Pharmacological targeting of NF-kappaB potentiates the effect of the topoisomerase inhibitor CPT-11 on colon cancer cells.

Abstract

NF-kappaB interferes with the effect of most anti-cancer drugs through induction of anti-apoptotic genes. Targeting NF-kappaB is therefore expected to potentiate conventional treatments in adjuvant strategies. Here we used a pharmacological inhibitor of the IKK2 kinase (AS602868) to block NF-kappaB activation. In human colon cancer cells, inhibition of NF-kappaB using 10 microM AS602868 induced a 30-50% growth inhibitory effect and strongly enhanced the action of SN-38, the topoisomerase I inhibitor and CPT-11 active metabolite. AS602868 also potentiated the cytotoxic effect of two other antineoplasic drugs: 5-fluorouracil and etoposide. In xenografts experiments, inhibition of NF-kappaB potentiated the antitumoural effect of CPT-11 in a dose-dependent manner. Eighty-five and 75% decreases in tumour size were observed when mice were treated with, respectively, 20 or 5 mg kg(-1) AS602868 associated with 30 mg kg(-1) CPT-11 compared to 47% with CPT-11 alone. Ex vivo tumour analyses as well as in vitro studies showed that AS602868 impaired CPT-11-induced NF-kappaB activation, and enhanced tumour cell cycle arrest and apoptosis. AS602868 also enhanced the apoptotic potential of TNFalpha on HT-29 cells. This study is the first demonstration that a pharmacological inhibitor of the IKK2 kinase can potentiate the therapeutic efficiency of antineoplasic drugs on solid tumours.

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Pharmacological targeting of NF-kB potentiates the effect of the
topoisomerase inhibitor CPT-11 on colon cancer cells
P Lagadec*,1,2, E Griessinger1,2, MP Nawrot1,2, N Fenouille1,2, P Colosetti1,2, V Imbert1,2, M Mari2,3,
P Hofman2,3, D Czerucka1,2, D Rousseau2,4, E Berard5, M Dreano6and JF Peyron1,2
1INSERM U526, Nice F-06000, France;2Faculte ´ de Me ´decine Pasteur (IFR50), Universite ´ Nice Sophia-Antipolis, Nice F-06107, France;3INSERM ERI
21, Nice F-06000, France;4INSERM U721, Nice F-06000, France;5CHU de Nice, Service de Pe ´diatrie, Ho ˆpital de l’Archet II, Nice F-06200, France;
6Merck-Serono International S.A., Geneva, Switzerland
NF-kB interferes with the effect of most anti-cancer drugs through induction of anti-apoptotic genes. Targeting NF-kB is therefore
expected to potentiate conventional treatments in adjuvant strategies. Here we used a pharmacological inhibitor of the IKK2 kinase
(AS602868) to block NF-kB activation. In human colon cancer cells, inhibition of NF-kB using 10mM AS602868 induced a 30–50%
growth inhibitory effect and strongly enhanced the action of SN-38, the topoisomerase I inhibitor and CPT-11 active metabolite.
AS602868 also potentiated the cytotoxic effect of two other antineoplasic drugs: 5-fluorouracil and etoposide. In xenografts
experiments, inhibition of NF-kB potentiated the antitumoural effect of CPT-11 in a dose-dependent manner. Eighty-five and 75%
decreases in tumour size were observed when mice were treated with, respectively, 20 or 5mgkg?1AS602868 associated with
30mgkg?1CPT-11 compared to 47% with CPT-11 alone. Ex vivo tumour analyses as well as in vitro studies showed that AS602868
impaired CPT-11-induced NF-kB activation, and enhanced tumour cell cycle arrest and apoptosis. AS602868 also enhanced the
apoptotic potential of TNFa on HT-29 cells. This study is the first demonstration that a pharmacological inhibitor of the IKK2 kinase
can potentiate the therapeutic efficiency of antineoplasic drugs on solid tumours.
British Journal of Cancer (2008) 98, 335–344. doi:10.1038/sj.bjc.6604082
Published online 8 January 2008
& 2008 Cancer Research UK
www.bjcancer.com
Keywords: CPT-11; chemoresitance; NF-kB; colorectal cancer; in vivo
??????????????????????????????????????????????????
Colorectal cancer (CRC) is the third commonest malignancy
worldwide with 954000 new cases and 492000 deaths in 2000
(Davies et al, 2005). These outcomes are largely due to the
poor clinical response of CRC to conventional drugs. CPT-11
(irinotecan) and its active metabolite, SN-38, are topoisomerase I
inhibitors that have shown efficacy in the treatment of advanced
and/or metastatic CRC (Wasserman et al, 2001). However, despite
the initial response, most patients treated with CPT-11 become
resistant and exhibit tumour progression (Calvo et al, 2002). CPT-
11 treatment has been shown to activate NF-kB (Xu and Villalona-
Calero, 2002; Janssens and Tschopp, 2006), which could be a
potential resistance mechanism in malignant cells (Wang et al,
1998; Baldwin, 2001; Nakanishi and Toi, 2005). Thus, reducing
NF-kB-mediated activation may help prevent CPT-11-induced
resistance to cell killing.
