Akt, MAPK (Erk1/2), and p38 Act in Concert to Promote
Apoptosis in Response to ErbB Receptor Family Inhibition*
James M.
Nelson
and
David W.
Fry
From Pfizer Global Research and Development, Ann Arbor, Michigan
48105
Received for publication, September 9, 2000, and in revised form, January 16, 2001
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ABSTRACT |
The ErbB receptor family is implicated in
the malignant transformation of several tumor types and is
overexpressed frequently in breast, ovarian, and other tumors. The
mechanism by which CI-1033 and gemcitabine, either singly or in
combination, kill tumor cells was examined in two breast lines,
MDA-MB-453 and BT474; both overexpress the ErbB-2 receptor. CI-1033, a
potent inhibitor of the ErbB family of receptor tyrosine kinases,
reduced levels of activated Akt in MDA-MB-453 cells. This effect alone,
however, did not induce apoptosis in these cells. Gemcitabine treatment
resulted in a moderate increase in the percentage of apoptotic cells
that was accompanied by activation of p38 and MAPK (ERK1/2). CI-1033
given 24 h after gemcitabine produced a significant increase in
the apoptotic fraction over treatment with either drug alone. During the combined treatment p38 remained activated, whereas Akt and activated MAPK were suppressed. Substitution of CI-1033 with the phosphatidylinositol 3-kinase inhibitor LY294002 and the MAPK/ERK kinase inhibitor PD 098059 in combination with gemcitabine produced the
same results as the combination of CI-1033 and gemcitabine. p38
suppression by SB203580 prevented the enhanced cell kill by CI-1033. In
contrast to MDA-MB-453, BT474 cells exhibited activated p38 under
unstressed conditions as well as activated Akt and MAPK. Treatment of
BT474 cells with CI-1033 inhibited both the phosphorylation of Akt and
MAPK and resulted in a 47% apoptotic fraction. Gemcitabine did not
cause apoptosis in the BT474 cells. These data indicate that
suppression of Akt and MAPK in the presence of activated p38 results in
cell death and a possible mechanism for the enhanced apoptosis produced
by the combination of CI-1033 and gemcitabine in MDA-MB-453 cells.
Furthermore, tumors that depend on ErbB receptor signaling for survival
and exhibit activated p38 in the basal state may be susceptible to
apoptosis by CI-1033 as a single agent.
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INTRODUCTION |
Signal transduction through the ErbB receptor family involves the
Raf-MEK-MAP1 kinase and the
PI 3-kinase-Akt signaling pathways. These pathways are often activated
simultaneously with apparently conflicting responses such as
apoptosis, proliferation, growth arrest, differentiation, and
senescence, depending on the cell type and the duration and strength of
the stimulus (1). The cytosolic serine/threonine protein kinase PKB/Akt
is considered the focal point of a survival pathway known to protect
cells from apoptosis during cytokine and growth factor stimulation.
Heregulin, a known ligand for ErbB3/4, has been reported to regulate
Akt in breast cancer cells (2) and when activated by signaling through
the epidermal growth factor receptor protects epithelial cells
against Fas-induced apoptosis (3). Activated Akt also has been shown to
inhibit the apoptotic effects of farnesyltransferase inhibitors (4) and
delay the onset of p53-mediated transcriptionally dependent apoptosis
(5). In addition, phosphorylation of Raf by Akt was shown to inhibit activation of the Raf-MEK-MAPK signaling pathway and shifted the cellular response in a human breast cancer cell line from cell cycle arrest to proliferation (6).
Recent evidence has pointed to a relationship between the Akt and MAPK
survival pathways and the structurally similar stress-activated members
of the MAPK family, which include the stress-activated protein
kinase/JNK and p38 pathways in terms of a balance between cell death
and proliferation. The precise role that signaling through the stress
pathways plays remains complex, however, and many times seems to result
in conflicting responses. For example, inhibition of the MAPK has been
shown to play a role in the activation of p38 and induces apoptosis in
HeLa cells (7). However, in trigeminal neurinoma cells, inhibition of
p38 reduced the activity of MAPK and Akt and induced apoptosis (8). Rat
mesangial cells conversely are resistant to tumor necrosis factor
-induced apoptosis that seem to be associated with an early
transient JNK activation (9).
