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Tumor Biology |
Department of Medical Oncology, Academic Hospital Vrije Universiteit Amsterdam, 1081HV Amsterdam, the Netherlands
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ABSTRACT |
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INTRODUCTION |
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Although the primary intracellular targets and the pharmacological
mechanisms of action of the anticancer drugs vary, it has become
evident that drug-induced cell kill is, at least partially, mediated by
programmed cell death or apoptosis (2)
. Relevant progress
has been made in the understanding of the events underlying
drug-induced apoptosis, and several molecules have been implicated in
this process. Among them, a family of aspartatic-specific
cysteine-proteases named caspases (3)
are the central
executors of the apoptotic process (4
, 5)
. Thus far, 14
family members have been cloned in mammals, but not all of them seem to
be directly involved in apoptosis (6)
. Caspases are
synthesized as inactive zymogens
(Mr 30,000–50,000) that
possess three domains: an NH2-terminal domain; a
large subunit (Mr 
20,000); and a
small subunit (Mr 10,000; Ref.
5
). In general, the process of caspase activation requires
a proteolytic processing between domains, followed by an assembly of
the subunits to generate an active tetramer (4, 5, 6)
.
Caspases are activated in a cascade fashion. It has been proposed that a proapoptotic signal can activate an initiator or upstream caspase, which usually possesses a long prodomain such as caspases-8, -9, and -10. In turn, these initiators can activate the effector or downstream caspases, including caspase-3, -6, and -7, which leads to the biochemical and morphological changes that are characteristic of apoptosis (7) . To date, two major caspase pathways or cascades, headed by caspases-8 and -9, have been described and shown to mediate distinct sets of signals (7, 8, 9, 10) . The cascade led by caspase-8 is involved in death-receptor-mediated apoptosis such as the one triggered by Fas, TNF, and TRAIL. On activation, these receptors recruit FADD, which in turn binds to procaspase-8, which leads to cleavage into its active form (11 , 12) . The subsequent activation of the effector caspases occurs either directly or after an amplification step involving mitochondria (13) . On the other hand, the caspase pathway headed by caspase-9 is thought to mediate chemical-induced apoptosis. This cascade is triggered by the cytochrome c release from mitochondria, leading to the formation of a complex with Apaf-1 that via its caspase recruitment domain (CARD) binds to procaspase-9 (14) . This complex, called apoptosome (15) in the presence of dATP can activate procaspase-9, which in turn activates effector caspases (16) . In contrast to the caspase-8 cascade, in which the role of mitochondria may vary between different cell types (13) , the caspase-9 pathway is clearly mitochondria-dependent.
The Bcl-2 family of proteins plays a crucial role in the function of mitochondria during the apoptotic process (17) . Overexpression of the antiapoptotic molecules Bcl-2 or Bcl-xL has been reported to cause resistance to anticancer drugs (18 , 19) . These molecules have the capacity to inhibit apoptosis by preventing permeability transition (PT) and/or by stabilizing the barrier function of the outer mitochondrial membrane (20 , 21) . Stabilization of the mitochondria pores abrogates the release of cytochrome c (22) , thereby preventing the activation of procaspase-9.
Besides the action of Bcl-2 family members, other more direct mechanisms of caspase inhibition have been identified. Viruses are known to produce potent caspase inhibitors, such as CrmA made by cowpox virus that preferentially inhibits caspase-1 and caspase-8 (23) . On the other hand, the baculovirus p35 protein is an example of a broad range inhibitor that blocks multiple caspases (24) . On the basis of sequence similarity toward p35, human homologues of IAPs have been identified such as XIAP, cIAP1, cIAP2 and survivin (25, 26, 27) . They have been shown to be able, in the case of XIAP, cIAP1, and cIAP2, to inhibit not only effector caspases but also the initiator caspase-9 (28) .
Current available data on the molecular mechanism underlying the sequential activation of caspases has led to a model in which caspase-9 is activated on chemotherapy and caspase-8 is activated by death receptor signaling (7, 8, 9, 10) . Nevertheless, some reports have described the activation of procaspase-8 after chemotherapy (29, 30, 31, 32, 33) . However, this activation seems to be a downstream and secondary event that follows caspase-9 activation (7 , 29, 30, 31) . In addition, it should be noted that most of these studies were performed in leukemia cell lines or in cell-free extracts, and that relatively little data are available on the activation of caspases in chemotherapy-induced apoptosis in solid tumor cells.