NF-kB complexes are composed of a variety of homo- or
heterodimers formed by five components: p50, p52, p65 (-RelA),
RelB, and c-Rel subunits. The p50–p65 complexes are the best-
characterized and most abundant dimers. In the absence of
stimulation, NF-kB is sequestered in the cytoplasm of most cells,
by binding to IkB inhibitory subunits. Upon stimulation,
IkB molecules are phosphorylated by the specific kinases
IKK(IkB kinase)1/a and IKK2/b, which together with NEMO
(NF-kB Essential Modulator) /IKKg form the IKK complex that
integrates signals for NF-kB activation. Serine phosphorylation is
followed by polyubiquitination, and subsequent degradation of
IkB by the proteasome reviewed in Karin (1999). Then, NF-kB
translocates into the nucleus where it controls the transcription of
numerous genes. Mechanisms by which topoisomerase-targeting
drugs induce IkB degradation to activate the NF-kB pathway have
to be elucidated. In response to DNA damage, NEMO appears to
translocate to the nucleus and undergo a series of post-
translational modifications. In the nucleus, NEMO establishes a
complex with p53-inducible protein with a death domain and
receptor interacting protein 1, allowing NEMO sumoylation
(Janssens and Tschopp, 2006). Then, sumo-NEMO is recognized
and phosphorylated by ATM (Ataxia Telangiectasia Mutated),
tagged by ubiquitination, which induces its release from ATM and
its cytoplasmic translocation allowing NF-kB activation (Wu et al,
2006). Thus, NEMO provides a means to link nuclear DNA damage
to the activation of the cytoplasmic IKK complex (Huang et al,
2003). Once activated, NF-kB promotes cell survival through
expression of genes coding for antiapoptotic proteins (c-IAP1,
c-IAP2, bfl-1, and Bcl-xl) and supports resistance of tumour cell to
treatments by inducing the expression of the multidrug resistance
Received 2 August 2007; revised 9 October 2007; accepted 12 October
2007; published online 8 January 2008
*Correspondence: Dr P Lagadec, INSERM U526, Faculte ´ de Me ´decine
Pasteur, Universite ´ de Nice Sophia Antipolis, 06107 Nice Cedex 2,
France; E-mail: lagadec@unice.fr
British Journal of Cancer (2008) 98, 335–344
& 2008 Cancer Research UKAll rights reserved 0007– 0920/08 $30.00
www.bjcancer.com
Translational Therapeutics

Page 2

proteins (Pahl, 1999). Furthermore, NF-kB could largely partici-
pate to thetumorigenic process
genes coding for growth factors and cell cycle regulators (Van
Antwerp et al, 1996; Wang et al, 1996) as well as it could promote
metastasis through induction of extracellular matrix-degrading
enzymes and angiogenesis through vascular endothelial growth
factor expression (Yu et al, 2004). The inhibition of NF-kB could
therefore affect tumour cells at different steps of their pathological
process.
The aim of our study was to evaluate the effect of inhibiting
NF-kB to potentiate the action of the topoisomerase poison
CPT-11 in colon cancer cells. We used a pharmacological inhibitor
of the IKK2 kinase (AS602868) that was previously shown to reveal
the apoptotic potential of TNF-a in Jurkat cells (Frelin et al, 2003)
and to induced apoptosis of primary human acute myeloid
leukaemia cells (Frelin et al, 2005). We show that both in vitro
and in vivo in HT-29 colon s.c. xenografts AS602868 potentiated
antitumour CPT-11 effectiveness by increasing CPT-11-induced
apoptosis of HT-29 tumour cells. This effect was associated with
decreased expression of antiapoptotic genes and a stimulation of
CPT-11 antiproliferative actions. The antitumoural effect of
AS602868 could also be due to its capacity to induce apoptosis
of HT-29 cells in the presence of TNFa whose intratumoural
concentration was increased upon CPT-11 treatment.
throughexpression of
MATERIALS AND METHODS
Drugs and antibodies
AS602868 is an anilino-pyrimidine derivative and ATP competitor
selected for its inhibitory effect in vitro on IKKee, a constitutively
active version of IKK2. The compound is covered by the patent
application PCT WO 02/46171. AS602868 has an in vitro inhibitory
concentration of 50% (IC50) of 60nM towards purified IKK2 and no
effect on IKK1 (IC50¼14mM) or on a large panel of recombinant
kinases. It has some inhibitory effect on JNK2 (IC50¼600nM).
AS602868 in sterile cyclodextrin solution was supplied by Merck-
Serono International SA (Geneva, Switzerland). CPT-11 was a gift
from Dr Pierre-Alain Vitte (Serono Pharmaceutical Research
Institute, Geneva, Switzerland). The pan caspase inhibitor z-VADfmk
(R&D Systems, Abington, UK) and SN-38 (a kind gift from Dr JL
Fischel, Antoine Lacassagne Oncology Center, Nice, France) were
prepared in DMSO and stock solutions were stored at ?201C.
Recombinant hTNFa was from PeproTech (Rocky Hill, NJ, USA).
Anti-Parp-a and anti-phospho IkB was purchased from Cell
Signaling (Beverly, MA, USA); anti-HSP60, anti-p65 p50, anti-
IkB, and anti-TNFa from Santa Cruz Biotechnology (Santa Cruz,
CA, USA); anti-caspases 3, 8, and 9 from Medical & Biological
Laboratories (Woburn, MA, USA); and anti-Ki-67 from DAKO-
Cytomation (Glostrup, Denmark).
Cell lines and cell drug treatments
The human colon cancer cell lines HT-29, SW-480, and SW-620
were obtained from the ATCC (Bethesda, MD, USA). Aliquots of
5?106viable cells in 10ml of DMEM medium containing 10%
fetal calf serum were plated into tissue culture dishes (100mm
diameter) for 24h, then stimulated for 72h before harvesting.
Xenograft growth assay
Animal experiments were performed in accordance with the
regulations of our institution’s ethics commission and with the
United Kingdom Co-ordinating Committee on Cancer Research
Guidelines (1998). Forty-five NMRI female nude mice (6–8 weeks
of age) were inoculated s.c. with 1?106tumour cells. Mice were
then dispatched into nine groups of 5. Treatments lasted 10 weeks
and consisted of five orally administrations of AS602868 (5 or
20mgkg?1), 5 days a week. CPT-11 (10 or 30mgkg?1) was
administered i.p. twice a week. In combination treatments,
AS602868 was given 4h before CPT-11 injections. Mice from
control group were administered with AS602868 vehicle (cyclo-
dextrin). Tumours were measured once a week with a caliper and
their volumes were calculated by the formula: (a?b2)/2, where ‘a’
and ‘b’ are, respectively, the larger and smaller diameter. At the
end of the treatments, the mice in each group have been killed with
CO2, 6 or 2h after the last AS602868 or CPT-11 administration
respectively. Tumours were removed, minced, put into liquid
nitrogen or RNA later (Ambion, Huntingdon, UK), and stored at
?801C.
