second spin,
the biotinylated oligodeoxynucleotide was precipitated with
ethanol-sodium acetate In control experiments, the previ-ously described NF-jB decoy ODN [30] was used
Preparation of liposomes
Liposomes were formulated using a cationic lipid, 3b-[N-(N¢,N¢,N¢-triethylaminopropane)-carbamoyl] cholesterol iodide (TEAPC-Chol) and neutral colipid dioleoyl phospha-tidylethanolamine, as previously described [25] Briefly, TEAPC-Chol and dioleoyl phosphatidylethanolamine were mixed at a ratio of 1 : 1 (w⁄ w) and dissolved in chloro-form The solution was dried in vacuum Sterile water was then added and the mixture was sonicated to clarity for 1 h
in cycles of 15 min Using light scattering, we found that the size distribution of the liposomes was unimodal The concentration of cationic lipid was monitored by UV spec-troscopy at 226 nm and the value was used to calculate the charge ratio, assuming one positive charge for each cationic lipid molecule
Transfection using liposomes
Cells were grown in four-well plates to a density of 0.5· 106
cellsÆmL)1 When the cells reached 50–60% con-fluence, they were transfected with hpST3dODN or the hairpin control ODN (0.5, 1 and 2 lg corresponding to
100, 200 and 400 nm, respectively) in 150 lL of DMEM medium (without stromal vascular fraction cells) combined with the liposomes (0.5, 1 or 2 lg of cationic lipid), thus yielding liposome : ODN ratios of 0.5 : 0.5, 2 : 2, 1 : 0.5 and 1 : 1 (lg⁄ lg) After 6 h at 37 C in a humidified 5%
CO2 incubator, the cells were placed in fresh serum-containing medium Expression was analysed after 48 h
In control experiments, the liposomes were used alone at the same lipid concentrations
Flow cytometry, cell viability
The uptake of FITC-labelled hpST3dODN was measured
by flow cytometry, gating on the FL1-positive signal on an EPICS XL Beckman-Coulter counter (Beckman Coulter, Villepinte, France) To measure the rate of cell death, cells were resuspended in annexin V-binding buffer, incubated with 5 lL of propidium iodide (BD Pharmingen, Morangis, France) and analysed in a EPICS XL Beckman-Coulter counter Cell viability was assessed using the trypan blue exclusion method
Luciferase activity
To measure the transcriptional activity of STAT3 and STAT1, cells were transfected with either the cyclin D1 luciferase 1745 promoter [44] (a generous gift of R Pestell, Kimmel Cancer Center, Jefferson University in
Trang 9Philadelphia, PA, USA) or the IRF-1 luciferase promoter
(a generous gift of P Kovarik, University of Vienna,
Austria) Cells were then transfected with hpST3dODN or
the hairpin control ODN (1 lg, corresponding to 200 nm)
combined with liposomes After 24 h of incubation, cells
were lyzed for 30 min on ice with lysis buffer (10 mm Tris–
HCl, pH 7.5, 1 mm EDTA, 100 mm NaCl, 1% NP40 and
1 mm dithiothreitol) In control experiments, the
transcrip-tional activity of NF-jB was analysed using the NF-jB-luc
0.4K-luc plasmid (a generous gift of A Israe¨l, Institut
Pasteur, Paris, France) The lysates were centrifuged at
18 000 g for 10 min at 4C Supernatants were collected
and assayed for luciferase activity using the Luciferase
Assay kit (Promega, Madison, WI, USA) and a
luminome-ter (Clarity, Fisher Bioblock Scientific, Illkirsch, France)
Protein concentrations were measured using the Bradford
method Luciferase activity was normalized as relative light
units per lg of total protein in the supernatant The
experi-ments were performed in triplicate
Immunofluorescence
Cellular uptake and subcellular localization of the
FITC-labelled hpST3dODN were analysed on cells grown on
glass slides (Lab-Tek; Nunc, Rochester, NY, USA) Cells
were washed twice in NaCl⁄ Pi, fixed in 3.7% formaldehyde
in NaCl⁄ Pifor 15 min, permeabilized in 0.1% Triton X-100
for 15 min and blocked with 5% FBS, 0.1% Tween in
NaCl⁄ Pi for 1 h Cells were incubated with the primary
antibody (anti-STAT3, anti-STAT1; Cell Signaling
Tech-nology, Beverly, MA, USA; dilution 1 : 100) for 2 h Alexa
Fluor 546-labelled secondary anti-rat serum
(Invitrogen-Molecular Probes, Carlsbad, CA, USA) at 1 : 250 was
added for 90 min After counterstaining with
4¢,6¢-diami-dino-2-phenylindole, coverslips were mounted onto glass
