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Tumor Biology |
-induced Mitogen-activated Protein Kinase and Nuclear Factor 
B Signaling Pathways1
Unité 6032, "Interactions entre Systèmes Protéiques et Différenciation dans la Cellule Tumorale," Centre National de la Recherche Scientifique, Faculté de Médecine, 13385 Marseille Cedex 5, France
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSAnalysis of IRS-1 Tyrosine...Flow Cytometric Analysis of...IL-8 AssayElectron MicroscopyStatistical MethodsRESULTSDISCUSSIONREFERENCES |
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/tumor necrosis factor-
(TNF) induced apoptosis.
Survival signaling initiated by IGF-I was not dependent on the
canonical survival pathway involving phosphatidylinositol
3'-kinase. In addition, neither pp70S6K nor protein kinase
C conveyed IGF-I antiapoptotic function. Inhibition of
mitogen-activated protein kinase (MAPK)/extracellular signal-regulated
kinase (ERK) with the MAPK/ERK kinase inhibitor PD098059 and MAPK/p38
with the specific inhibitor SB203580 partially reversed, in a
nonadditive manner, the IGF-I survival effect. Inhibition of nuclear
factor 
B (NF-B) activity by preventing degradation of the
inhibitor of NF-B (IB-) with BAY 11-7082 also blocked in part
the IGF-I antiapoptotic effect. However, the complete reversal of the
IGF-I effect was obtained only when NF-B and either MAPK/ERK or
MAPK/p38 were inhibited together. Because these pathways are also those
used by TNF to signal inflammation and survival, these data point to a
cross talk between IGF-I- and TNF-induced signaling. We further report
that TNF-induced IL-8 production was indeed strongly enhanced upon
IGF-I addition, and this effect was totally abrogated by both MAPK and
NF-B inhibitors. The IGF-I antiapoptotic function was
stimulus-dependent because Fas- and IFN/Fas-induced apoptosis was not
efficiently inhibited by IGF-I. This was correlated with the weak
ability of Fas ligation to enhance IL-8 production in the presence or
absence of IGF-I. These findings indicate that the antiapoptotic
function of IGF-I in HT29-D4 cells is based on the enhancement of the
survival pathways initiated by TNF, but not Fas, and mediated by
MAPK/p38, MAPK/ERK, and NF-B, which act in concert to suppress the
proapoptotic signals. In agreement with this model, we show that it was
possible to render HT29-D4 cells resistant to Fas-induced apoptosis
provided that IGF-I and TNF receptors were activated simultaneously. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSAnalysis of IRS-1 Tyrosine...Flow Cytometric Analysis of...IL-8 AssayElectron MicroscopyStatistical MethodsRESULTSDISCUSSIONREFERENCES |
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The best characterized death factors Fas ligand and
TNF4
bind to ubiquitously expressed members of the TNFR superfamily. Fas
ligand binds to Fas/CD95/APO-1 receptor, and TNF binds to two
receptors, p55 (TNFR1) and p75 (TNFR2), that do not share any homology
within their cytoplasmic domain. Both Fas and TNFR1 can activate
apoptotic signaling pathways through a similar mechanism, recruiting
directly or indirectly Fas-associated death domain protein and
pro-caspase 8 (1, 2, 3)
. However, both TNFR1 and TNFR2 also
associate with molecules that do not interact with Fas, especially the
TRAF family of adaptor proteins and receptor-interacting protein. These
molecules activate additional signaling pathways including NF-B and
the MAPK cascades (especially, JNK and p38), these latter being
involved in the stimulation of AP-1 activity (7, 8, 9)
. TNF
recruitment of both NF-B and AP-1 transcription factors is pivotal
to regulate many genes, especially those involved in expression of
inflammatory cytokines and cell survival (10, 11, 12, 13, 14)
. Thus,
TNF transmits one signal eliciting cell death and another that protects
against cell death, this latter being closely linked to the
proinflammatory signaling. In contrast, Fas-mediated signal appears to
be simpler and does not lead to direct and efficient NF-B and AP-1
activation. This may explain why Fas activation generally results in a
more efficient apoptotic response (15)
.
The phenomenon of resistance by tumor cells to death factor-induced apoptosis is of major concern in cancer therapy. It contributes in a great part to the limited effectiveness of naturally occurring as well as peptide/cytokine-driven antitumor immune response generally observed in cancer patients. Moreover, this resistance may also antagonize the efficiency of chemotherapeutic drugs because many of them induce apoptosis of tumor cells by activating death factor/receptor systems, particularly the Fas/Fas ligand system (16 , 17) .
Several growth factors have been identified as regulators of cell survival (18) , and among them IGF-I, IGF-II, and insulin have been reported to have a potent ability to protect a broad range of cells from a variety of proapoptotic challenge (19) . The biological functions of the IGFs and insulin are pleiotropic and mediated by specific membrane receptors designated IGF-IR and IR, respectively. These receptors are heterotetrameric proteins with a highly homologous intracellular tyrosine kinase domain. An earliest step in signal transduction by both IGF-IR and IR is the extensive tyrosine phosphorylation of IRS-1, which initiates several distinct signaling pathways such as PI-3'K and MAPK cascades. In addition, the actions of IGFs, but not of insulin, are regulated by interactions with IGF binding proteins that modulate the IGF bioavailability to cell surface IGF-IR (reviewed in Refs. 20 and 21 ).
The gastrointestinal tract is one of the most responsive target tissues for IGFs (22) , and several studies have shown that the IGF system contributes to homeostasis and functional integrity of the intestinal epithelium by regulating several basic cellular functions, such as proliferation and differentiation (23) . Moreover, alterations of the IGF-I signaling have been reported to be associated with colorectal carcinoma both in vivo and in vitro, suggesting a role for the IGF axis in the pathogenesis of this disease (24, 25, 26) . In this way, we have shown previously that two key processes, enterocyte-like differentiation (27 , 28) and cell migration (29) , were under the control of the IGF system in the human colon carcinoma cell line HT29-D4.