The ErbB receptor family is considered an important target in the
treatment of cancer. Overexpression of one or more of these receptors
occurs frequently in human tumors and often correlates with poor
prognosis and shorter survival time (10, 11). Clinical validation for
employing this receptor family as a target in cancer treatment has been
achieved recently through specific antibody therapies directed toward
ErbB2 (12) or the epidermal growth factor receptor (13). Lately, there
has been a significant effort to identify specific inhibitors of the
ErbB family of tyrosine kinases (14). CI-1033 is a potent and specific
irreversible inhibitor of the ErbB receptor family (15) and is
currently in phase I clinical trials (Fig.
1). This agent efficiently inhibits all
signal transduction mediated via epidermal growth factor or heregulin
(16) and exhibits broad spectrum antitumor activity in several human
tumor xenograft models (17). Cancer agents are used frequently in
combinations in the clinic, and recently protein kinase inhibitors have
been shown to enhance the therapeutic effect of more conventional
cytotoxic therapies (18).

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Fig. 1.
Structure of CI-1033
(N-[4-(3-chloro-4-fluorophenylamino)-7-(3-morpholin-4-yl-propoxy)-quinazolin-6-yl]-acrylamide
dihydrochloride).
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The present study examines the molecular mechanism by which CI-1033
induces apoptosis either as a single agent or in combination with
gemcitabine (2',2'-difluorodeoxycytidine, dFdC), an analog of
deoxycytidine, with clinical activity against several types of cancer
(19). The results indicate that the apoptotic response to CI-1033
and gemcitabine is caused by the inhibition of the survival pathways,
MAPK and Akt, in concert with the activation of the p38 stress pathway.
These studies further suggest that those tumors where survival signals
are mediated through the ErbB family of receptors and exhibit
constitutively activated p38 may be susceptible to CI-1033 as a single agent.
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EXPERIMENTAL PROCEDURES |
Reagents--
LY294002, SB203580, FITC-phalloidin, RNase, and
propidium iodide were purchased from Sigma. CI-1033 and PD 098059 were
synthesized at Pfizer, and gemcitabine was from Lilly. The antibodies
used for Western blotting, phospho-Akt (Ser-473), Akt, phospho-MAPK, MAPK, phospho-p38, and p38 were purchased from New England Biolabs (Beverly, MA), and p27 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Cultured Cells--
The human breast carcinomas, MDA-MB-453 and
BT474 cells, were obtained from the American Type Culture Collection
(Rockville, MD). Cells were grown as monolayers at 37 °C in 5%
CO2 in air in Dulbecco's modified Eagle's
medium/F12, 50:50 (Life Technologies, Inc.) containing 10% fetal
bovine serum (Sigma) and gentamicin (Life Technologies, Inc.).
Western Blots--
Cells were grown in 100 × 20-mm dishes
(Falcon, Becton Dickinson, Lincoln Park, NJ) seeded at 1.5 × 106 cells/dish and allowed to grow 24 h before
treatment as described under "Results." Media and cells were
collected and centrifuged at 200 × g for 8 min. The
supernatant was removed and the pellet was washed once in PBS. The
cells were lysed in 1 ml of ice-cold lysis buffer (50 mM
HEPES (pH 7.5), 150 mM NaCl, 10% glycerol, 1 mM EDTA, 1% Triton X-100, 10 mM
-glycerol
phosphate, 0.1 mM sodium vanadate, 1 mM NaF, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 50 µg/ml
4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochloride, 1 mM dithiothreitol). The lysate was transferred to a
microcentrifuge tube, allowed to sit for 15 min on ice, and
centrifuged for 20 min at 10,000 × g at 4 °C. The
supernatant was transferred to a clean microcentrifuge tube and stored
at
80 °C. Protein concentrations were determined by the BCA
protein assay (Pierce), and 50 µg of total protein was resuspended
with 3× Laemmli buffer (20), heated to 100 °C for 5 min, and loaded
onto a 4-20% Tris/glycine polyacrylamide gel electrophoresis gel
(Novex, San Diego, CA). Proteins in the gel were transferred to
nitrocellulose (Novex), and the membrane was blocked for 1 h in
blocking buffer (3% bovine serum albumin, 50 mM Tris (pH
8.0), 0.15 M NaCl, 0.1% Tween 20, 10 mM
-glycerol phosphate, 1 mM NaF, 0.1 mM sodium
orthovanadate). The membrane was blotted for 24 h in blocking
buffer at 4 °C with the primary antibody, washed once for 15 min and
three times for 5 min in wash buffer (PBS, 0.05% Tween 20) at
room temperature, and blotted for 1 h at room temperature with
horseradish peroxidase-conjugated secondary antibody (Bio-Rad) in
blocking buffer. Blots were washed in wash buffer once for 15 min and
three times for 5 min. Proteins were detected by ECL (Amersham
Pharmacia Biotech) on BioMax MR-1 film (Sigma). The membranes were
stripped using the Re-blot Western blot recycling 1 kit (Chemicon,
Temecula, CA). Representative blots from duplicate experiments are
shown in Figs. 3 and 4.