Statistical analysis
Statistical significance of in vivo drug treatment effectiveness on
tumour growth was calculated using ANOVA and the protective
least significant difference using Fisher test. A probability of less
than 0.05 was considered as significant. Additive or synergistic
effect of drug combinations in vitro was evaluated using a non-
constant ratio isobologram analysis with the CompuSyn software
(ComboSyn Inc., New York, NY, USA). The combination index
values were interpreted as follows: o1.0, synergism; 1.0, additive;
and 41.0 antagonism.
Cytotoxicity assay
Cytotoxic studies were carried out using an MTT assay (van de
Loosdrecht et al, 1994), representing the percentage of growth
inhibition induced by treatments. One thousand HT-29 cells were
plated per well in 96-well plates with medium and various
concentrations of AS602868±SN-38 for 5 days.
EMSA and gel mobility shift assays
Nucleic extracts of HT-29 cells and tumours were prepared
according to the method described by Dignam et al (1983). Briefly,
5?106cells were trypsinized, washed in PBS, and pelleted.
Tumours were crushed in 500ml PBS and pelleted (1000g, 5min,
41C). Cell pellets or tumours were then resuspended in 50–100ml
of hypotonic buffer A. They were incubated for 10min on ice,
vortexedand centrifuged(10000g,
supernatants (cytosolic extracts) were collected, cell pellets and
tumours were suspended in 40–70ml of buffer B and centrifuged
(13000g, 10min, 41C). Supernatants (nuclear extracts) were
collected and diluted in 50–80ml of buffer C. EMSA were
performed as described previously. For supershift assays, anti-
bodies against p65 or p50 or rabbit IgG (4mg) were added 10min
before the labelled probe.
2min, 41C).Tumour
Apoptosis and cell proliferation assays
Apoptosis was measured after a 5-day stimulation of HT-29 cells,
plated as described for cytotoxicity assay using the cell death
detection ELISAplusKit (Roche Diagnostics, Meylan, France). Cell
proliferation was measured using the ELISA BrdU Kit from
Roche Diagnostics. Assays were performed in triplicate following
manufacturer’s instructions.
Western blot analysis
Total HT-29 cell extracts were prepared in lysis buffer as described
previously (Frelin et al, 2003), incubated for 30min on ice and
centrifuged (10000g, 10min, 41C). HT-29 cell or tumour extracts
were separated by sodium dodecyl sulphate-polyacrylamide gel
electrophoresis on polyacrylamide gels and blotted on immobilon
membranes (Millipore, Bedford, MA, USA). Primary antibodies
were revealed with secondary peroxidase-conjugated antibody
Tumour cell chemosensitization by IKK2 inhibition
P Lagadec et al
336
British Journal of Cancer (2008) 98(2), 335–344
& 2008 Cancer Research UK
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(DakoCytomation) followed by enhanced chemiluminescence
detection (Amersham Pharmacia, Saclay, France).
Reverse transcriptase-polymerase chain reaction
Total RNA from HT-29 cells or tumours was prepared in 2–4ml of
Trizol reagent (Invitrogen, Amsterdam, The Netherlands) accord-
ing to Chomczynski and Sacchi (1987). A total of 1mg RNA was
reverse transcripted using SuperScript II reverse transcriptase
(Invitrogen) following manufacturer’s instructions and resus-
pended in 12ml final volume. Two microlitre of the reverse-
transcribed material were amplified by polymerase chain reaction
(PCR) in 20ml reactions containing 0.5ml sense and antisense
primers (Eurogentec, Angers, France); 0.6ml dNTP (20mM); 2ml of
Taq polymerase (New England Biolabs, Saint Quentin, France) at
5000Uml?1of commercial buffer for a total of 30 cycles consisting
of 941C for 60s, 551C for 60s, and 701C for 60s. Ten microlitre
amplification products were analysed by electrophoresis in
ethidium bromide-stained agarose gels. Primer sequences are
available upon request.
Flow cytometric analyses
Cell cycle analysis was performed by quantifying DNA content
using propidium iodide staining and analyzing by flow cytometry,
as described previously (Vindelov et al, 1983).
0
25
50
75
100
Growth inhibition (% of control)
SW-480 cellsSW-620 cells
41
76.5
24
72.5
20.5
31
10 0 10 10
0 3 3 0 3 3
0 10
+SN-38 (nM)
0
25
50
75
100
Growth inhibition (% of control)
0.3 1
AS602868 (?M)
3 10
3 10 30 100
SN-38 (nM)
3 0
0 3
3 0
3
3
10 10 +SN-38 (nM)
0
53
3
16
11
42
86
90
14
6
28
38
79
HT-29 cells
0
25
50
75
100
Growth inhibition (% of control)
3 0 3 3 0 3 3 0 3
0 1 1 0 1 1 0
Etoposide (?M)5-FU (?M)
1 1 +
HT-29 cells
Oxaliplatin (nM)
17
22.6
50.7
13.7
16.3
47.5
13.3
51.2
49.5
∗
∗
∗
∗
∗
AS602868 (?M)
AS602868 (?M)
AS602868 (?M)
Figure 1
AS602868, SN-38, or both compounds simultaneously. (C) HT-29 cells were incubated for 5 days with 5-FU, etoposide, or oxaliplatin±AS602868 for 5
days. Cytotoxicity was evaluated using the MTT assay. Data are expressed as mean±s.d. of quadruplicates of one representative experiment out of 8 (A), 3
(B), and 3 (C). * indicates detection of the synergistic effect of AS602868 and SN-38, 5-FU, etoposide, or oxaliplatin on cell viability by using the non-
constant ratio isobologram method.