We report in this study that engagement of IGF-IR and IR induces a full
resistance against IFN/TNF-induced apoptosis in HT29-D4 cells. The
findings further indicate that the antiapoptotic function of IGF-I is
mediated via enhancement of the inflammatory/survival signaling
pathways generated by the TNF activation itself and involves activation
of MAPK/ERK and MAPK/p38 in combination with NF-B. This model of
resistance to cell death is further supported by the observation that
IGF-I, which is unable to prevent Fas- and IFN/Fas-induced apoptosis,
became able to prevent this apoptosis when TNF is simultaneously added.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSAnalysis of IRS-1 Tyrosine...Flow Cytometric Analysis of...IL-8 AssayElectron MicroscopyStatistical MethodsRESULTSDISCUSSIONREFERENCES |
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-IR3) was purchased
from Oncogene Science (Uniondale, NY), and the anti-phosphotyrosine mAb
(PY20) was from ICN Biomedical (Aurora, OH). The antihuman FAK Ab
(A-17) was from Santa Cruz Biotechnology (Santa Cruz, CA), and the mAb
raised against ß-catenin was from Transduction Laboratories
(Lexington, KY). Nitrocellulose sheets (Hybond-C extra), horseradish
peroxidase-coupled antimouse secondary Ab, and enhanced
chemiluminescence detection reagents were purchased from Amersham
(Aylesbury, United Kingdom).
Cell Culture and Induction of Apoptosis
The HT29-D4 human colon adenocarcinoma cell line was cultured
routinely in DMEM supplemented with 10% FCS as reported elsewhere
(27
, 28) . For each experiment, HT29-D4 cells were seeded
at a density of 2.5 x 105
cells/cm2 in six-well tissue culture dishes.
After 24 h, the cells were washed with HBSS and further incubated
in FCS-free DMEM containing 0.1% BSA (serum-free DMEM) for 24 h
at 37°C. After washing, cells were incubated with or without IFN (40
ng/ml) for 5 min at 37°C and then washed again, and apoptosis was
induced by adding TNF (4 ng/ml) or CH-11 anti-Fas Ab (500 ng/ml) in the
presence or absence of various concentrations of IGF family peptides.
Whenever used, metabolic and kinase inhibitors were added to cells at
the concentrations indicated under the Figure 6
, Figure 7
, Figure 8
, Figure 9
, 60 min prior
to the addition of des-(1, 2, 3)
-IGF-I. At the end of incubation,
nonadherent and adherent cells (recovered by 0.53 mM
EDTA/0.05% trypsin) were combined and assayed for apoptosis as
described below. All of the experiments were made in duplicate and
repeated at least three times.
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Flow Cytometric Determination of DNA Fragmentation.
To quantify cells with advanced DNA fragmentation, we used the
technique described by Nicoletti et al. (30)
.
Briefly, cells (1 x 106/ml) were
treated by an hypotonic fluorochrome solution containing 0.1% Triton
X-100, 0.1% sodium citrate, and 50 µg/ml PI. Flow cytometric
analysis was done on a FacSort (Becton Dickinson, San Jose, CA) for
quantifying the proportion of hypodiploid nuclei
(pre-G1 peak). Ten thousand events were examined
for each determination, and the data were analyzed using the Cell Quest
software package (Becton Dickinson).
Double Staining with FITC-conjugated Annexin V and PI.
This method was used to detect both apoptosis and necrosis from the
same cell samples (31)
. Cells (1 x 106/ml) were simultaneously stained with
FITC-annexin V and PI as recommended by the supplier (Immunotech) and
subjected to flow cytometric analyses to detect the percentage of
apoptotic (FITC-stained) and necrotic (PI-stained) cells in a given
population. A minimum of 10,000 cells was examined for each sample.
Immunoblotting for FAK and ß-Catenin Cleavage.
Cells were lysed by incubation in 50 mM HEPES (pH 7.5)
containing 100 mM NaCl, 1% Triton X-100, 1 mM
EDTA, 1 mM DTT, 1 mM phenylmethylsulfonyl
fluoride, and a mixture of protease inhibitors (aprotinin, leupeptin,
iodoacetamin, and pepstatin, 1.0 µg/ml each). Lysates were clarified
by centrifugation (12,000 x g for 3 min),
and equal amounts of proteins were subjected to SDS-PAGE on 7.5%
polyacrylamide slab gels, then electrophoretically transferred onto
nitrocellulose sheet for 1 h at 100 V, and analyzed by
immunoblotting with anti-FAK and anti-ß-catenin Abs. Immunoreactive
proteins were identified by horseradish peroxidase-conjugated secondary
antibody, followed by enhanced chemiluminescence reagents, with the
technique recommended by the manufacturer.
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Analysis of IRS-1 Tyrosine Phosphorylation |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSAnalysis of IRS-1 Tyrosine...Flow Cytometric Analysis of...IL-8 AssayElectron MicroscopyStatistical MethodsRESULTSDISCUSSIONREFERENCES |
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Flow Cytometric Analysis of Fas and TNFR |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSAnalysis of IRS-1 Tyrosine...Flow Cytometric Analysis of...IL-8 AssayElectron MicroscopyStatistical MethodsRESULTSDISCUSSIONREFERENCES |
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IL-8 Assay |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSAnalysis of IRS-1 Tyrosine...Flow Cytometric Analysis of...IL-8 AssayElectron MicroscopyStatistical MethodsRESULTSDISCUSSIONREFERENCES |
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Electron Microscopy |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSAnalysis of IRS-1 Tyrosine...Flow Cytometric Analysis of...IL-8 AssayElectron MicroscopyStatistical MethodsRESULTSDISCUSSIONREFERENCES |
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Statistical Methods |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSAnalysis of IRS-1 Tyrosine...Flow Cytometric Analysis of...IL-8 AssayElectron MicroscopyStatistical MethodsRESULTSDISCUSSIONREFERENCES |
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSAnalysis of IRS-1 Tyrosine...Flow Cytometric Analysis of...IL-8 AssayElectron MicroscopyStatistical MethodsRESULTSDISCUSSIONREFERENCES |
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indicates that HT29-D4 cells constitutively expressed Fas,
TNFR1 and TNFR2. However, as shown in Fig. 1B
, incubation of
cells with TNF alone (up to 50 ng/ml) did not induce a significant
increase in cell death above basal levels. In contrast, incubation of
cells with anti-Fas Ab (CH-11) induced a 38% cell death at 24 h.