Apoptosis--
Several methods were used to measure apoptosis.
Cell rounding and detachment were noted during the treatment of the
cells, and chromatin condensation was observed with a
4,6-diamidino-2-phenyl-indole stain. Poly(ADP-ribosyl)
polymerase cleavage was determined by Western blotting, and
caspase activation was measured using a kit from BioVision, Inc. (Palo
Alto, CA). Apoptosis was determined also by actin/DNA staining using a
modification of the procedure reported by Endresen et al.
(21). Briefly, ~1.5 × 106 cells were collected and
centrifuged at 200 × g for 8 min, the medium was
removed, and the cells were fixed by adding 3 ml of ice-cold 70% EtOH
while gently vortexing. After a minimum of 1 h the cells were
centrifuged at 200 × g for 8 min at 10 °C and the
EtOH was aspirated. The actin was stained by resuspending the pellet in
20 µl of 10 µg/ml FITC-phalloidin in PBS (Sigma) for 1 h in
the dark at room temperature and washing once with PBS. The DNA was
stained with 500 µl of propidium iodide (50 µg/ml in PBS) and 25 µl of RNase (600 kunitz units/ml) for 30 min at 37 °C in
the dark. In the original protocol, the cells were fixed in
paraformaldehyde; this was interfering with the propidium iodide staining so we used 70% EtOH, which prevented us from determining what
phase of the cell cycle the cells were apoptosing from. The fluorescence was measured on a Coulter Elite flow cytometer and analyzed using Winlist (Verity Software House, Inc., Topsham, ME). This
method is based on the knowledge that actin is cleaved when cells enter
apoptosis (22). All of the methods used to determine apoptosis gave the
same apoptotic profile in each of the treatment regimens (data not
shown). The apoptotic data presented here were obtained using
actin/DNA staining.
Treatment Studies--
In experiments involving treatment of
MDA-MB-453 and BT474 cells, CI-1033 (0.5 µM in
H2O) and gemcitabine (50 nM in saline) were
added as single agents or in combination for 3 days. The treatment
protocols for the combination studies spanned 3 days and included 1)
simultaneous addition, 2) addition of gemcitabine 24 h after
CI-1033, and 3) addition of CI-1033 24 h after gemcitabine. LY294002 (10 µM), a PI 3-kinase inhibitor, and PD 098059 (1 µM), an MEK inhibitor, were made up in
Me2SO and substituted for CI-1033. When cells were
pretreated for 24 h with SB203580 (10 µM in
Me2SO), a p38 inhibitor, experiments spanned 4 days. All
experiments were done in duplicate.
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RESULTS |
The ability of CI-1033 to induce apoptosis either alone or in
combination with gemcitabine was examined in two human breast carcinomas, the MDA-MB-453 and the BT-474. Both tumors express high
levels of ErbB2; however, each exhibited markedly different responses
to treatment with the two drugs. CI-1033 alone did not induce apoptosis
in the MDA-MB-453 cells, whereas ~45% of the cell population was
apoptotic in the presence of gemcitabine under the conditions described
in Fig. 2. When these cells were exposed to CI-1033 and gemcitabine simultaneously, there was a significant enhancement of apoptosis and an even greater effect (82%) if the cells were treated sequentially first with gemcitabine and then CI-1033. This enhanced effect was not seen if cells were treated first
with CI-1033 and then gemcitabine (Fig. 2), and in fact this schedule
reduced the apoptotic response below that seen for gemcitabine alone.
