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Experimental Therapeutics |
Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80262 [P. L. R., D. J. A.], and Department of Veterans Affairs Medical Center, Denver, Colorado 80220 [P. L. R., R. J. G., E. C. R., L. J. D., D. J. A.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Sulindac is a prodrug that is rapidly metabolized to two major derivatives; the sulfide metabolite is an active NSAID that inhibits both COX-1 and COX-2 and, thereby, inhibits prostaglandin synthesis (12) . By contrast, the sulfone metabolite is not an NSAID because it does not inhibit either COX-1 or COX-2 and does not inhibit prostaglandin synthesis (6 , 12 , 13) Both sulindac and its sulfone metabolite have chemopreventive activity; both drugs prevent tumor formation in chemically induced animal models of colon (4, 5, 6) , breast (14 , 15) , and lung cancer (16 , 17) . These observations originally suggested that COX inhibition was not necessary for the chemopreventive activity of some of the NSAIDs and related compounds.
Several lines of evidence suggest that the biological mechanism of the chemopreventive action of sulindac is the selective induction of apoptosis, or programmed cell death, in neoplastic cells. Both sulindac sulfide and sulindac sulfone induce apoptosis in many cancer cell lines in vitro (12 , 18, 19, 20, 21, 22, 23) . Sulindac treatment also increases the rate of apoptosis in adenomas from FAP patients 3-fold, with no apparent effect on cell proliferation (24) . Whereas apoptosis primarily accounts for the growth inhibitory effects of sulindac, less is known about the biochemical mechanisms of apoptotic induction by sulindac. NSAIDs are classically defined by their ability to inhibit COX-1 and -2. However, NSAIDs including sulindac can induce apoptosis in cell lines such as HCT15 that have no COX-1 or -2 expression (13) . In addition, the sulfone metabolite of sulindac, which does not inhibit COX-1 or -2, induces apoptosis of cancer cells in vitro and prevents tumor formation in animal models of colon (6) , breast (14 , 15) , and lung cancer (16 , 17) .
Sulindac sulfone and related compounds inhibit cGPDE, increase cGMP concentrations, and increase PKG activity in colon cancer cell lines with a dose and time dependence that suggests that cGPDE inhibition is the proximate mediator of growth inhibition and apoptosis of these drugs (25) . PKG is known to affect several signal transduction pathways that could mediate apoptosis. However, the mechanism of apoptotic cell death that follows cGPDE inhibition by these drugs is not known.
Mutation of the K-ras proto-oncogene is a common, early event in colon cancer development (26) . Activated Ras recruits c-Raf to the plasma membrane where it activates MEK1/2, which then phosphorylates its only known substrate, ERK1/2. Activation of ERK1/2 culminates in phosphorylation of transcription factors responsible for regulating genes that enhance cell proliferation and protect cells from apoptosis (27) . Cancer cells with ras mutations appear to be more susceptible to the growth inhibitory effects of sulindac than those with wild-type ras. In a rat model of azoxymethane-induced colonic neoplasia, sulindac preferentially inhibited the growth of tumors with K-ras mutations from 90 to 36% of all tumors (28) . Similarly, sulindac sulfone preferentially suppressed the number of mammary tumors containing H-ras mutations in a methylnitrosourea-induced model of rat mammary carcinogenesis (15) . Sulindac sulfide has been reported to physically bind to Ras protein in vitro, and this interaction prevented binding of c-Raf to Ras (29) . In addition, PKG can phosphorylate c-Raf and inhibit its interaction with Ras (30) . Taken together, these data suggest the hypothesis that the biochemical mechanism of apoptosis by sulindac metabolites may be attributable to effects on Ras signaling pathways.