To obtain more insight in the molecular mechanisms underlying drug sensitivity in NSCLC cells, here we examined the caspase cascades involved in triggering apoptosis in the NSCLC cell line H460. Our results—obtained by means of various biochemical assays, including caspase cleavage and activity assays, and by using selective peptide and molecular inhibitors of caspases—clearly point to a central role for caspase-8 in chemotherapy-induced apoptosis in H460 cells.
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MATERIALS AND METHODS |
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were freshly diluted in culture medium to the
indicated final concentration. The caspase-inhibitors
Z-VAD-fmk, used at a final concentration ranging from 50 to 100
µg/ml and the preferential caspase-8 inhibitor IETD-fmk, added to a
final concentration of 20–40 µg/ml, were used 2 h prior to drug
exposure. Both of the inhibitors were purchased from Enzyme System
Products (Livermore, CA). In some experiments, the caspase-3-like
inhibitor DEVD-CHO (Calbiochem, San Diego, CA) was used at a
final concentration of 50–100 µg/ml.
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Cell Lines and Transfection.
The human NSCLC cell lines NCI-H460 (H460), NCI-H322 (H322), and SW1573
and Jurkat-T-leukemia cells were used in the experiments. Cells were
cultured in RPMI 1640 supplemented with 10% heat-inactivated FCS (Life
Technologies, Inc., Breda, the Netherlands), 2 mM
L-glutamine, 50 IU/ml penicillin, and 50 µg/ml
streptomycin, and grown at 37°C in a humidified atmosphere with 5%
CO2. The cell lines were tested regularly for the
absence of Mycoplasma infection. Cells from exponentially
growing cultures were used in all of the experiments.
For the generation of stable transfectants, H460 cells were transfected with 10 µg cDNA, using Superfect reagent (Life Technologies, Inc.) according to the manufacturer’s protocol. After 24 h, cells from each transfection were split into five separate culture dishes to ensure that independent lines were established. Selection was made using increasing concentrations of puromycin (Sigma, Zwijndrecht, the Netherlands) ranging from 1 to 2 µg/ml or 200–400 µg/ml Geneticin (Life Technologies, Inc.), depending on the transfected plasmid used. Independent clones were collected and allowed to grow in six-well plates. Clones were tested for the expression of the constructs by Western blotting and were selected for use in the desired experiments.
Cell Death Measurement.
Cells were plated at a density of 5 x 106 cells in 75-cm2
tissue
culture flasks (Costar, Cambridge, MA) 24 h before treatment.
Cells were incubated for 4–72 h with IC50s or
IC80s of cisplatin, topotecan, or gemcitabine
(Table 1)
in the presence or the absence
of peptide inhibitors of caspases when indicated. The analysis of
apoptotic cells was performed as described previously
(38)
. Briefly, the extent of cell death was determined by
PI staining of hypodiploid DNA or by annexin V-FITC and 7-AAD
double staining. For the PI staining, 3 x 105 cells were resuspended in Nicoletti buffer as
described previously (39)
and analyzed by FACScan (Becton
Dickinson, Mount View, CA). The fraction of cells with sub-G1 DNA
content was assessed by the Lysis program (Becton Dickinson). Annexin V
staining was performed according to the manufacturer’s protocol
(Nexins Research, Kattendijk, the Netherlands). After incubation with
annexin V, 10 µl of 7-AAD (PharMingen, San Diego, CA) was added and
analysis was performed on FACScalibur using CELLQuest software (Becton
Dickinson). The percentage of specific apoptosis was calculated by
subtracting the percentage of spontaneous apoptosis of the relevant
controls from the total percentage of apoptosis.
Growth-Inhibition Assays.
Cytotoxicities to cisplatin, topotecan, and gemcitabine were assessed
by MTT assay basically as described previously (38
, 41) .
In brief, a suspension of 10,000 cells/100 µl of medium was added to
each well of flat- or U-bottomed 96-well plates (Costar, Corning, NY)
and allowed to grow. Twenty-four h later, drugs were made up in medium
and eight different concentrations were added to the plates at a volume
of 100 µl per well, and plates were incubated for 72 h with
drugs. Then 20 µl of a solution of 5 mg/ml MTT (Sigma Chemicals, St.
Louis, MO) were added to each well and incubated for another
4 h at 37°C. Plates were then centrifuged at 1000 rpm at 4°C
for 5 min, and the medium was carefully discarded. The formazan
crystals were dissolved in 100 µl of DMSO (ACROS Organics, Geel,
Belgium) and absorbance was read at 540 nm using Spectra Fluor (Tecan,
Salzburg, Austria). Absorbance values were expressed as a percentage of
untreated controls and concentrations resulting in
IC50 and IC80 were
calculated. The IC50 and
IC80 values represent the means of at
least three independent experiments.