In vitro effect of AS602868 combined with SN-38 on cell viability. (A, B) HT-29 cells, SW-480, and SW-620 cells were incubated for 5 days with
Tumour cell chemosensitization by IKK2 inhibition
P Lagadec et al
337
British Journal of Cancer (2008) 98(2), 335–344
& 2008 Cancer Research UK
Translational Therapeutics

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TUNEL analyses
Frozen tumour sections (7mm) were rehydrated in PBS, fixed for
20min at room temperature using 3.7% formaldehyde and then
permeabilized for 2min in 0.1% Triton X-100 in 0.1% sodium
citrate solution at 41C. They were mounted in Fluoromount-G
solution (Southern Biotechnology Associates Inc., Birmingham,
AL, USA) and processed following the protocol described in the In
Situ Cell Death Detection Kit (Roche Diagnostics). Analyses were
performed using an LSM 510 confocal laser-scanning microscope
(Carl Zeiss AG, Jena, Germany).
Histology
Tumour sections (3mM) were incubated with an anti-Ki-67 (clone
MIB-1) or anti-TNFa at room temperature for 30min. After washing
in PBS, a peroxydase-conjugated antibody was added for 30min at
room temperature and reaction developed using an AEC Kit
(DakoCytomation). After haematoxylin counterstaining, slides were
permanently mounted in an aqueous medium (Aquatex, Merck,
Darmstadt, Germany) and analysed for the presence and the
distribution of the immunostaining. For morphological studies,
sections were stained with haematoxylin/eosin/safran (HES).
RESULTS
Inhibition of HT-29 cell viability in vitro by AS602868 in
combination with SN-38
After 5 days incubation, increasing concentrations of AS602868 or
SN-38 resulted in a decrease in HT-29 cell viability (Figure 1A) in a
dose-dependent manner, with a maximal effect for 10mM AS602868
and for 100nM SN-38 (53 and 90% inhibition, respectively). As 3
and 10ngml?1SN-38 are sub-lethal doses for HT-29 cells, these
concentrations have been chosen in combined experiments. In the
presence of both AS602868 (3mM) and SN-38, an additive (SN-38,
3nM) or synergistic (SN-38, 10nM) cytotoxic effect could be
observed: 28 and 79% of cytotoxicity respectively. The IC50for SN-
38 on HT-29 cells estimated at B25nM decreased to B10nM in the
presence of 3mM AS602868. A different sequencing order of the two
drugs (AS602868 added 24h before or after SN-38) had compar-
able effects on HT-29 cell viability to the simultaneous treatment
(not shown). The potentiating effect of AS602868 on SN-38-
mediated cytotoxicity was also observed on SW-480 and SW-620
tumour cells (Figure 1B). A synergistic cytotoxic effect was also
observed when HT-29 cells were incubated with AS602868 plus
5-fluorouracil (5-FU) or etoposide but not in the presence of
oxaliplatin (Figure 1C).
Dose-dependent potentiation of CPT-11 antitumour
activity by NF-jB inhibition with AS602868 in xenograft
experiments
Mice (5/group) were inoculated s.c. with HT-29 human colon
tumour cells. Treatments started when the mean tumour volume
was150±44mm3. Clinically
AS602868 (5 or 20mgkg?1) was administered either alone, or in
combination with CPT-11 (10 or 30mgkg?1) (Figure 2). No signs
of visible toxicity (diarrhea, weight lost, apathy, hair or skin
problems, etc.) were observed with any of the treatments. After 6
weeks, no significant differences in tumour size were observed
between control mice and mice treated with 20 or 5mgkg?1
AS602868 (Figure 2A–D respectively). These mice had to be killed
for ethical reasons. After 10 weeks, CPT-11 (30mgkg?1) strongly
achievableconcentrationsof
CPT-11 (10 mg kg–1)
AS602868 (20 mg kg–1)
AS602868 (5 mg kg–1)
0
1000
2000
3000
4000
5000
6000
01234
Time (weeks)
56789 10 11
Tumour volume (mm3)
Tumour volume (mm3)
Tumour volume (mm3)
Tumour volume (mm3)
NS
CPT-11 (30 mg kg–1)
AS + CPT-11
CPT-11
c
AS602868
AB
CD
0
1000
2000
3000
4000
5000
6000
01234
Time (weeks)
5678910 11
NS
CPT-11 (10 mg kg–1)
c
AS602868
CPT-11
AS + CPT-11
0
1000
2000
3000
4000
5000
6000
01234
Time (weeks)
5678910 11
NS
CPT-11 (30 mg kg–1)
AS + CPT-11
c
AS602868
CPT-11
0
1000
2000
3000
4000
5000
6000
01234
Time (weeks)
56789 10 11
NS
NS
c
AS602868
CPT-11
AS + CPT-11
P<0.0053
P<0.0386
P<0.0083
Figure 2
Nude mice received daily oral injections of AS602868, 5 days a week (
(
). Data are the mean±s.d. of tumour measurements using 5 mice/group and are representative of three other experiments. Statistically significant
differences between control and AS602868-treated groups on the 6th week and between CPT-11 and CPT-11þAS602868-treated groups on the 10th
week are indicated on each figure. NS, not significant.
In vivo effect of AS602868 combined with CPT-11 on the development of s.c. HT-29 xenografts. (A–D) Evolution of HT-29 tumour volume.
), and ()/or ( ) CPT-11 i.p. injections twice a week, or vehicle buffer
Tumour cell chemosensitization by IKK2 inhibition
P Lagadec et al
338
British Journal of Cancer (2008) 98(2), 335–344
& 2008 Cancer Research UK
Translational Therapeutics

Page 5

delayed tumour development (Po0.0001) (Figure 2A and C) and
appeared 50% less efficient (Po0.0051) when administrated at
10mgkg?1(Figure 2B and D). Addition of AS602868 20mgkg?1
(Figure 2A) or 5mgkg?1(Figure 2C) significantly potentiated the
effect of 30mgkg?1CPT-11 (Po0.0053 and 0.0386 respectively).