Pretreatment of cells with IFN (40 ng/ml) resulted in induction of
sensitivity to killing by TNF (4 ng/ml) and enhancement of cell death
induced by anti-Fas Ab (500 ng/ml). Preliminary dose-response
relationship with IFN, TNF, and anti-Fas Ab revealed that these
concentrations were optimal for induction of cell death measured by
counting floating dead cells (not shown). Using these concentrations,
stimulation of TNFR and Fas resulted in 84 and 69% cell death,
respectively, at 24 h (Fig. 1B)
. It should also be
noted that a 5-min pulse of IFN treatment was sufficient for an optimal
sensitizing effect of cells to TNF- and anti-Fas Ab-mediated apoptosis.
Longer exposures to IFN did not significantly improve death
factor-mediated apoptosis but induced 
15% cell death after 24 h in the absence of any additional proapoptotic stimuli (not shown).
Fig. 1C
, left panel, indicates that TNFR1 Ab inhibited
IFN/TNF-induced apoptosis by 45%. This incomplete inhibition was in
fact attributable to the agonistic activity of this Ab (Fig. 1C
,
right panel). In contrast, addition of TNFR2 Ab, alone or in
combination with TNFR1 Ab, did not interfere with cell death (Fig. 1C
, both panels). Fig. 1D
shows a flow cytometric
analysis of PI-stained nuclei, which was used as an alternative method
to quantify apoptosis. About 60 and 38% of the cells contained <2N
DNA content (pre-G1 peak) after a 24-h treatment
with IFN/TNF and IFN/anti-Fas Ab, respectively.
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also shows that treatment of
cells with des-(1, 2, 3)
-IGF-I at 50 ng/ml induced a potent resistance
(>85%) to IFN/TNF-induced apoptosis, whatever the assay used. In
contrast, des-(1, 2, 3)
-IGF-I did not significantly inhibit apoptosis
driven by Fas cross-linking and provided only a poor protection
(<25%) against apoptosis induced by IFN/anti-Fas Ab. Des-(1, 2, 3)
-IGF-I
was used instead of IGF-I because this NH2
terminally truncated IGF-I analogue binds to IGF-IR with the same
affinity as native IGF-I but does not bind to any of the IGF binding
proteins secreted by the HT29-D4 cells (27)
.
We next sought to confirm that the IGF-I-mediated cytoprotective effect
was in apoptotic and not merely in necrotic cells. For this purpose, we
used a simultaneous labeling of cells with annexin V-FITC and PI after
a 8-h stimulation of the cells with IFN/TNF or IFN/anti-Fas Ab (Fig. 2A)
. Annexin V bound to phosphatidylserine that translocated
to the outer leaflet of the plasma membrane during the early phase of
apoptosis, whereas these apoptotic cells with intact cell membranes did
not stain with PI (Fig. 2A
, quadrant R2). This early stage
of apoptosis was rapidly followed in vitro by the loss of
membrane integrity and PI staining of the cells during a secondary
necrotic stage (Fig. 2A
, quadrant R1). Because of the
dynamic nature of this process, the percentage of trapped
annexin-positive, PI-negative apoptotic cells was lower than the ones
measured with assays based on cell death or DNA fragmentation,
i.e., 34 and 22% for IFN/TNF- and IFN/anti-Fas Ab-induced
apoptosis, respectively. Fig. 2A
also indicates that
addition of des-(1, 2, 3)
-IGF-I totally suppressed the appearance of cells
that were actively undergoing apoptosis (quadrant R2) when
IFN/TNF was the proapoptotic stimuli. In contrast, des-(1, 2, 3)
-IGF-I
could not do it for apoptotic cells driven by IFN/anti-Fas Ab (Fig. 2A)
. To confirm these results by another approach, we
evaluated the ability of des-(1, 2, 3)
-IGF-I to prevent the
caspase-dependent cleavage of FAK (32)
and ß-catenin
(33)
. Fig. 2B
shows that des-(1, 2, 3)
-IGF-I
totally inhibited these proteolytic cleavages when apoptosis was
induced by IFN/TNF but not IFN/anti-Fas Ab. Finally, it is important to
note that all of these early- and late-stage markers of apoptotic
events, including the unique morphological features of apoptotic cells
(Fig. 2C)
, were observed in matrix-detached cells only. Not
one of these markers was detected in the remaining adherent cells, even
after a 24-h apoptotic process.
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shows the dose-response curves examining the survival of
IFN-sensitized cells after an incubation with TNF or anti-Fas Ab
for 24 h in the presence of graded concentrations of
des-(1, 2, 3)
-IGF-I and insulin. Des-(1, 2, 3)
-IGF-I inhibited TNF-mediated
cell apoptosis with an IC50 at 2.5 ng/ml. The
survival effect elicited by insulin was slightly less potent with an
IC50 at 10 ng/ml. Quite similar rates of
protection were obtained when TNFR1 Ab was used as an agonistic Ab
instead of TNF to induce apoptosis (not shown). In contrast, apoptosis
induced by Fas cross-linking was minimally inhibited by
des-(1, 2, 3)
-IGF-I (25% at 100 ng/ml), whereas insulin did not
promote any significant survival effect. Des-(1, 2, 3, 4, 5, 6)
-IGF-II was as
potent as des-(1, 2, 3)
-IGF-I to prevent IFN/TNF-induced apoptosis,
whereas IGF-I and IGF-II were slightly less protective with an
IC50 at 5.0 and 10 ng/ml, respectively (not
shown). IGF-binding proteins secreted by HT29-D4 cells may explain this
slight lower potency of IGFs compared with their truncated counterparts
(27
, 28)
.