As a downstream effector of Ras signaling, ERK1/2 phosphorylation is often up-regulated in cancers harboring ras mutations. Colon tumors displayed especially high levels of ERK1/2 phosphorylation in a screening study of >100 tumors (31) . Inhibition of ERK1/2 phosphorylation in vitro by a synthetic MEK1/2 inhibitor, PD184352, decreased soft agar growth and inhibited the transformed phenotype of colon 26 cells. In vivo, PD184352 suppressed the growth of mouse and human colon tumor xenografts (32) . We, therefore, tested the hypothesis that sulindac metabolites cause apoptosis by down-regulating ERK1/2 signaling in human colon cancer cells. This was determined by Western blotting with phospho-specific antibodies raised against ERK1/2 and MEK1/2. Both of the sulindac metabolites caused the inhibition of ERK1/2 phosphorylation at times and doses consistent with cleavage of caspase-3 and -7 and nuclear morphology indicative of apoptotic cell death. To further examine the role of ERK1/2 down-regulation and apoptotic induction, colon cancer cells were treated with the MEK1/2 inhibitor, U0126. U0126 substantially inhibited ERK1/2 phosphorylation, induced apoptosis alone, and potentiated apoptosis induced by both of the sulindac metabolites. These results indicate that sulindac metabolites inhibit ERK1/2, and this inhibition is sufficient to cause programmed cell death in human colon cancer cells, which suggests that this is one mechanism of the chemopreventive effect of sulindac.
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MATERIALS AND METHODS |
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Tissue Culture.
HCT116 human colon cancer cells were purchased from American Type Cell
Culture (Manassas, VA) and maintained in RPMI 1640 supplemented with
10% fetal bovine serum and 1% penicillin/streptomycin/fungizone
solution. Medium was replaced two or three times per week, and
cells were passaged at subconfluency. The cells were grown in a
humidified atmosphere of 5% CO2-95% air. Cells
were plated and grown to 80–100% confluency before treatment with
vehicle or drug in the experiments described below.
Morphological Apoptosis Assay.
Apoptosis and viability were quantified by staining cells with acridine
orange and ethidium bromide, then by assaying for nuclear morphology, a
hallmark of apoptosis (33
, 34)
. For each determination,
three separate 100-cell counts were scored. Apoptosis was expressed as
a percentage calculated from the number of cells with apoptotic nuclear
morphology divided by the total number of cells examined. Statistics
reported in text are averages of at least three experiments, determined
from triplicate wells for each experiment ± SE;
Ps were calculated using Student’s t test.
Western Blot Analyses.
For Western blot analysis, cells were scraped from plates, pelleted,
resuspended in lysis buffer [15 mM Tris; 2 mM
EDTA; 50 mM 2-mercaptoethanol; 20% glycerol; 0.1% Triton
X-100; 1 mM phenylmethylsulfonyl fluoride; 1 mM
sodium fluoride; 1 mM sodium orthovanadate; and 1 µg/ml
each aprotinin, leupeptin, and pepstatin (pH 7.5)], incubated 10 min
on ice, then sonified for 12 s on ice. Lysates were centrifuged at
10,000 rpm (14,000 x g) for 10 min at 4°C,
and supernatant was collected. Protein concentrations were determined
by the method of Lowry et al. (35)
. Lysates
were prepared for SDS-PAGE, and 50 µg total protein were separated
and electrotransferred overnight onto Immobilon-P polyvinylidene
fluoride membranes (Millipore). Blots were blocked for 30 min in
Tris-buffered normal saline with 1% (w/v) dry milk and 0.05%
Tween 20, then incubated with phospho-ERK1/2 (1 µg/ml),
phospho-MEK1/2 (1:5000), cleaved caspase-3 (1:1000), or cleaved
caspase-7 (1:1000) primary antibody overnight at 4°C. Immunoreactive
protein was detected by incubating blots with horseradish
peroxidase-conjugated secondary antibody for 1 h followed by
chemiluminescent substrate for 1 min. Immunoreactive proteins were
visualized by exposure to film. Quantitation of protein levels was
determined by densitometry using a computerized visual imaging system
(Bio-Rad, Hercules, CA). After the first Western blot using
phospho-specific antibodies, the membranes were stripped for 30 min in
10% sodium dodecyl sulfide, 67 mM Tris (pH 6.7),
and 0.8% 2-mercaptoethanol, while rocking at 50°C, and washed twice
for 10 min each in Tris-buffered normal saline with 0.05%
Tween 20. Blots were then reblocked and probed with pan-ERK1/2 (1
µg/ml) or pan-MEK1/2 (1:5000) primary antibodies as described above.