These effects were in sharp contrast to those obtained in the BT-474
breast tumor model in which CI-1033 alone induced significant apoptosis
(47%) but gemcitabine had no effect (Table
I). Moreover, treatment of these cells
with a combination of CI-1033 and gemcitabine, regardless of the
sequence of addition, produced no enhanced cytotoxic response.

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Fig. 2.
Histograms showing the apoptotic fraction
in black and the nonapoptotic fraction in
white in MDA-MB-453 cells treated with CI-1033 and
gemcitabine. MDA-MB-453 cells (1.5 × 106) were
seeded into 100-mm plates. Twenty-four hours later, gemcitabine (50 nM) and CI-1033 (0.5 µM) were added as single
agents, in combination (simultaneously), or in combination 24 h
apart for a total drug exposure time of 72 h for all treatment
regimens. The cells were collected as described under "Experimental
Procedures," stained for actin, DNA (with FITC-phalloidin), and
propidium iodide, respectively, and analyzed on the flow cytometer.
Gating on the lower fluorescent population stained with
FITC-phalloidin, the cleaved actin population (gate 1) with
the color eventing on black, allowed us to identify the same
population of cells stained with propidium iodide, which show up as the
apoptotic sub-G1 population. The nonapoptotic population
(gate 2), with color eventing on white,
identifies the uncleaved actin stained with FITC-phalloidin with
increased fluorescence. This correlates with the nonapoptotic DNA
histogram stained with propidium iodide. The Winlist software was used
to analyze these data. These data are representative of duplicate
experiments.
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Table I
Analysis of the apoptotic fraction in BT474 cells treated with CI-1033
and gemcitabine
BT474 cells (1.5 × 106) were seeded into 100-mm plates.
Twenty-four hours later, gemcitabine (50 nM) and CI-1033
(0.5 µM) were added as single agents in combination
(simultaneously) or in combination 24 h apart for a total drug
exposure time of 72 h for all treatment regimens. Cells were
stained for flow cytometry and analyzed as described under
"Experimental Procedures." These data are representative of
duplicate experiments.
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The biochemical mechanism by which CI-1033 alone or in combination with
gemcitabine causes cells to enter apoptosis was explored. This was
looked at in the context of the major signaling pathways associated
with the ErbB family of receptors, as well as the potential contribution of stress-activated pathways and modulation of the WAF
family of cyclin-dependent kinase inhibitors, p27 and p21 (23). ErbB family signaling through the Ras/Raf/MAPK pathway and PI
3-kinase/Akt survival pathways has been well established (24), and
recent studies indicate that this receptor system also signals via the
stress-activated protein kinase/JNK and p38 pathways (25). Modulation
of these pathways was investigated with regard to the molecular
mechanism by which CI-1033 and gemcitabine synergize and induce cell
death. CI-1033 alone significantly suppressed constitutively activated
Akt, MAP kinase, and increased p27 expression in the MDA-MB-453 cells
(Fig. 3, A, B, and
D). These effects alone, however, did not induce apoptosis
(Fig. 2). Gemcitabine (50 nM) as a single agent had no
effect on Akt but increased the phosphorylation of p38 as well as the
total p38 protein expression (Fig. 3C) and also increased
the level of activated MAP kinase (Fig. 3B). These effects
were associated with 45% of the cell population being apoptotic (Fig.
2). CI-1033 in combination with gemcitabine inhibited Akt, prevented
the increased activation of MAPK phosphorylation, and stimulated p38
phosphorylation in MDA-MB-453 cells (Fig. 3). Apoptosis was most
prominent (82% (Fig. 2)) when CI-1033 was added 24 h after
gemcitabine and was associated with maximum inhibition of Akt,
decreased activation of MAPK by gemcitabine, and sustained activation
of p38 by gemcitabine. An increase in apoptosis also occurred when
gemcitabine and CI-1033 were added simultaneously but was 10% less
than when gemcitabine was added 24 h before CI-1033. When the
order was reversed and CI-1033 was added 24 h before gemcitabine,
the fraction of cells that were apoptotic was only 36% (Fig. 2). Under
this schedule no activation of the p38 occurred (Fig. 3C),
and there was incomplete suppression of Akt (Fig. 3A). In
addition p27 expression was not reduced to the levels seen when
gemcitabine was given as a single agent, simultaneously, or 24 h
prior to CI-1033 treatment (Fig. 3D). p21 levels did not change under any of the treatment conditions (data not shown).