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-IR3 and B6 mAbs raised against IGF-IR and IR, respectively. We have
shown previously that these Abs inhibited the binding of
125I-labeled IGF-I and
125I-labeled insulin to their cognate receptors
(28)
. Fig. 3B
shows that neither -IR3 nor B6
Ab reversed the cell survival effect elicited by IGF-I and insulin. In
fact, -IR3 and B6 Abs acted as IGF-I- and insulin-mimetic Abs in
term of induction of survival; at a concentration of 10.0 µg/ml,
-IR3- and B6-mediated cell survival was as large as that seen with
des-(1, 2, 3)
-IGF-I and insulin at 10 and 50 ng/ml, respectively.
Fig. 4A
indicates that the protective effect of des-(1, 2, 3)
-IGF-I
was maximum when it was added to the cells prior to or within 2 h
of IFN/TNF addition. Addition of des-(1, 2, 3)
-IGF-I at later time points
offered less cell protection. However, the prevention of cell death was
yet highly effective (80% of the maximal protective effect) when
des-(1, 2, 3)
-IGF-I was added with a delay of 6 h after IFN/TNF
stimulation, a time that correlated with the beginning of cell-matrix
detachment. As shown on Fig. 4B
, it is interesting to note
that a 1-min pulse of des-(1, 2, 3)
-IGF-I treatment was quite sufficient
to induce a potent survival effect against IFN/TNF-mediated apoptosis.
Pulse treatment prior to or after IFN cell sensitization did not modify
the rate of IGF-I protection (not shown).
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180,000 identified as IRS-1 (Ref. 28
; Fig. 5A
). Preincubation of the cells for 1 h with TNF or
anti-Fas Ab alone did not induce any tyrosine phosphorylation of IRS-1
(Fig. 5B
, Lanes b and c). In addition, neither
TNF nor anti-Fas Ab significantly altered the extent of IGF-I-induced
IRS-1 tyrosine phosphorylation (Fig. 5B
, Lanes e and
f). However, preincubation with TNF caused a delay in the
electrophoretic mobility of IRS-1 (Fig. 5B
, Lane e),
presumably because of serine phosphorylation of IRS-1
(34)
. IGF-I also induced tyrosine phosphorylation of
proteins at Mr 97,000 and
Mr 102,000 identified as two isoforms
of IGF-IR ß-subunits in HT29-D4 cells (35)
. However,
neither the extent of phosphorylation nor the mobility of these bands
was altered by pretreatment of cells with TNF and anti-Fas Ab (not
shown).
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shows that BIM, a PKC inhibitor affecting all PKC isoforms,
inhibited by 50% IFN/TNF-induced apoptosis. In contrast, CPHC, a
specific inhibitor of diacylglycerol-dependent (conventional and novel)
PKC isoforms did not, suggesting the involvement of an atypical PKC.
Because we detected the atypical PKC 
/
in HT29-D4 cells
(29)
, we suggest that it is a likely candidate for
participating in the apoptotic pathway induced by IFN/TNF in these
cells. Both BIM and CPHC were, however, unable to reverse the IGF-I
protective effect on IFN/TNF-induced apoptosis (Fig. 6A)
,
thus indicating that activation of PKC is not necessary to convey the
IGF-I antiapoptotic signaling.
The PI3'K pathway has been widely reported to play a central role in
signal transduction by IR and IGF-IR (19, 20, 21)
. Moreover,
this kinase has been shown to sustain survival in many cell models via
activation of the downstream serine/threonine kinase PKB/AKT (37
, 38)
. Fig. 6A
shows that two specific PI3'K
inhibitors, LY294002, a synthetic inhibitor (39)
, and WMN,
a fungal protein, enhanced the degree of apoptosis induced by IFN/TNF.
However, when the rate of protection conferred by the addition of
des-(1, 2, 3)
-IGF-I in combination with LY294002 was calculated with
respect to the level of apoptosis induced by IFN/TNF plus
LY294002, it was not significantly different from the one measured in
the absence of the drug (68% versus 80%; Fig. 6A
). The use of WMN as a PI3'K inhibitor gave essentially
the same results. Finally, Fig. 6A
shows that rapamycin, a
specific inhibitor of pp70S6K (40)
,
a kinase lying downstream from PI3'K, did not interfere with the IGF-I
antiapoptotic effect. Thus, the PI3'K pathway does not appear to play a
key role in the protection conferred by IGF-I against IFN/TNF-induced
apoptosis in HT29-D4 cells.
The MAPK/ERK pathway mediates many of the known effects of IGF-I
(19, 20, 21)
. To block this pathway, we used PD098059, a
specific inhibitor of the activation of MAPK/ERK kinase by Raf-1, thus
suppressing ERK activation (41)
. Two other MAPK
subfamilies, the JNK and p38 kinases, have been also reported to be
involved in the regulation of TNF-induced apoptosis
(7, 8, 9, 10, 11)
. The specificity of the SB203580 inhibitor
(42)
allowed us to define the specific requirement for
MAPK/p38 as a mediator of IGF-I survival signaling. Fig. 6B
shows that PD098059 increased by 30%, whereas SB203580 decreased by
40%, the extent of apoptosis induced by IFN/TNF. Thus, MAPK/ERK and
MAPK/p38 pathways convey opposite signals on survival during IFN/TNF
exposure. PD098059 and SB203580 partially blocked the protective effect
of des-(1, 2, 3)
-IGF-I on IFN/TNF-induced apoptosis (55 and 50%,
respectively, versus. 80%; Fig. 6B
). These
results suggest that the MAPK/ERK and MAPK/p38 pathways convey in part
the IGF-I-induced antiapoptotic signaling. Reversal of the IGF-I
antiapoptotic effect was not further improved by incubation of the
cells with PD098059 and SB203580 together (Fig. 6B)
. This
latter observation suggests that common downstream survival targets are
used by these two MAPK subfamilies.