Independent experiments validated that this stripping procedure did not
lead to loss of signal.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, as determined by examining nuclear morphology after
staining with acridine orange and ethidium bromide. Low levels of
apoptosis were seen at 24 h at the highest dose (120
µM) of sulindac sulfide; both doses resulted in
progressively increased levels of apoptosis at 48, 72, and 120 h.
Sulindac sulfide caused a similar time- and dose-dependent inhibition
of ERK1/2 phosphorylation (Figs. 1B
and 2A and B)
, as determined by Western blotting with a
monoclonal antibody raised against phosphorylated ERK1 (the same
peptide sequence is present in phosphorylated ERK2). Significant
inhibition of ERK1 and ERK2 phosphorylation occurred by 12 h and
persisted through 72 h after treatment (P < 0.05). Decreased phospho-ERK1/2 was detected by 12 h, but
not earlier than 8 h after sulindac sulfide treatment (data not
shown). Phosphorylation of both ERK1 (p44) and ERK2 (p42) was reduced
to similar extents. The inhibitory effect of sulindac on ERK1/2
activity was confirmed using an in vitro ERK1/2 kinase assay
using Elk-1 as a substrate (data not shown). The amount of total ERK1/2
protein expression was similar to vehicle-treated cells except at
72 h of treatment, when ERK1 was significantly reduced compared
with vehicle control (P < 0.05; Figs. 1B
and 2
, C and D).
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. Sulindac resulted in inhibition of phospho-ERK1/2
expression by 12 h of drug treatment that persisted through
72 h (Figs. 3B
and 4A and B)
, without decreased expression of total
ERK1/2 protein, except for total ERK1 at 72 h after treatment
(Figs. 3B
and 4
, C and D). The ERK1/2
inhibitory effect of sulindac sulfone was confirmed with an in
vitro ERK1/2 kinase assay using Elk-1 as a substrate (data not
shown). Similar apoptotic induction and ERK1/2 inhibition were seen for
both sulindac sulfide and sulindac sulfone using two additional human
colon cancer cell lines, HT29 and SW480 (data not shown).
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Sulindac Metabolites Inhibit MEK1/2 Phosphorylation in Human
Colon Cancer Cells.
The only characterized upstream activator of ERK1/2 is MEK1/2
(27)
. We, therefore, determined whether sulindac
metabolites inhibit ERK1/2 phosphorylation by inhibiting its upstream
activator, MEK1/2. Like ERK1/2, MEK1/2 is activated by phosphorylation,
and phospho-specific antibodies were used to assay MEK1/2 activity by
Western blotting. Sulindac sulfide (Fig. 1B)
and sulindac
sulfone (Fig. 3B)
inhibited MEK1/2 phosphorylation with a
time and dose dependence similar to that of ERK1/2 inhibition,
which indicated that sulindac metabolites inhibit ERK1/2
phosphorylation by acting upstream of, and not at the level of, ERK1/2.
Unlike total ERK1/2 expression, total MEK1/2 expression, as determined
by Western blotting with pan-MEK1/2 antibody, was decreased after
treatment with sulindac sulfide (Fig. 1B)
or sulindac
sulfone (Fig. 3B)
. However, inhibition of MEK1/2
phosphorylation, apparent by 12 h after treatment, preceded the
loss of total MEK1/2 protein, which occurred after 24 h of
treatment.
More Potent cGPDE Inhibitors CP248 and CP461 Inhibit ERK1/2
Phosphorylation.