Clonogenic assays were performed essentially as described previously
(40)
. H460 cells were seeded in triplicate into six-well
plates at a concentration of 300 cells per well. After 24 h, drugs
(cisplatin, topotecan, or gemcitabine) were added at final
concentrations of IC50 or
IC80 (Table 1)
for 24–72 h. After drug removal,
cells were washed twice with PBS and allowed to proliferate in fresh
medium. Colonies were counted when they reached the size of 50–100
cells, after staining with 0.1% crystal violet in 0.9% saline for 30
min at room temperature. The number of colony-forming units in treated
cultures was expressed as the percentage of untreated controls.
Electrophoresis and Western Blotting.
Western blot analysis was performed essentially as described previously
(38)
. In brief, from each sample, 25 µg of protein per
lane were separated on 8–15% SDS-PAGE and electroblotted onto
polyvinylidene difluoride membranes (Amersham, Braunschweig, Germany).
Subsequently, membranes were incubated overnight at 4°C in a solution
of PBS supplemented with 5% nonfat dry milk. For immunodetection, the
following antibodies were used: anti-caspase-3, anti-caspase-7, and
anti-FADD mouse mAbs (Transduction Laboratories, Lexington, KY);
anti-caspase-8 mAb (Immunotech, Marseille, France); anti-Flag mAb
(Stratagene, La Jolla, CA); anti-cytochrome c mAb and
anti-caspase-9 polyclonal antibody (PharMingen, San Diego, CA);
anti-Myc mAb and anti-Bcl-xL polyclonal antibody
(Santa Cruz Biotechnologies, Santa Cruz, CA), Bcl-2 mAb (Dako, Santa
Barbara, CA), and rabbit Apaf-1 polyclonal antibody (provided by Dr.
Xiadong Wang). After 2-h incubation with the primary antibody in
dilutions that ranged from 1:500 to 1:2000, membranes were washed in
TBST [10 mM Tris-HCl (pH 8.0), 0.15
M NaCl, and 0.05% Tween 20], followed by
horseradish peroxidase-conjugated goat-antimouse or goat-antirabbit
antibody. ECL (Amersham, Braunschweig, Germany) was used for
detection, and protein expression was quantified by densitometry of
autoradiographs (Bio-Rad, Model GS-690, Imaging densitometer,
Richmond, CA). Protein loading equivalence was assessed by the
expression of ß-actin.
Preparation of Cytosolic Extracts.
Cytosolic extracts were prepared essentially as described by Deveraux
et al. (28)
. Briefly, cells were pelleted by
centrifugation after being washed once with ice-cold buffer A
[20 mM HEPES (pH 7.5), 10 mM KCl, 1.5
mM MgCl2, 1 mM EDTA, and 1
mM DTT]. Subsequently, cell pellets were resuspended in
two volumes of buffer A, incubated for 20 min on ice, and disrupted by
20 passages through a 26-gauge needle. Cell extracts were clarified of
mitochondria by centrifugation at 20,000 x g
for 30 min at 4°, and the harvested cytosolic extracts were stored at
-80°C.
Fluorimetric Assay for Caspase Activity.
Spectrofluorimetric assays of proteolytic activity were carried out
using synthetic fluorogenic substrates DEVD-AFC to measure
caspase-3-like or effector caspase activity and IETD-AFC to assess
initiator caspase activity. Both kits were purchased from Clontech
Laboratories Inc. (Palo Alto, CA). Assays for caspase-9 activity were
performed using the synthetic fluorogenic substrate LEHD-AFC
(MBL Co., Nagoya, Japan). Experiments were performed according
to the manufacturer’s protocols. Fluorescence was detected using a
fluorometer equipped with a 400-nm excitation and a 505-nm
emission filter (Spectra Fluor Tecan, Salzburg, Austria).
Fold-increase in the protease activity was determined by comparing the
levels of the treated cells with untreated controls. The values
obtained are depicted in Figs. 1E
, 4F
, 6C
, and 7B
as fluorescence units
(F.U. x 106).
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RESULTS |
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and exposed to cisplatin.