An essential role for NF-B in preventing cell death induced by TNF
has been reported in several cell models (10
, 12, 13, 14)
. To
determine the role of NF-B in the mediation of the IGF-I
antiapoptotic effect, we used BAY 11-7082, a drug inhibiting IB
phosphorylation (43)
, which prevents proteasome-mediated
IB degradation and in turn release of free NF-B. Fig. 6B
shows that treatment of the cells with BAY 11-7082
increased the extent of IFN/TNF-induced apoptosis by 40% but
also partially blocked the des-(1, 2, 3)
-IGF-I antiapoptotic effect (47%
versus 80%; Fig. 6B
). Thus, activation of the
NF-B pathway appears to be required for both TNF- and IGF-I-mediated
survival signaling.
Lastly, we determined whether MAPK- and NF-B-dependent antiapoptotic
signals may act synergistically to mediate the IGF-I antiapoptotic
function. Fig. 6B
shows that the simultaneous blocking of
these pathways by adding BAY 11-7082 plus PD098059 or SB203580 indeed
induced a total reversal of the IGF-I antiapoptotic effect. In
contrast, no additive effect was observed when the PI3'K inhibitor,
LY294002, was added with inhibitors of either NF-B or MAPK pathways
(not shown). These results indicate that the coordinate activation of
MAPK and NF-B pathways is required for IGF-I to confer a full
resistance against IFN/TNF-induced apoptosis in HT29-D4 cells.
Fig. 7
shows protein synthesis requirement for IGF-I to induce survival
signaling. As reported previously (36)
, addition of CHX
(10 µg/ml) allowed TNF to induce apoptosis to a death rate of 42%.
Surprisingly, the sensitizing effect of IFN on TNF-induced apoptosis
was not altered by CHX. In contrast, the antiapoptotic capacity of
des-(1, 2, 3)
-IGF-I was totally abrogated in the presence of CHX (Fig. 7)
.
Thus, new protein synthesis is a prerequisite for IGF-I to function as
an antiapoptotic factor in HT29-D4 cells.
IGF-I Enhances TNF-induced IL-8 Production.
TNF has been reported to stimulate in colon cancer cells the synthesis
of the proinflammatory chemokine IL-8 (44, 45, 46)
. IL-8
expression requires the recruitment, among others, of the transcription
factors NF-B and AP-1 (47)
. Because the above reported
results suggest that NF-B and MAPK/AP-1 pathways are essential to
convey the IGF-I-antiapoptotic effect, we next examined the ability of
des-(1, 2, 3)
-IGF-I to modulate IL-8 production. As previously reported
for the parental HT29 cells (44, 45, 46)
, HT29-D4 cells
produced low levels of IL-8 in the absence of added stimuli, and this
production was slightly up-regulated by the addition of
des-(1, 2, 3)
-IGF-I (Fig. 8)
. The basal production of IL-8 was markedly increased by the addition
of TNF, and TNF-induced IL-8 production was further strongly increased
(2.5-fold) upon addition of des-(1, 2, 3)
-IGF-I. The sensitization of
the cells with IFN also enhanced the TNF-induced IL-8 production, and
addition of des-(1, 2, 3)
-IGF-I further increased this synthesis by a same
2-fold factor (not shown). As shown on Fig. 8
, treatment with PD098059,
SB203580, and BAY 11-7082 completely abrogated basal (not shown),
TNF-induced, and TNF-induced/IGF-I-enhanced IL-8 production (Fig. 8)
.
In contrast, LY294002 had no appreciable effect on IL-8 production.
Thus, IGF-I is a potent stimulator of TNF-induced MAPK/ERK-, MAPK/p38-,
and NF-B-dependent IL-8 production in HT29-D4 cells.
The Simultaneous Triggering of IGF-IR- and TNFR1-dependent Survival
Pathways Induces Resistance against Fas-induced Apoptosis.
In view of the above reported results, we hypothesized that the poor
capacity of IGF-I to protect HT29-D4 cells against IFN/Fas- and
Fas-mediated apoptosis (Fig. 1)
could be attributable to an
insufficient triggering of the MAPK- and NF-B-dependent
inflammatory/survival pathways by Fas cross-linking. We therefore
investigated the ability of anti-Fas Ab to modulate, with or without
IGF-I, the production of IL-8, used as a marker of activation of
MAPK/AP-1 and NF-B pathways. Fig. 9A
shows that anti-Fas Ab did induce only a weak stimulation
of IL-8 production, and addition of des-(1, 2, 3)
-IGF-I raised this level
to 5% of that induced by the combination of TNF and IGF-I (compare
Figs. 9A
and 8
). This ratio remained identical when the
assays were performed with IFN-sensitized cells (not shown). In
addition, Fig. 9A
indicates that the signaling requirement
for Fas- and TNF-mediated IL-8 production was identical. Thus, an
insufficient level of activation of the MAPK and NF-B signaling
pathways may be the reason for the incapacity of IGF-I to protect
HT29-D4 cells from Fas-induced apoptosis. To further address this
issue, cells were exposed to anti-Fas Ab in combination with TNF and/or
des-(1, 2, 3)
-IGF-I. These experiments were done in the absence of IFN to
activate the TNF-dependent survival but not the TNF-dependent
proapoptotic signaling. Fig. 9B
shows that individually
addition of either TNF or des-(1, 2, 3)
-IGF-I to anti-Fas Ab-stimulated
cells did not alter the extent of apoptosis. However, the simultaneous
addition of TNF and des-(1, 2, 3)
-IGF-I together induced a potent
resistance against Fas-induced cell apoptosis. Taken together, these
data suggest that resistance to Fas-induced apoptosis requires the
synergistic activation of TNF- and IGF-I-dependent antiapoptotic
signals in HT29-D4 cells.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSAnalysis of IRS-1 Tyrosine...Flow Cytometric Analysis of...IL-8 AssayElectron MicroscopyStatistical MethodsRESULTSDISCUSSIONREFERENCES |
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B. This antiapoptotic mechanism
is further strengthened by the capacity to induce cell resistance
against Fas-mediated apoptosis provided that TNFR1- and
IGF-IR-dependent antiapoptotic signals are simultaneously triggered. Our experiments show that HT29-D4 cells, which contain a nonfunctional p53 protein (48) , express in a constitutive manner Fas, TNFR1 and TNFR2. However, preincubation of the cells with IFN was necessary to induce apoptosis in response to TNF, which correlates well with several reports done with the parental HT29 cells (36 , 44 , 49 , 50) . Although HT29-D4 cells express about 2-fold more TNFR2 than TNFR1, only the TNFR1 could mediate TNF-induced apoptosis, which agrees with most of the reports using cells expressing physiological TNFR2 levels (51) . In contrast, HT29-D4 cells were susceptible to anti-Fas Ab-induced apoptosis, which indicates that their intracellular death pathway is functional. However, IFN further increased the cell sensitivity to Fas-mediated apoptosis. Strikingly, a very short pulse of IFN treatment (<5 min) was sufficient to induce an optimal sensitization to TNFR1- and Fas-mediated apoptosis, and this effect was not abrogated by treatment with CHX. Thus, the IFN-induced proapoptotic sensitization, in contrast to other systems (36 , 49) , does not appear to regulate the expression of select genes in HT29-D4 cells. Such a yet unknown signaling pathway might use select signal transducer and activator of transcription factors exerting a function unrelated to gene expression, e.g., adaptor function, as reported recently (52) .