The more potent inhibitors of cGPDE, CP248 and CP461, induce apoptosis
of cancer cells at substantially lower doses than do sulindac sulfide
and sulindac sulfone (25)
. To determine whether ERK1/2
inhibition and apoptotic induction were common mechanisms of
cGPDE-inhibitory agents, colon cancer cells were treated with CP248 and
CP461 and analyzed for phospho-ERK expression. CP248 (5 and 10
µM) and CP461 (10 and 50 µM)
caused a dose- and time-dependent induction of apoptosis (data not
shown) and inhibition of ERK1/2 phosphorylation (Fig. 5)
. This suggests that inhibition of ERK1/2 phosphorylation may be a
common mechanism of action of this class of antineoplastic agents, and
that the effect is not limited to sulindac metabolites alone. There was
a similar level of ERK1/2 inhibition by CP248 and CP461 compared with
sulindac doses that induced equivalent apoptotic responses.
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at times preceding morphological apoptosis. Cleavage of caspase-3 and
-7 was detectable as early as 12 h after treatment, whereas
changes in nuclear morphology were usually not apparent until at least
24 h and were maximal at 5 days after treatment. Cleavage of both
caspase-3 and caspase-7 occurred at similar times and to the same
extent in both sulindac sulfide- and sulfone-treated cells. In all of
the experiments, inhibition of ERK1/2 phosphorylation was seen
concomitant with caspase cleavage.
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. A persistent dose-dependent inhibition of ERK phosphorylation was
seen at 24 and 48 h after treatment. At these doses, U0126 induced
cleavage of caspase-3 and caspase-7 at 48 h (Fig. 7B)
,
which indicated that persistent down-regulation of ERK1/2
phosphorylation is able to induce apoptosis. Apoptosis was confirmed by
nuclear morphology (data not shown).
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. Total ERK1/2 protein levels were not decreased in
U0126-treated compared with control lysates. U0126 treatment alone
induced apoptosis of HCT116 cells and potentiated the apoptotic effect
of sulindac sulfide and sulindac sulfone (Fig. 8B)
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although apoptotic induction of cultured cells by chemical inhibition of ERK1/2 has been described in other systems (37) , to our knowledge, this is the first report that ERK1/2 inhibition can cause apoptotic cell death in cultured human colon cancer cells. ERK activation is commonly thought to protect many different types of cells from apoptosis, predominantly in neuronal models of growth factor withdrawal (37) . One mechanism by which cancer cells maintain their growth advantage may be via activation of antiapoptotic pathways such as ERK1/2. This may be particularly relevant to colon cancer because mutation of K-ras occurs commonly during colon cancer development, and these mutations result in activation of the ERK1/2 arm of the Ras signaling pathway (26) . ERK1/2 activity is higher in colon cancer cells with mutant K-ras than in those with wild-type K-ras (38) , and ras mutant cell lines are differentially sensitive to the apoptotic effects of NSAIDs (39) . Cells that have become dependent on high levels of K-ras signaling for survival may be more affected by inhibition of ERK1/2. Our results indicate that persistent inhibition of the pro-proliferative ERK1/2 pathway is sufficient to cause the colon cancer cells in culture to undergo apoptotic cell death.
Our results are also the first indication that inhibition of ERK1/2 signaling plays a role in sulindac-induced apoptosis. Both of the sulindac metabolites inhibit ERK1/2 phosphorylation at times and doses that are consistent with apoptotic induction, without causing an equivalent decrease in total ERK1/2 protein expression. The time course of effects indicate that the onset of ERK inhibition occurs before the earliest morphological evidence of apoptotic cell death and concomitant with the earliest evidence of caspase cleavage. These results suggest that ERK inhibition may not be the result of apoptotic cell death and that it occurs in a time frame such that it could be inducing the apoptotic process.