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Fig. 3.
Western blot analysis of Akt, MAPK, p27, and
p38 modulation in MDA-MB-453 cells treated with CI-1033 and
gemcitabine. MDA-MB-453 cells (1.5 × 106) were
seeded into a 100-mm plate. Twenty-four hours later, gemcitabine (50 nM) and CI-1033 (0.5 µM) were added as single
agents, in combination (simultaneously), or in combination 24 h
apart for a total drug exposure time of 72 h for all treatment
regimens. Cell lysates containing equal amounts of proteins were run on
4-20% SDS-polyacrylamide gel electrophoresis and transferred to
nitrocellulose membranes. The membranes were blotted with antibodies
against the phosphorylated forms of Akt (Ser-473), MAPK, and p38. The
blots were stripped and reprobed with antibodies against total protein
for Akt, MAPK, and p38.
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In contrast to the MDA-MB-453 cells, the BT474 cells exhibited
constitutively activated p38 as well as highly activated Akt and MAPK.
CI-1033 inhibited Akt and MAPK phosphorylation within 30 min in the
BT474 cells, which in this tumor model was associated with a 47%
increase in apoptosis by 72 h (Fig.
4 and Table I). In further contrast to
the MDA-MB-453 cells, treatment of BT474 cells with gemcitabine alone
did not induce apoptosis, and no cytotoxic synergy was observed in
combination studies with gemcitabine and CI-1033.

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Fig. 4.
Western blot analysis of Akt, MAPK, and p38
modulation in BT474 cells treated with CI-1033. BT474 cells
(1.5 × 106) were seeded into 100-mm plates.
Twenty-four hours later CI-1033 (0.5 µM) was added at the
indicated time points. Cell lysates containing equal amounts of
proteins were run on 4-20% SDS-polyacrylamide gel electrophoresis and
transferred to nitrocellulose membranes. The membranes were blotted
with antibodies against the phosphorylated forms of Akt (Ser-473),
MAPK, and p38. The blots were stripped and reprobed with antibodies
against total protein for Akt, MAPK, and p38.
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To provide evidence that the enhancement of gemcitabine-mediated
apoptosis by CI-1033 in the MDA-MB-453 cells was mediated through
inhibition of both the PI 3-kinase/Akt and MAPK pathways, CI-1033 was
substituted with a specific PI 3-kinase inhibitor, LY294002, and a
specific MEK inhibitor, PD098059. When both the PI 3-kinase inhibitor
and the MEK inhibitor were used in place of CI-1033 in combination with
gemcitabine, an apoptotic profile occurred similar to that seen with
CI-1033 as a single agent or in combination with gemcitabine (Table
II). These results indicate that
inhibition of both Akt and MAPK is at least partially responsible for
induction of apoptosis in combination with gemcitabine. The requirement
that activation of the p38 pathway occur for maximum induction of
apoptosis in the presence of the drugs was established through the use
of the p38 inhibitor SB203580 (26). Preincubation of the MDA-MB-453
cells with SB203580 prevented nearly all apoptosis in the presence of
gemcitabine alone and reduced the percentage of apoptotic cells treated
with gemcitabine followed by CI-1033 from 82 to 24% (Fig.
5). The signaling profiles in the
presence of CI-1033 and gemcitabine obtained from both cell lines imply that maximum induction of apoptosis depends on both the suppression of
survival pathways, which signal through Akt and MAPK, and simultaneous activation of the stress pathway through p38.
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Table II
Analysis of the apoptotic fraction in MDA-MB-453 cells treated with
gemcitabine and (PD098059 + LY294002) in place of CI-1033
Cells (1.5 × 106) were seeded into a 100-mm plate.