When CPT-11 was injected at 10mgkg?1, AS602868 significantly
improved (Figure 2B) CPT-11 antitumour effect at 20mgkg?1
(Po0.0083) but not at 5mgkg?1(Figure 2D). The combination of
CPT-11 (10mgkg?1) plus AS602868 (20mgkg?1) was as efficient
as 30mgkg?1CPT-11. Potentiation of CPT-11 antitumour activity
by AS602868 was also observed in two other colon xenograft
models using SW-480 and SW-620 cell lines (not shown).
Inhibition of CPT-11/SN-38-induced NF-jB pathway
activation by AS602868 in vitro and in vivo
Western blotting experiments (Figure 3A) performed on HT-29
cells (left panel) or tumours (right panel) showed that IkB-a
phosphorylation was increased (upper row) while total levels were
reduced (intermediate row) upon CPT-11 stimulation (lanes 3 and
30compared, respectively, to lanes 1 and 10). IkB-a phosphoryla-
tion was reduced and total IkB-a enhanced when AS602868 was
added (lanes 4 and 40compared, respectively, to lines 3 and 30). As
a control, we checked that no changes in Hsp60 levels were
observed (lower row). Subsequent NF-kB DNA-binding activity
was then studied.
As shown by EMSA (Figure 3B), 1h in vitro stimulation of HT-
29 cells with SN-38 (3 and 10nM) induced NF-kB activation in a
dose-dependent manner (lanes 4 and 6 compared to lane 2). This
was dramatically decreased after incubation with AS602868 (3mM)
(lanes 4 and 6 compared, respectively, to lanes 5 and 7). AS602868
also inhibited the weak constitutive activity of NF-kB observed in
HT-29 cells (lane 3 compared to 2). SN38 had less of an affect on
NF-kB activation compared to PMA (lane 6 vs 1). Similar results
were observed in tumours (Figure 3C, left panel). The specificity of
the NF-kB DNA-binding activity was demonstrated by competitive
inhibition in the presence of a 100ng excess of unlabelled
probe (not shown). When EMSAs were performed in the presence
PMA0
0
3
0
0
3
3
3
03
(10 ng ml–1)1010
NF-?B
NF-?B
HT-29 cells
Rabitt IgG p50 p65
0
0
200 20
30
0 20
30 30
0 20
30 30
0 20
30 300 30
HT-29 tumours (EMSA) HT-29 tumours (supershift)
HT-29 cellsHT-29 tumours
0
0
3
0
030
0
20
0
0 20
30 10 1030
pI?B-?
I?B-?
HSP60
37 kDa
37 kDa
60 kDa
1 2 3 4 5 6 7
1 2 3 4 1′ 2′ 3′ 4′
1′ 2′ 3′ 4′
CPT-11 (mg kg–1)
AS602868 (mg kg–1)
CPT-11 (mg kg–1)
AS602868 (mg kg–1)
AS602868 (?M)
SN-38 (nM)
AS602868 (?M)
SN-38 (nM)
Figure 3
western blotting either on lysates of HT-29 cells that were stimulated 30min with indicated concentrations of AS602868 and SN-38 or in tumours from
mice treated as indicated. HSP60 was used as loading control. (B–C) NF-kB activation was visualized by EMSA. HT-29 cells were treated with indicated
concentrations of AS602868, 30min before stimulation with SN-38 (3 and 10nM) for 1h or with PMA (10ngml?1) for 1h as positive control. These results
correspond to one representative experiment from 3. In supershift experiments, nuclear protein extracts of tumours from CPT-11 and AS602868±CPT-
11-treated mice were incubated with anti-p50 and anti-p65 antibodies or rabbit IgG as negative control.
In vitro and in vivo effect of AS602868 combined with CPT-11/SN-38 on the NF-kB pathway. (A) IkB-a phosphorylation levels were studied by
Tumour cell chemosensitization by IKK2 inhibition
P Lagadec et al
339
British Journal of Cancer (2008) 98(2), 335–344
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of anti-p52, c-Rel, Rel-B, p50, and p65 antibodies in tumour
extracts from mice treated with CPT-11 or CPT-11 plus AS602868,
supershifts were obtained only in the presence of anti-p50 and
anti-p65 antibodies (Figure 3C, right panel). Therefore, CPT-11
appears to mobilize classical p50–p65 NF-kB complexes.
Induction and potentiation of SN-38-mediated apoptosis of
HT-29 colon tumour cells by AS602868
Quantification by ELISA (Figure 4A) of mono- and oligonucleo-
somes showed that AS602868 (3mM) or SN-38 (3 and 10nM) alone
induced HT-29 cell apoptosis. A higher effect was produced by
combining the two drugs: 3mM AS602868 (two-fold medium OD), 3
and 10nM SN-38 (1.5 and 2-fold medium OD, respectively),
compared to (three-fold medium OD) obtained with 3mM
AS602868þ3nM SN-38 and (3.2-fold) with 3mM AS602868þ10nM
SN-38. Similar results were obtained in vivo (Figure 4B). TUNEL
experiments revealed nearly no apotosis in tumours from mice of
the control group, and an increasing level in tumours from mice
treated with AS602868, CPT-11, and with the combined treatment.
Figure 4C indicates that the cytotoxic effect of AS602868 and
SN-38 is only partly caspase-dependent since z-VADfmk(a pan
caspase inhibitor) could only prevent the decrease in viability by
40% on average. Thus, the induction of apoptosis cannot
completely explain AS602868 and SN-38 cytotoxic effect.