The antiapoptotic function of insulin and IGFs peptide family against
IFN/TNF-stimulated apoptosis was powerful and obtained for quite
physiological concentrations. It was observed whatever the early- and
late-stage apoptotic marker we used. These observations also correlate
well with the ability of IR-3 and B6 Abs, which interfere with the
binding to their cognate receptors of IGF-I and insulin, respectively,
to exert a potent agonist antiapoptotic effect. Therefore, we conclude
that endogenous IGF-IR (25,000/cell) and IR (5,000/cell; Ref.
35
) are effective for survival signaling in HT29-D4
colonic cancer cells. The IR-dependent antiapoptotic function is in
contrast to several studies (40)
, which report a survival
effect of insulin exclusively at high concentrations thought to trigger
the IGF-IR. It is, however, in agreement with recent reports
demonstrating that IR can protect cells from apoptosis in several
models (19
, 53)
. This finding may also be related to the
emerging concept suggesting a role for hyper-insulinemia in colon
cancer pathogenesis (54)
. The capacity of IGF-I to prevent
IFN/TNF-stimulated apoptosis was rapidly and irreversibly delivered
because a 1-min IGF-I pulse treatment was sufficient to induce an
optimal antiapoptotic effect. This correlates well with the delay
required for IGF-I to optimally induce tyrosine phosphorylation of
IRS-1, the most proximal substrate in the IGF-I signaling pathway
(20
, 21)
. TNF, at the concentrations here used to induce
apoptosis, had no significant effect on IGF-I-induced tyrosine
phosphorylation of IRS-1, but induced its serine phosphorylation.
Because serine phosphorylation of IRS-1 by TNF has been reported to
impair insulin and IGF-I biological actions (34)
, the
mechanism of protection by the IGF-IR, at this point, is obscure.
However, D’Ambrosio et al. (55)
have reported
that IGF-I was quite able to protect cells from apoptosis induced by
okadaic acid, a drug inducing serine phosphorylation of IRS-1, provided
the drug was used at a concentration that does not decrease the
IGF-I-induced IRS-1 tyrosine phosphorylation. Although the full
significance of these observations awaits additional experimentation,
we suggest that unaltered IGF-I-induced tyrosine phosphorylation of
IRS-1 by TNF allows IGF-I to exert a potent protective action in our
system, even in the face of a substantial TNF-induced serine
phosphorylation of IRS-1. Alternatively, protection by IGF-I may be
mediated via signaling pathways that are distinct from those requiring
IRS-1 because it was suggested by using IGF-IR and IR mutated in their
cytoplasmic domain (55
, 56)
.
Although the aim of this study was not to examine the intracellular
mechanisms by which IFN/TNF signals apoptosis in the absence of IGF-I,
we report that inhibition of atypical PKC, presumably the /
isoform (29)
, and MAPK/p38 markedly decreased
IFN/TNF-induced apoptosis. This suggests that these enzymes are
required for a successful apoptotic response. In contrast, inhibitors
of NF-B, MAPK/ERK, and PI3'K enhanced apoptosis induced by IFN/TNF,
thus suggesting that these signaling molecules are mediators of
endogenous protective signals. However, it was not possible to
determine whether these sets of molecules belong to IFN- or
TNF-dependent signaling pathways or both, because neither TNF nor IFN
stimulated apoptosis in the HT29-D4 system.
Taking into account the reported role for the PI3'K/PKB pathway for
signaling several IGF-I/insulin-induced biological responses (20
, 21)
and survival against a wide variety of stimuli (37
, 38 , 40
, 57, 58, 59)
, it was surprising to find that the PI3'K
inhibition failed to block the ability of IGF-I to protect cells from
IFN/TNF-induced apoptosis. These findings are, however, in agreement
with reports showing that PI3'K/PKB-independent survival signaling
pathways may also be used by IGF-I (56
, 60)
. It is
therefore likely that context-dependent parameters should influence the
nature of the pathway that conveys the IGF-I antiapoptotic message.
Furthermore, no PKC activity was involved in the IGF-I antiapoptotic
signaling. Because we recently reported that PI3'K, PKC 
and PKC
were involved in IGF-I-induced HT29-D4 cell migration
(29)
, the current data illustrate the complexity of the
signaling pathways used by IGF-I to exert a balanced control on a
variety of biological functions in the same cell.