slides in Vectashield (Vectorlabs, Clinisciences, Montrouge,
France) Fluorescence images were digitally acquired using
a Zeiss Axioplan2 Deconvolution microscope (CarlZeiss,
Le Pecq, France) and analysed with Metafer4
(Metasys-tems, Altlussheim, Germany)
Oligodeoxynucleotide pull-down assays and
western blotting
Nuclear protein extracts were obtained as follows: 20
mil-lion cells were resuspended in lysis buffer (20 mm Hepes,
pH 7.4, 1 mm MgCl2, 10 mm KCl, 0.3% NP40, 0.5 mm
dithiothreitol, 0.1 mm EDTA, protease inhibitors;
Compete; Boehringer Ingelheim GmbH, Ingelheim
Germany) at 4C for 5 min The lysates were centrifuged
at 14 000 g for 5 min at 4C, and the supernatants
con-taining the cytoplasmic proteins were discarded The pellets
were resuspended in the cell lysis buffer adjusted with 20%
glycerol and 0.35 m NaCl for 30 min at 4C After
centri-fugation at 14 000 g for 5 min at 4C, the supernatants
were stored at )80 C For pull-down assays, 100–200 lg
of nuclear protein extracts were incubated for 30 min at
4C in binding buffer (1% NP40, 50 mm Hepes, pH 7.6,
140 mm NaCl) containing salmon sperm DNA (1 lg per assay) and 1 lg of biotinylated hairpin decoy ODN or mutated control ODN The complexes were captured by incubation with 50 lL of avidin-sepharose beads (neutravi-din; Pierce) for 2 h at 4C, washed three times with NaCl ⁄ Tris (20 mm NaCl, 500 mm Tris–HCl, pH 8), and once with NaCl⁄ Tris-0.1% Tween After resuspension in sample buf-fer, complexes were separated on a SDS-polyacrylamide (10%) gel, and subjected to immunoblotting using anti-STAT3 (Cell Signaling Technology) Results were analysed by chemiluminescence (LumiGLO; Cell Signaling Technology) and autoradiography (X-Omat R; Eastman Kodak, Rochester, NY, USA)
Acknowledgements
A.T.H was supported in part by the Fondation Martine Midy This work was supported in part by grants from the Association de recherche contre le cancer (ARC, grant 3133) and RFBR 06-04-49196
References
1 Benekli M, Baer MR, Baumann H & Wetzier M (2003) Signal transducer and activator of transcription proteins
in leukemias Blood 101, 2940–2954
2 Zhong Z, Wen Z & Darnell JE Jr (1994) Stat3: a STAT family member activated by tyrosine phosphorylation in response to epidermal growth factor and interleukin-6 Science 264, 95–98
3 Park OK, Schaefer TS & Nathans D (1996) In vitro activation of Stat3 by epidermal growth factor recep-tor kinase Proc Natl Acad Sci USA 93, 13704– 13708
4 Coffer PJ & Kruijer W (1995) EGF receptor deletions define a region specifically mediating STAT transcrip-tion factor activatranscrip-tion Biochem Biophys Res Commun
210, 74–81
5 Shao H, Cheng HY, Cook RG & Tweardy DJ (2003) Identification and characterization of signal transducer and activator of transcription 3 recruitment sites within the epidermal growth factor receptor Cancer Res 63, 3923–3930
6 Fujitani Y, Nakajima K, Kojima H, Nakae K, Takeda
T & Hirano T (1994) Transcriptional activation of the IL-6 response element in the junB promoter is mediated
by multiple Stat family proteins Biochem Biophys Res Commun 202, 1181–1187
7 Bromberg J & Darnell JE Jr (2000) The role of STATs
in transcriptional control and their impact on cellular function Oncogene 19, 2468–2473
Trang 108 Garcia R, Bowman TL, Niu G, Yu H, Minton S,
Muro-cacho CA, Cox CE, Falcone R, Fairclough R,
Parsons S et al (2001) Constitutive activation of Stat3
by the Src and JAK tyrosine kinases participates in
growth regulation of human breast carcinoma cells
Oncogene 20, 2499–2513
9 Kanda N, Seno H, Konda Y, Marusawa H, Kanai M,
Nakajima T, Kawashima T, Nanakin A, Sawabu T,
Uenoyama Y et al (2004) STAT3 is constitutively
activated and supports cell survival in association with
survivin expression in gastric cancer cells Oncogene 23,
4921–4929
10 Bromberg JF, Wrzeszczynska MH, Devgan G, Zhao Y,
Pestell RG, Albanese C & Darnell JE (1999) STAT3 as
an oncogene Cell 98, 295–303
11 Epling-Burnette PK, Zhong B, Bai F, Jiang K, Bailey
RD, Garcia R, Jove R, Djeu JY, Loughran TP Jr &
Wei S (2001) Cooperative regulation of Mcl-1 by Janus
kinase⁄ stat and phosphatidylinositol 3-kinase contribute
to granulocyte-macrophage colony-stimulating