The observation that sulindac metabolites inhibit phosphorylation of MEK1/2, the upstream activator of ERK1/2, suggests that sulindac metabolites inhibit ERK1/2 activity by decreasing MEK1/2-dependent phosphorylation. These effects are likely to be at the level of MEK1/2 or upstream. The ability of sulindac sulfide to bind Ras protein in vitro and inhibit association with c-Raf (29) , the upstream activator of MEK1/2, is a possible mechanism of the effects of sulindac on ERK1/2. The decrease in total MEK1/2 protein at later times indicates that sulindac induces proteolytic cleavage or inhibits the transcription or translation of MEK1/2 protein.
cGPDE is a direct biochemical target for non-NSAID apoptotic agents such as sulindac sulfone, CP248, and CP461 (25) . We found that several of the cGPDE inhibitors have in common the ability to inhibit ERK activity at doses that induce apoptotic cell death. PKG activation that follows cGPDE inhibition by sulindac-like compounds may result in down-regulation of ERK1/2 signaling. Of interest, c-Raf-1 is a substrate for activated PKG, and PKG-mediated phosphorylation of c-Raf-1 results in the inhibition of Raf activity and dissociation from Ras (30) . The inhibition of c-Raf-1 activity would down-regulate MEK1/2 and, therefore, ERK1/2 signaling.
Treatment of colon cancer cells with U0126 led to a profound transient
inhibition of ERK1/2 phosphorylation followed by a dose-related
persistence of the effect that was associated with the induction of
apoptotic cell death. Thus, to the extent that U0126 has selective
effects on MEK and ERK, this result suggests that ERK inhibition is
sufficient to induce apoptotic cell death in colon cancer cells. The
time course suggests that the duration of ERK inhibition may be
important for the apoptotic effect in that both the sulindac
metabolites and the higher doses of U0126 led to comparable ERK
inhibition that lasted 24–48 h and led to the induction of apoptotic
cell death. U0126 substantially potentiated the apoptotic effect of
both of the sulindac metabolites, which suggests that these compounds
together lead to greater inhibition of ERK1/2-mediated signaling.
However, the level of ERK1/2 inhibition did not correlate well with the
level of apoptotic cell death by these structurally unrelated
compounds. For example, 20 µM U0126 and 200
µM sulindac sulfone induced similar amounts of apoptosis,
whereas U0126 was much more effective at inhibiting ERK1/2
phosphorylation than sulindac sulfone (Fig. 8)
. This implies that
sulindac metabolites also have effects on additional apoptotic
pathways, which have an additive or synergistic effect on the induction
of apoptotic cell death. Inhibition of ERK1/2 may, therefore, account
for part, but not all, of the apoptotic effect of sulindac. Sulindac
affects additional signaling pathways that may induce apoptotic
cell death, including cGPDE (25)
, nuclear
factor-
B (40)
, and lipoxygenase (41)
. It
is possible that some of these separate biochemical effects are in fact
interdependent. Future studies will address possible interactions
between ERK1/2 and these alternate biochemical targets of sulindac.
Establishing the mechanism of apoptotic induction by sulindac and its metabolites is an important step in understanding the potent chemoregressive activity of this drug in colonic adenomas. The results presented in this paper provide direct evidence of a link between one biochemical effect of sulindac (ERK1/2 inhibition) and the induction of apoptosis by the drug. Although it is possible, even likely, that sulindac affects more than one apoptotic pathway, we believe that we have identified ERK1/2 inhibition as at least one of the biochemical mechanisms of apoptotic activation by sulindac and its metabolites. Chemopreventive drugs that maintain this ERK inhibitory activity may be more potent than those that have more transient effects.
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FOOTNOTES |
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1 Supported by the Department of Veterans Affairs
Merit Review Program and NIH Training Grant 5T32DK07038 and by the
Immunology and Flow Cytometry Cores of the University of Colorado
Cancer Center. 
2 To whom requests for reprints should be
addressed, at Division of Gastroenterology, A009–151 School of
Medicine, University of Colorado Health Sciences Center, 4200 East
Ninth Avenue, Denver, CO 80262.
3 The abbreviations used are: NSAID, nonsteroidal
anti-inflammatory drug; FAP, familial adenomatous polyposis; COX,
cyclooxygenase; cGPDE, cyclic GMP-dependent phosphodiesterase; PKG,
protein kinase G; ERK, extracellular signal-regulated kinase; MAPK,
mitogen-activated protein kinase; MEK, mitogen-activated protein kinase
kinase.
Received 7/27/00. Accepted 12/13/00.
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