The data reported in the present study show that ERK and p38 MAPK mediate in part the IGF-I-antiapoptotic effect. The absence of additive effect we obtained by simultaneously inhibiting MAPK/ERK together with MAPK/p38 suggests that these MAPK cascades converge at the level of common downstream survival effectors as reported previously (8, 9, 10, 11) . It is also likely that this observation accounts for the MAPK/JNK that the pharmacological approach here used does not permit us to discriminate. In agreement with our observations, IGF-I has been reported recently to interfere with p38 and JNK MAPK (61 , 62) . The role of MAPK cascades in the regulation of apoptosis is, however, full of contradictions. Depending on the cell type, its state of activation, and the context, the activities of these kinases are seen as a cause of apoptosis, a consequence of stress, or a survival force (7, 8, 9, 10, 11) . Such a versatility was also observed in HT29-D4 cells, where the MAPK/p38 appears to be able to convey both IFN/TNF-induced proapoptotic stimuli and the IGF-I-mediated antiapoptotic signal.
Several reports (12, 13, 14)
have shown that activation of
NF-B protects numerous tumor and embryonic cells from death induced
by TNF and various stimuli including Fas ligation, ionizing radiation,
and chemotherapeutic drugs. In HT29-D4 cells, our results indicate that
blocking NF-B activation by inhibiting IB phosphorylation
prevented a substantial part of the protection afforded by IGF-I. Thus,
induction by IGF-I of NF-B-driven protective programs appears to
also be a way for the colonic cancer cells to acquire resistance
against IFN/TNF-induced cell death. Activation of the NF-B pathway
was reported recently to be involved in the IGF-I-mediated neuronal
survival against oxidative stress (63)
and the insulin
antiapoptotic activity in IR-overexpressing CHO cells
(53)
.
A particularly interesting finding is that the combined inhibition of
MAPK and NF-B-dependent antiapoptotic signals synergistically
induced the complete reversal of the IGF-I capability to rescue cells
from IFN/TNF-induced apoptosis. To our knowledge, this is the first
report showing that the combined activation of MAPK and NF-B
pathways is a key regulatory mechanism to protect cells after IGF-IR
engagement. At this point, it should be reminded that activation of
MAPK/AP-1 and NF-B pathways are also key events by which TNFR1
initiates the proinflammatory signaling and the protection against
apoptosis (1, 2, 3
, 10
, 14)
. Thus, it should be noticed that
IGF-IR and TNFR1 share common signaling pathways. This overlap was
further confirmed in this study by showing that IGF-I strongly enhanced
TNF-induced IL-8 production whether cells were pretreated or not with
IFN. Moreover, IL-8 production was totally abrogated by blocking either
MAPK or NF-B pathway, thus confirming its dependence on activation
of AP-1 and NF-B transcription factors (47)
. IGF-IR
activation alone did induce only a weak increase in IL-8 production.
Thus, we conclude that IGF-I functions as a potent enhancer of
TNF-induced NF-B- and MAPK/AP-1-dependent inflammatory/survival
signaling in HT29-D4 cells. Although a PI3'K dependence of
IGF-I-induced NF-B (63)
and MAPK/JNK (61)
activities has been reported by others, we observed no alteration in
IGF-I-enhanced IL-8 production by inhibiting PI3'K. This indicates that
in HT29-D4 cells, this pathway is not involved in the IGF-I activation
of MAPK and NF-B and agrees with the above-discussed incapacity of
PI3'K inhibitors to block the IGF-I antiapoptotic effect. The
synergistic roles of MAPK and NF-B we observed in mediating
IGF-I-induced cytoprotection suggest that the IGF-I protective signals
are separately conveyed by MAPK and NF-B, and that convergence of
these two pathways exists at a more distal point. This is in agreement
with the finding that TRAF2/MAPK and NF-B initiate distinct
antiapoptotic pathways that act in concert to prevent TNF-induced
apoptosis (14)
. We suggest that such a convergence may
operate on the regulation of the expression of inflammatory/survival
genes controlled by NF-B and AP-1 transcription factors. The fact
that the antiapoptotic effect of IGF-I required new protein synthesis
is in favor of such a gene-driven protective mechanism.
The constitutive activation of MAPK/ERK in colon tumors
(64)
and increased NF-B activation in inflamed colonic
epithelial cells (65)
have been reported. We also observed
in HT29-D4 cells a constitutive MAPK/ERK
activity.5
In addition, as shown in this study and reported by others
(44, 45, 46)
, HT29 cells produce small amounts of IL-8 in the
absence of added stimuli. Thus, one would have thought that the
constitutive activation of MAPK and NF-B constitutes a significant
factor in resistance of colon cancer cells to TNF-induced apoptosis.
However, this hypothesis can be rejected because inhibition of MAPK
and/or NF-B basal activities resulted only in a modest apoptosis in
response to TNF. Moreover, such a constitutive activation was also
insufficient to prevent anti-Fas Ab-induced apoptosis.
In agreement with the above reported model, the incapacity of IGF-I to
mediate a successful resistance against Fas- and IFN/Fas-induced
apoptosis is consistent with the minimal capacity of anti-Fas Ab, with
or without IGF-I, to enhance IL-8 production used as a marker of
MAPK/AP-1 and NF-B activation. This observation is in agreement with
our general understanding of Fas signaling, which initiates the
Fas-associated death domain protein-dependent apoptotic pathway, but
unlike TNFR1, does not recruit TRAF molecules, thus being unable to
efficiently lead to NF-B and MAPK activation (1, 2, 3)
. We
have further investigated whether the activation state of MAPK and
NF-B would be able to influence Fas-induced apoptosis. Indeed,
sensitization to apoptosis by NF-B blockade has been reported to be
restricted to the context of TNF signaling in some systems (14
, 66)
but not in others (13)
. In HT29-D4 cells, we
report that although treatment with TNF or IGF-I did not alter Fas-
induced apoptosis, the combination of these stimuli induced a marked
protective effect. Thus, both MAPK and NF-B pathways, provided they
achieve a sufficient state of activation, can protect colonic cancer
cells from death factor-induced apoptosis in general. It remains to
test other proapoptotic stimuli to generalize this conclusion.