Induction and potentiation of SN-38-mediated pro-caspase
cleavage in HT-29 cells and tumours by AS602868
In HT-29 cells, AS602868 induced a dose-dependent cleavage of
pro-caspases 3 and 8, had a slight effect on pro-caspase 9, and no
effect on the caspase substrate Parp-a (Figure 5A: lanes 2 and 3).
SN-38 (10nM) induced cleavage of Parp-a and pro-caspase 9 and
had a weak effect on pro-caspase 8 (lanes 4 and 5). Combining
AS602868 with SN-38 resulted in a higher proteolysis of pro-
capases 3, 8, and 9 and of Parp-a (lane 6). Overall, results were
comparable to those found in tumour extracts (lanes 10–40), but
for unknown reasons it has not been possible to detect Parp-a in
tumour extracts, whatever the protocol used. Thus, in vivo and in
vitro inhibition of NF-kB activity using AS602868 allowed the
potentiation of the processing of pro-caspases 3, 8, and 9.
AS602868 inhibits expression of NF-jB anti-apoptotic
target genes in vitro and in vivo
In HT-29 cells, AS602868 decreased the expression of Bcl-xl,
c-IAP1, and survivin but not that of c-IAP2 (Figure 5B: lane 2
compared to lane 1). SN-38 had nearly no effect on the expression
of theses genes except a slight increase in survivin gene expression
(lane 3). However, combining AS602868 with SN-38 further
decreased Bcl-xl expression and to a lesser extent that of c-IAP1
and survivin (lane 3 compared to lane 4). In tumours (Figure 5B:
lanes 10–40), each compound alone had minor effect on expression
of these genes except a strong increase in Bcl-xl expression was
observed after CPT-11 treatment. The combination therapy
dramatically decreased CPT-11-induced Bcl-xl expression, below
baseline level. The levels of c-IAP1 and 2 and survivin were also
decreased (lane 40).
Inhibition of necrosis, tumour cell proliferation, and cell
cycle progression in HT-29 cells and tumours by AS602868
Histological examination of HT-29 xenografts (Figure 6A) revealed
an extensive necrosis in tumours (HES staining) from control and
AS602868 groups that decreased by two-fold in tumours from
the CPT-11 group and by four-fold in tumours from the
AS602868þCPT-11 group. Ki-67 staining revealed a two-fold
decrease in tumour cell proliferation in the AS602868þCPT-11
0
0.1
0.2
0.3
0.4
OD (490 nm)
0
20
40
60
80
100
Growth inhibition (% of control)
– z-VAD
+ z-VAD (50 ?M)
AS602868
(3 ?M)
AS602868
(1 ?M)
ControlAS602868
CPT-11
AS602868
CPT-11
+
Control0
0
3
0
0
3
03
3
3
1
0
0
3
01
3 10
1
10
3
0
0
3
0
10
3
3
3
10
1010
AS602868 (?M)
SN-38 (nM)
AS602868 (?M)
SN-38 (nM)
Figure 4
incubated for 5 days with AS602868±SN-38 at indicated concentrations. Data are expressed as mean OD±s.d. of duplicates of one representative
experiment from 4. The positive control is a DNA–histone complex included in the kit. (B) Tumours were removed from mice treated with AS602868
vehicle (control group), with AS602868 (20mgkg?1) alone or combined with CPT-11 (30mgkg?1). Then, they were minced, put into liquid nitrogen, and
stored at ?801C. Frozen tumour sections (7mm) were mounted in Fluoromount-G solution and processed following the protocol described in the In situ
Cell Death Detection Kit (Roche Diagnostics). Analyses were performed using an LSM 510 confocal laser-scanning microscope with an oil objective ?40.
(C) HT-29 cells were treated with AS602868±SN-38±z-VADfmk(50mM) for 5 days. Cell viability was evaluated using the MTT assay. Data are expressed
as means±s.d. of quadruplicates of one representative experiment from 3.
In vitro and in vivo effect of AS602868 combined with CPT-11/SN-38 on apoptosis. (A) Apoptosis was measured by ELISA. HT-29 cells were
Tumour cell chemosensitization by IKK2 inhibition
P Lagadec et al
340
British Journal of Cancer (2008) 98(2), 335–344
& 2008 Cancer Research UK
Translational Therapeutics

Page 7

group compared to the other groups. BrdU incorporation
(Figure 6B) showed that the combination of suboptimal doses of
AS602868 (1 and 3mM) and SN-38 (3 and 10nM) had additive effect
to inhibit cell proliferation with a maximum of 85% inhibition with
3mM AS602868 plus 10nM SN-38.
Cell cycle analysis (Figure 6C) revealed that AS602868 or SN-38
alone induced a slight increase in the number of cells in S phase
(6.8 and 8.4, respectively, compared to 4.6%). SN-38 also strongly
blocked HT-29 cells in G2/M (37.6 compared to 12.9%).
Combining AS602868 (3mM) with SN-38 (10nM) resulted in a
synergistic blockade in S phase (29.7%) and an additive blockade
in G2/M phase (46.9%). The number of cells in S phase was
increased by 6.5-fold compared with 1.5- or 1.8-fold with AS602868
or SN-38 alone, respectively.
Increase of TNFa concentration by CPT-11 in HT-29
tumours and induction of TNFa apoptosis potential in
HT-29 cells by AS602868
Histological examination of HT-29 xenografts (Figure 7A) revealed
the presence of low concentrations of TNFa in tumours from
control mice and AS602868-treated groups. The CPT-11 treatment
induced a three-fold increase in intratumoural TNFa concentra-
tion, which was decreased to two-fold upon co-treatment with
AS602868.
NF-kB is known to inhibit apoptosis induced by TNFa. Alone,
TNFa (10ngml?1) induced a minimal (5%) decrease in viability of
HT-29 cells (Figure 7B). However, addition of AS602868 at 3mM
(18% cytotoxicity) had an additive effect (30.8%) while at 10mM
(40% toxicity) it produced a synergistic effect with TNFa (65%).