Twenty-four hours later, gemcitabine (50 nM) and (PD098059
(1 µM) + LY294002 (10 µM)) were added as
single agents in combination (simultaneously) or in combination 24 h apart for a total drug exposure time of 72 h for all treatment
regimens. Cells were stained for flow cytometry and analyzed as
described under "Experimental Procedures." These data are
representative of duplicate experiments.
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Fig. 5.
Analysis of the apoptotic fraction in
MDA-MB-453 cells pretreated for 24 h with the p38 inhibitor
(SB203580) prior to gemcitabine and CI-1033 treatment. MDA-MB-453
cells (1.5 × 106) were seeded into 100-mm plates.
Twenty-four hours after seeding the cells, SB203580 (10 µM) was added 24 h prior to gemcitabine (50 nM) as a single agent and in combination with gemcitabine
24 h prior to CI-1033 (0.5 µM) for a total time of
96 h. The cells were collected as described under "Experimental
Procedures," stained for actin, DNA with FITC-phalloidin, and
propidium iodide, respectively, and analyzed on the flow cytometer.
Gating on the lower fluorescent population stained with
FITC-phalloidin, the cleaved actin population (gate 1) with
the color eventing on black, allowed us to identify the same
population of cells stained with propidium iodide, which show up as the
apoptotic sub-G1 population. The nonapoptotic population (gate
2), with color eventing on white identifies the
uncleaved actin stained with FITC-phalloidin with increased
fluorescence. This correlates with the nonapoptotic DNA histogram
stained with propidium iodide. The Winlist software was used to analyze
these data. These data are representative of duplicate
experiments.
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DISCUSSION |
The balance between cell survival and cell death is a complex
issue, and there is considerable effort to understand how tumor cells
regulate the decision points between these critical pathways. A
significant number of clinically useful anticancer agents have been
shown to induce apoptosis in tumor cells (27), and this process is
believed to be a major component of their therapeutic mechanism. The
pathways through which these agents trigger apoptosis in cancer cells
reportedly have been varied and tend to depend on both the nature of
the drug and the genetic background and origin of the tumor (28). The
present study provides evidence that modulation of both survival and
stress-activated pathways plays a role in the strong synergistic
interaction between gemcitabine and CI-1033 in terms of enhanced
apoptosis in tumor cells. CI-1033 is a specific inhibitor of the ErbB
family of receptor tyrosine kinases (16), which has been shown to be a
viable target in cancer chemotherapy (14). Inhibitors of both the
kinase activity and neutralizing antibodies to the extracellular domain
are currently in clinical trials with encouraging results (14, 15).
Gemcitabine is a nucleoside analog that, after phosphorylation, is
incorporated into DNA and is a clinically approved agent for pancreatic
cancer (19).
Signal transduction through the ErbB family of receptors has been shown
to be mediated through two major pathways resulting in activation of
Akt and the MAP kinases (Erk1/2), which have both been associated with
mitogenesis and cell survival (24). CI-1033 significantly reduced the
constitutively activated components of both pathways in the MDA-MB-453
breast tumor; however, this effect alone did not induce cell death.
Gemcitabine alone, in contrast, caused a considerable percentage of the
cell population to commit to apoptosis, and the major effect on
signaling pathways was an accompanying elevation of activated p38. The
addition of CI-1033 to cells treated with gemcitabine, however, caused
maximum apoptosis above and beyond what occurred in the presence of the two agents alone. The implication here is that p38 activation is an
essential component of the apoptotic response (29) and that
simultaneous inhibition of the two major survival pathways by CI-1033
enhances this process to a maximum. Further evidence for the
involvement of these particular pathways was obtained with inhibitors
of the Akt and MAP kinase pathways, LY294002 and PD 098059, respectively, both of which together mimicked the action of CI-1033,
and the apoptotic response could be tempered by the p38 inhibitor,
SB203580. No activation of JNK was seen under any of the described
treatments and is probably not essential for the induction of apoptosis
in this cell (data not shown).
The notion that activated p38 is necessary for CI-1033 to induce
apoptosis is consistent with the ability of this drug to stimulate
apoptosis in the BT-474 human breast carcinoma as a single agent. This
cell exhibits highly activated p38, Akt, and Erk1/2 in the basal state
and thus mimics the conditions seen in the MDA-MB-453 cells in the
presence of gemcitabine. The latter agent in this case did not induce
apoptosis in the BT-474 model presumably because these cells have
learned to survive in the presence of activated p38 and die only when
their survival pathways are suppressed as when treated with CI-1033.