In this study, the IGF-I antiapoptotic function has been assayed in most of the experiments in IFN-sensitized cells. Therefore, it could be argued that the IGF-I survival signaling interferes with IFN-mediated rather than TNF-mediated pathways. Although this question cannot be directly assessed because of the incapacity of TNF and IFN to individually induce apoptosis, we propose that IGF-I specifically interferes with the TNF antiapoptotic signaling for the following reasons: (a) IGF-I was able to potentiate the TNF-mediated proinflammatory pathway, even in the absence of IFN; (b) IGF-I was unable to protect cells against Fas-induced apoptosis, even in IFN-sensitized cells; and (c) the combination of IGF-I and TNF did not require IFN sensitization to protect cells from Fas-mediated apoptosis.
Finally, an interesting finding of this study is that IGF-I, in addition to its antiapoptotic function, is also a potent inflammatory mediator contributing to the amplification of the TNF-induced secretion of IL-8 in colonic cancer cells. The role of IGF-I should be then in vivo more complex because the enhanced release of inflammatory mediators by colon cancer cells may promote further inflammation, neutrophil migration, and tumor destruction. The result of such a dual effect on tumor growth may be an important determinant for the outcome of colon cancer cells in patients. Whether IGF-I enhances secretion of other inflammatory cytokines in intestine epithelial cells also remains an open question.
Taken together, the findings reported in this study underline the synergism between the signaling pathways shared by IGF-IR and TNFR1 to induce resistance in colon cancer cells against death factors. Here exists a therapeutic challenge because the IGF-I and IGF-II autocrine/paracrine loops, which have been reported to be key components in the intestine epithelial cell physiology (24 , 27, 28, 29) , may exert a dual proinflammatory and survival-promoting effect interfering with: (a) the undesirable cell loss observed during the inflammatory bowel diseases; and (b) the expected efficiency of immune- and drug-based anticancer therapies.
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ACKNOWLEDGMENTS |
|---|
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FOOTNOTES |
|---|
1 This work has been partially supported by a
grant from Association pour la Recherche sur le Cancer. 
2 Present address: Laboratory of Experimental
Cancerology, University Hospital, B 9000 Gent, Belgium.
3 To whom requests for reprints should be
addressed, at Unité 6032, Centre National de la Recherche
Scientifique, Faculté de Médecine, 27 Boulevard J. Moulin,
13385 Marseille Cedex 5, France. Phone: 33-4-91-32-44-14; Fax:
33-4-91-25-89-70; E-mail: pommier{at}medecine.univ-mrs.fr
4 The abbreviations used are: TNF, tumor necrosis
factor ; TNFR, TNF receptor; TRAF, TNFR-associated factor; MAPK,
mitogen-activated protein kinase; ERK, extracellular signal-regulated
kinase; JNK, c-Jun NH2-terminal kinase; IGF, insulin-like
growth factor; IGF-IR, IGF-I receptor; IR, insulin receptor; IRS-1,
insulin receptor substrate-1; PI3'K, phosphatidylinositol 3'-kinase;
PKC, protein kinase C; PKB/AKT, protein kinase B/product of the
oncogene v-akt; pp70S6K, pp70 ribosomal
protein S6 kinase; AP-1, activator protein-1; NF-B, nuclear factor
B; IB, inhibitor of NF-B; FAK, focal adhesion kinase; mAb,
monoclonal antibody; PI, propidium iodide; CHX, cycloheximide; BIM,
bisindolylmaleimide; CPHC, calphostin C; WMN, wortmannin; IL,
interleukin; IFN, IFN-.
Received 8/ 9/99. Accepted 2/ 3/00.
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J.-i. Okano and A. K. Rustgi Paclitaxel Induces Prolonged Activation of the Ras/MEK/ERK Pathway Independently of Activating the Programmed Cell Death Machinery J. Biol. Chem., May 25, 2001; 276(22): 19555 - 19564. [Abstract] [Full Text] [PDF]
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A.-M. Khatib, G. Siegfried, A. Prat, J. Luis, M. Chretien, P. Metrakos, and N. G. Seidah Inhibition of Proprotein Convertases Is Associated with Loss of Growth and Tumorigenicity of HT-29 Human Colon Carcinoma Cells. IMPORTANCE OF INSULIN-LIKE GROWTH FACTOR-1 (IGF-1) RECEPTOR PROCESSING IN IGF-1-MEDIATED FUNCTIONS J. Biol. Chem., August 10, 2001; 276(33): 30686 - 30693. [Abstract] [Full Text] [PDF]
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 A. Anwar, A.A. Zahid, K.J. Scheidegger, M. Brink, and P. Delafontaine Tumor Necrosis Factor-{alpha} Regulates Insulin-Like Growth Factor-1 and Insulin-Like Growth Factor Binding Protein-3 Expression in Vascular Smooth Muscle Circulation, March 12, 2002; 105(10): 1220 - 1225. [Abstract] [Full Text] [PDF]
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) or treated with TNF (4 ng/ml) without (
) or with
(
) des-(1–3)-IGF-I (50 ng/ml; IGF), and the
percentage of dead cells was determined 24 h later as described in
"Materials and Methods." #, P < 0.01
versus IFN/TNF-treated control cells (-). To determine
the percentage of IGF-I protection, the number of dead cells in the
control samples (
) CHX (10 µg/ml) 30-min before TNF (4 ng/ml) addition in the
presence or absence of des-(1–3)-IGF-I (IGF) at 50
ng/ml, and the percentage of dead cells was quantified 24 h later
as described in "Materials and Methods." *,
P < 0.05; **,
P < 0.005. Bars, SD.
). *,
P < 0.001 versus control
cells; **, P < 0.001
versus TNF- and TNF + IGF-treated cells.
Bars, SD.
) and
des-(1–3)-IGF-I (•) on IFN/TNF (
) was added alone (P) or
with either anti-IGF-IR (
)
was added at 10 µg/ml to IFN/TNF-treated cells; 