A comparable synergistic effect was observed when 10nM SN-38
was added to 10ngml?1
TNFa (31.7 compared to 51.7%
cytotoxicity) or in the presence of the three compounds (83% of
cytotoxicity). TNFa induced a small decrease in Parp-a and
pro-caspase 3 but not in HSP60 levels (Figure 7C, lane 3).
AS602868, which had no effect by itself (lane 2), increased the
cleavage of Parp-a and pro-caspase 3 by TNFa (lane 4 vs lane 3). As
showed by EMSA (Figure 7D), AS602868 inhibited TNFa-induced
NF-kB activation in a dose-dependent manner (lanes 5, 6
compared to 4).
DISCUSSION
NF-kB activation by antineoplastic drugs is one of the mechanisms
for tumour resistance to chemotherapy (Baldwin, 2001; Nakanishi
and Toi, 2005). In the present study, we show that pharmacological
inhibition of the NF-kB pathway by the IKK2 inhibitor AS602868
potentiates the antitumoural effect of CPT-11 in vivo and that of
its active metabolite SN-38 in vitro. Interestingly, in xenograft
experiments, the combined treatment allowed a three-fold decrease
in CPT-11 concentration without any loss in efficiency. Inhibition
of NF-kB was also observed to reveal an apoptotic action of TNFa
on HT-29 cells, whose intratumoural concentrations were in-
creased upon CPT-11 treatment. Furthermore, in agreement with
previous data (Wang et al, 2003; Voboril et al, 2004), inhibition of
NF-kB also augmented sensitivity of HT-29 tumour cells to 5-FU,
the most common antimetabolite used for the treatment of CRC
and other types of solid tumours.
In haematopoietic malignancies, inhibition of abnormal con-
stitutive NF-kB activity frequently results in the death of leukaemic
cells (Frelin et al, 2005; Garcia et al, 2005). In solid tumours like
CRC, however, a combined therapy with antineoplastic drugs
appears necessary to get similar results. Inhibition of NF-kB
through the intratumoural adenoviral delivery of a super repressor
form of IkBa, in combination with CPT-11, led to a considerable
growth suppression of Lovo colon tumours associated with an
enhanced apoptotic response (Wang et al, 1999). In the same
tumour model, i.v. administration of the proteasome inhibitor
PS-341 prior to CPT-11 inhibited NF-kB activation, resulting in a
marked decrease in tumour size (Cusack et al, 2001). The level of
apoptosis reached 80–90% in the group receiving combined
treatments compared with 10% in tumours treated with single
agents. Recently, the new proteasome inhibitor NPI-0052 has also
been demonstrated to significantly improve the tumouricidal
response of chemotherapy when orally administered in Lovo
xenograft-bearing mice, by increasing apoptosis and shifting cells
towards G2 cell cycle arrest (Cusack et al, 2006). NPI-0052 effects
resulted in a 1.8-fold increase in response to CPT-11, 5-FU, and
leucovorin triple drug combination; a 1.5-fold increase in response
to the oxaliplatin, 5-FU, and leucovorin triple drug combination;
and a 2.3-fold greater response to the CPT-11, 5-FU, leucovorin,
and Avastin regimen. Reduction of endogenous p65 by siRNA
treatment in HCT-116 colon cancer cells significantly impaired
CPT-11-mediated NF-kB activation, enhanced apoptosis, de-
creased colony formation in soft agar and when administered in
vivo, reduced HCT-116 tumour formation in the presence but not
in the absence of CPT-11 (Guo et al, 2004). Our results appear
consistent with these studies and show that the inhibition of NF-kB
AS602868 (?M)
SN-38 (nM)
AS602868 (?M)
SN-38 (nM)
03 10 03 100200 20
AS602868 (mg kg–1)
CPT-11 (mg kg–1)
000 10 10 1000 30
30
CPT-11 (mg kg–1)
HT-29 cellsHT-29 tumours
Parp-?
Pro-casp. 3
Pro-casp. 8
Pro-casp. 9
HSP60
1 2 3 4 5 6
1′ 2′ 3′ 4′
32 kDa
55 kDa
45 kDa
60 kDa
116 kDa
89 kDa
0 3 0 3 020 020AS602868 (mg kg–1)
0 0 10 10 0 0 30 30
Bcl-xl
c-IAP1
HT-29 cellsHT-29 tumours
Bcl-xl
c-IAP1
c-IAP2
Survivin
Actin
c-IAP2
Survivin
Actin
Figure 5
caspase cleavage and anti-apoptotic gene expression in HT-29 cells and
tumours. (A) Cleavage of Parp-a and pro-caspases was demonstrated by
western blotting either on lysates of HT-29 cells incubated for 72h with
indicated concentrations of AS602868±SN-38 or in cytoplasmic protein
extracts of HT-29 tumours from mice treated as indicated. HSP60 was
used as loading control. (B) Anti-apoptotic gene expression was studied by
RT-PCR analysis on RNA extracted either from HT-29 cells stimulated for
72h with indicated concentrations of AS602868±SN-38 or from HT-29
tumours. Experiments were performed on 1mg RNA and amplification of
cDNA was of 30 cycles. Actin was used as an invariant control.
Effect of AS602868 combined with CPT-11/SN-38 on pro-
Tumour cell chemosensitization by IKK2 inhibition
P Lagadec et al
341
British Journal of Cancer (2008) 98(2), 335–344
& 2008 Cancer Research UK
Translational Therapeutics

Page 8

by AS602868, easy to use, can potentiate chemotherapeutic drug
efficiency in CRC.
The potentiating effect of AS602868 on HT-29 tumours
appears to be partly due to enhanced CPT-11-induced apoptosis.