The pattern whereby apoptosis occurs only when Akt and MAP kinase
pathways are suppressed in the presence of activated p38 is also
consistent with results from the order of addition experiments. When
MDA-MB-453 cells were exposed to CI-1033 first and then to gemcitabine,
the resulting percentage of cells in apoptosis was markedly lower than
with exposure to gemcitabine alone. The diminished apoptotic effect is
consistent with the requirement for p38 activation because this
sequence of drug additions did not result in activation of this enzyme
(Fig. 3C). CI-1033, when added first, may indirectly inhibit
p38 activation by gemcitabine by suppressing signaling through the ErbB
receptor family, which is consistent with reports that p38 can be
activated in cells stimulated with heregulin (NDF), a ligand for ErbB3
and ErbB4 (25). Alternatively, MDA-MB-453 cells treated with CI-1033
show a decrease in S phase cells as measured by bromodeoxyuridine
uptake (data not shown). Thus gemcitabine may require cells to be
cycling to be effective; gemcitabine-treated cells that are pretreated
with the p38 inhibitor (SB203580) arrest in S phase (Fig. 5), where
normally they would eventually enter apoptosis. However, if the cells
were blocked earlier in the cell cycle by CI-1033, which is consistent
with the observed increase in p27 levels, this could prevent the
cytotoxic effects of gemcitabine.
The results from this analysis are consistent with studies in other
cell systems that point to a combination of p38 activation in the
absence of one or more survival pathways as a necessary condition to
promote apoptosis. Inhibition of the MAP kinase pathway in HeLa cells
by the MEK inhibitor PD098059 in the absence of serum stimulated the
p38 pathway and initiated apoptosis (7). Cell death could be abrogated
by serum, which apparently turned on the PI 3-kinase/Akt pathway
as was demonstrated by the use of specific inhibitors and transfection
of dominant negative mutants. Similarly, serum deprivation in Rat1
fibroblasts or nerve growth factor withdrawal from PC12 cells
resulted in apoptosis, which depended on activation of the p38 pathway
(30). These observations are also consistent with the hypersensitive
response that the C-26 murine colon carcinoma exhibits to the MEK
inhibitor, CI-1040. The C-26 have a highly activated p38 pathway in the
basal state and were exceptionally sensitive to inhibition of the MAP
kinase pathway by CI-1040 as determined by clonogenic assays (31).
The above results suggest that elimination of the survival pathways
involving Akt and Erk1/2 by inhibition of signaling through the ErbB
family of receptors per se may not be a lethal condition for
tumors and indicate that activated p38 may be required for cells to
commit to apoptosis. Therapeutically then, these studies imply that
simple assessment of the basal activation state of the stress-activated
and survival pathways could identify tumors that are sensitive to
single-agent inhibitors of the ErbB family as well as other signaling
pathways. Furthermore, these studies indicate that the synergistic
interaction between gemcitabine and CI-1033 is not because of the
intrinsic biochemical mechanism of gemcitabine but perhaps because of
the resulting stress response in general. That concept then suggests
that inhibitors of the ErbB family have the potential to produce an
enhanced cytotoxic response with any compound or condition that
stimulates p38 activation and may have implications in the choice of
drugs, or combinations of drugs, as well as patient selection for
clinical trials.
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FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.:
734-622-7610; Fax: 734-622-7158; E-mail:
James.Nelson@Pfizer.com.
Published, JBC Papers in Press, February 7, 2001, DOI 10.1074/jbc.M008786200
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ABBREVIATIONS |
The abbreviations used are:
MEK, mitogen-activated protein kinase/extracellular signal-regulated
kinase kinase;
MAP, mitogen-activated protein;
MAPK, mitogen-activated
protein kinase;
CI-1033
(N-[4-(3-chloro-4-fluorophenylamino)-7-(3-morpholin-4-yl-propoxy)-quinazolin-6-yl]-acrylamide
dihydrochloride, PI, phosphatidylinositol;
JNK, c-Jun
NH2-terminal kinase;
FITC, fluorescein isothiocyanate;
PBS, phosphate-buffered saline.
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