factor-delayed apoptosis in human neutrophils J Immunol
166, 7486–7495
12 Adachi Y, Aoki C, Yoshio-Hoshino N, Takayama K,
Curiel DT & Nishimoto N (2006) Interleukin-6 induces
both cell growth and VEGF production in malignant
mesotheliomas Int J Cancer 119, 1303–1311
13 Wang T, Niu G, Kortylewski M, Burdelya L, Shain K,
Zhang S, Bhattacharya R, Gabrilovich D, Heller R,
Coppola D et al (2004) Regulation of the innate and
adaptive immune responses by Stat-3 signaling in tumor
cells Nat Med 10, 48–54
14 Grandis JR, Drenning SD, Zeng Q, Watkins SC,
Melhem MF, Endo S, Johnson DE, Huang L, He Y &
Kim JD (2000) Constitutive activation of Stat3
signaling abrogates apoptosis in squamous cell
carcinogenesis in vivo Proc Natl Acad Sci USA 97,
4227–4232
15 Konnikova L, Kotecki M, Kruger MM & Cochran BH
(2003) Knockdown of STAT3 expression by RNAi
induces apoptosis in astrocytoma cells BMC Cancer 3,
23
16 Nagel-Wolfrum K, Buerger C, Wittig I, Butz K,
Hoppe-Seyler F & Groner B (2004) The interaction of
specific peptide aptamers with the DNA binding
domain and the dimerization domain of the
transcrip-tion factor Stat3 inhibits transactivatranscrip-tion and induces
apoptosis in tumor cells Mol Cancer Res 2, 170–182
17 Turkson J, Ryan D, Kim JS, Zhang Y, Chen Z, Haura
E, Laudano A, Sebti S, Hamilton AD & Jove R (2001)
Phosphotyrosyl peptides block Stat3-mediated DNA
binding activity, gene regulation, and cell
transforma-tion J Biol Chem 276, 45443–45455
18 Leong PL, Andrews GA, Johnson DE, Dyer KF, Xi S,
Mai JC, Robbins PD, Gadiparthi S, Burke NA,
Wat-kins SF et al (2003) Targeted inhibition of Stat3 with a
decoy oligonucleotide abrogates head and neck cancer cell growth Proc Natl Acad Sci USA 100, 4138–4143
19 Barton BE, Murphy TF, Shu P, Huang HF, Meyenhofer
M & Barton A (2004) Novel single-stranded oligonucleo-tides that inhibit signal transducer and activator of transcription 3 induce apoptosis in vitro and in vivo in prostate cancer cell lines Mol Cancer Ther 3, 1183–1191
20 Tomita T, Takano H, Tomita N, Morishita R, Kaneko
M, Shi K, Takahi K, Nakase T, Kaneda Y, Yoshikawa
H et al (2000) Transcription factor decoy for NFkappaB inhibits cytokine and adhesion molecule expressions in synovial cells derived from rheumatoid arthritis Rheumatology (Oxford) 39, 749–757
21 Nakamura H, Aoki M, Tamai K, Oishi M, Ogihara T, Kaneda Y & Morishita R (2002) Prevention and regres-sion of atopic dermatitis by ointment containing NF-kB decoy oligodeoxynucleotides in NC⁄ Nga atopic mouse model Gene Ther 9, 1221–1229
22 Liu X, Li J & Zhang J (2007) STAT3-decoy ODN inhibits cytokine autocrine of murine tumor cells Cell Mol Immunol 4, 309–313
23 Bromberg JF, Horvath CM, Wen Z, Schreiber RD & Darnell JE Jr (1996) Transcriptionally active Stat1 is required for the antiproliferative effects of both inter-feron alpha and interinter-feron gamma Proc Natl Acad Sci USA 93, 7673–7678
24 Bromberg JF, Fan Z, Brown C, Mendelsohn J & Dar-nell JE Jr (1998) Epidermal growth factor-induced growth inhibition requires Stat1 activation Cell Growth Differ 9, 505–512
25 Chin YE, Kitagawa M, Kuida K, Flavell RA & Fu XY (1997) Activation of the STAT signaling pathway can cause expression of caspase 1 and apoptosis Mol Cell Biol 17, 5328–5337
26 Lin Q, Lai R, Chirieac LR, Li C, Thomazy VA, Gram-matikakis I, Rassidakis GZ, Zhang W, Fujio Y, Kunis-ada K et al (2005) Constitutive activation of
JAK3⁄ STAT3 in colon carcinoma tumors and cell lines: inhibition of JAK3⁄ STAT3 signaling induces apoptosis and cell cycle arrest of colon carcinoma cells Am J Pathol 167, 969–980
27 Kunnumakkara AB, Anand P & Aggarwal BB (2008) Curcumin inhibits proliferation, invasion, angiogenesis and metastasis of different cancers through interaction with multiple cell signaling proteins Cancer Lett 269, 199–225
28 Lin JK (2007) Molecular targets of curcumin Adv Exp Med Biol 595, 227–243
29 Schust J, Sperl B, Hollis A, Mayer TU & Berg T (2006) Stattic: a small-molecule inhibitor of STAT3 activation and dimerization Chem Biol 13, 1235–1242
30 Laguillier C, Hbibi AT, Baran-Marszak F, Metelev V, Cao A, Cymbalista F, Bogdanov A Jr & Fagard R (2007) Cell death in NF-kappaB-dependent tumour cell lines as a result of NF-kappaB trapping by