AS602868 alone had moderate effects on apoptosis by itself but
activation of caspases 3, 8, and 9 could be easily detected when
the inhibitor was combined with CPT-11 or SN-38. Thus, the two
cellular apoptotic pathways appeared mobilized by combined
treatments. A decrease in the transcription of several anti-
apoptotic NF-kB target genes such as Bcl-xl, survivin, and to a
lesser extent c-IAP1 and c-IAP2 was observed which could reflect
an overall decrease in survival influences preceding caspase
activation. AS602868 has been previously shown to induce
apoptosis of human acute myeloid leukaemia cells (Frelin et al,
2005), effects that were associated with disruption of the
mitochondrial potential and by activation of pro-caspases 9 and
3. It has been demonstrated that p65 siRNA enhanced CPT-11-
mediated apoptosis by increasing caspase 3 activity and lowering
c-IAP 1 and c-IAP 2 protein levels (Guo et al, 2004). Moreover,
antisense Bcl-xl downregulation in HCT-11 colon cancer cells
switched the response to topoisomerase 1 inhibition from
senescence to apoptosis, enhancing global cytotoxicity (Hayward
et al, 2003). These results suggest that NF-kB likely supports cell
survival by different means depending on cell types and/or
underlying oncogenic mechanisms.
The level of necrosis in HT-29 tumours was inversely propor-
tional to tumour size indicating that necrosis cannot account for
AS602868/CPT-11 effects. On the other hand, autophagy could be a
possible mechanism of action for AS602868 since it has recently
been shown that a direct cross-talk exists between autophagy and
NF-kB reviewed in Xiao (2007). The combined treatment also
reduced tumour cell proliferation. In agreement with previous data
(Ohwada et al, 1996; Xu and Villalona-Calero, 2002), we found
that SN-38 alone markedly inhibited HT-29 cell proliferation by
arresting cells mainly at the G2/M phase and, to a lesser extent, at
the S phase. AS602868 for its part only induced a modest arrest at
the S phase, but in combination with SN38 increased the number
of cells in both S and G2/M phases. Thus, the enhanced antitumour
effect of the combined therapies could also be explained by the
ability of AS602868 to increase the number of cells in S phase as
these cells are 100–1000 times more sensitive to CPT-11 (Li et al,
1972).
Invalidation of the genes coding for IKK2, NEMO, or RelA
resulted in early embryo death from TNF-dependent liver
apoptosis demonstrating the important anti-apoptotic functions
of NF-kB (Karin and Lin, 2002). Moreover, the expression of a
non-phosphorylatable form of the IkB-a subunit, which acts as a
super-repressor of NF-kB activation, increased apoptotic re-
sponses in various cell lines stimulated by TNFa (Van Antwerp
et al, 1998). As CPT-11 treatment resulted in an increase in TNFa
intratumoural concentration and as AS602868 revealed the
apoptotic potential of TNFa in HT-29 colon cancer cells as well
as in Jurkat leukaemic cells (Frelin et al, 2003), this mechanism
could also be involved in AS602868/CPT-11 antitumoural effect.
Furthermore, the suppressor protein p53 is mutated in HT-29 cells
(Goldberg et al, 1996; Gobert et al, 1999) and recent data showed
that p53 mutations may promote cancer progression by augment-
ing NF-kB activation in the context of chronic inflammation
(Weisz et al, 2007).
To our knowledge, none of the many NF-kB signaling inhibitors
described so far (Gilmore and Herscovitch, 2006) have been shown
in vivo to increase chemotherapy efficiency in CRC. Taken
Ki-67HES
Controls
AS602868
(20 mg kg–1)
CPT-11
(30 mg kg–1)
AS602868
+ CPT-11
0
25
50
75
100
Inhibition of proliferation (% of control)
6
8
30
64
80
10
8
40
64
85
AS602868 (?M)
SN-38 (nM)
0
10
3
1
3
0
10
1
10
3
0
0
33
33
10 10
0
0
25
50
75
100
% cell cycle distribution
AS602868
G2 phase
G1 phase
S phase
∗
71.4
4.6
12.9
75.2
6.810.1
38.6
8.4
37.6
17
29.7
46.85
0
3 ?M
3 ?M
+ 10 nM SN-38
10 nM
SN-38
AS602868
AS602868
(1 ?M)
AS602868
(3 ?M)
Figure 6
examination of HT-29 tumour xenografts after 6 (control and AS602868 groups) or 10 weeks of treatment (CPT-11 and CPT-11þAS60286 groups).
Tumour sections were stained with HES (necrosis) or anti-Ki-67 (proliferation staining). Magnification: ?320. (B) Quantification of AS602868±SN-38
effect on HT-29 cancer cell proliferation by ELISA based on BrdU incorporation in DNA. (C) Measure of AS602868±SN-38 effect on cell cycle progression
by flow cytometric analyses using propidium iodide staining. Cells were stimulated for 72h after adhesion. * indicates detection of the synergistic effect of
AS602868 and SN-38 by using the non-constant ratio isobologram method.
Effect of AS602868 combined with CPT-11 on necrosis, cell proliferation, and cell cycle progression in HT-29 cells and tumours. (A) Histological
Tumour cell chemosensitization by IKK2 inhibition
P Lagadec et al
342
British Journal of Cancer (2008) 98(2), 335–344
& 2008 Cancer Research UK
Translational Therapeutics

Page 9

together, our results provide a rationale for using the IKK2
inhibitor AS602868 combined with CPT-11 as a promising
therapeutic strategy for clinical testing in CPT-11 refractory CRC
and probably other solid tumours. Of course the toxicity/efficacy
ratio will be a crucial factor for the therapeutical use of AS602868
molecule. However, preliminary results warrant that clinical trials
will be performed.
ACKNOWLEDGEMENTS
We thank Drs Sandrine Marchetti, Pierre-Alain Vitte, and Arnaud
Jacquel for helpful discussions and Dr Samantha Sarno for
critical review of the manuscript. This work was supported by an
institutional funding from INSERM and Universite ´ de Nice Sophia
Antipolis and by a grant from Merck-Serono International SA.
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