Interestingly, as shown in Fig. 1B
, caspase-9S did not block
drug-induced apoptosis in H460 cells (P = 0.72). In line with this observation, overexpression of the caspase-9
inhibitor XIAP led only to minimal protection against drug-induced
apoptosis (Fig. 1B
; P = 0.30). The
lack of effect of overexpression of caspase-9S and XIAP on apoptosis
was independent of the anticancer agent (cisplatin, topotecan, and
gemcitabine), drug concentration (IC50 and
IC80), duration of exposure (4–72 h), or clone
used in the experiments (data not shown). Moreover, the caspase-3-like
or effector activity induced by these drugs was not affected in cells
overexpressing caspase-9S (P = 0.27), and
only slightly decreased in cells transfected with XIAP (Fig. 1C
; P = 0.20), which suggests that
the effector activity triggered by chemotherapy is
caspase-9-independent in H460 cells. The small percentage of inhibition
provided by the overexpression of XIAP on drug-induced apoptosis and
DEVD-AFC cleavage, compared with the absence of blockade achieved by
the overexpression of caspase-9S, might be interpreted as an additional
effect of XIAP on caspases-3 and -7, also known targets of this IAP
member (28)
. As shown in Fig. 1D
, H460 cells
express similar levels of caspase-9 and Apaf-1 as compared with
Jurkat-T-leukemia cells, used as a control due to their
well-established dependency on caspase-9 activation for
chemical-induced apoptosis (28)
. However, upon exposure to
chemotherapy, no substantial cleavage of LEHD-AFC, a preferential
substrate for caspase-9 (42)
, was observed above the
control levels in H460 cells (P = 0.2),
whereas, in Jurkat-T-leukemia cells, a 6-fold increase was found (Fig. 1E
; P = 0.005). Interestingly, as
shown in Fig. 1A
, the overexpression of caspase-9S in H460
cells was accompanied by a reduction in the levels of endogenous
caspase-9. Nevertheless, the overexpression of caspase-9S and the
reduction on the caspase-9 protein had no effect on the levels of
LEHD-AFC cleavage induced by chemotherapy in these cells when compared
with H460 cells transfected with an empty vector (data not shown).
Taken together, these results indicate that, in H460 cells, caspase-9
is not instrumental in mediating chemotherapy-induced apoptosis.
Inhibition of Caspase-8 Suppresses Drug-induced Apoptosis.
In a previous study, we observed chemotherapy-induced caspase-8
activation that was independent of Fas/FasL signaling in NSCLC cells
(38)
. To further examine the relation between caspase-8
activation and drug-induced apoptosis, we engineered H460 cells to
overexpress the caspase-1 and -8 inhibitor CrmA, or a mutated and
inactive derivative, CrmA-mut (32)
, as a control (Fig. 2A)
. CrmA has been shown to be
a potent inhibitor of apoptosis induced by the TNF family of receptors
but a poor inhibitor of apoptosis triggered by other stimuli, such as
chemotherapeutic agents (32
, 43)
. In contrast to these
reports, an analysis of apoptosis in H460 cells overexpressing CrmA
demonstrated at least 90% protection against cisplatin-induced cell
death (P = 0.002), whereas no protection was
observed in cells transfected with the loss-of-function CrmA-mut
(P = 0.65), or with the empty vector (Fig. 2B)
. The inhibitory effect of CrmA are known to be
mediated by pseudosubstrate sequences in the protein that are
preferential for caspase-1 and -8 (32)
; however, some
effects on caspases-9, -10, and -4 cannot be completely discarded
(44)
. Therefore, we examined the effect of expression of
DN-acting caspase 8, a more specific inhibitor of caspase-8
(35)
, on chemotherapy-induced apoptosis in H460 cells
(Fig. 2C)
. Stable transfection with caspase-8-DN blocked
70% of drug-induced apoptosis (Fig. 2
D;
P = 0.0031), thus further substantiating the
results obtained with CrmA and pointing to a crucial role of caspase-8
in this process. In both CrmA- and caspase-8-DN-overexpressing H460
cells, a similar inhibitory pattern was observed on use of other drugs
(topotecan and gemcitabine), drug concentration
(IC50), or prolonged times of exposure (data not
shown). Furthermore, the results were consistent in independently
selected transfectants that overexpressed either CrmA or caspase-8-DN,
or when the annexin V and 7-AAD double-staining method was used to
assess apoptosis (results not shown).
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). As
expected, FADD-DN blocked more than 80% of Fas-induced apoptosis in
H460 cells; however, it provided no protection against
chemotherapy-induced cell death (P = 0.22;
Fig. 3B
). Furthermore, as determined by Western blotting,
cleavage of caspase-8 was not suppressed in cells overexpressing
FADD-DN (Fig. 3C)
. These findings demonstrate that caspase-8
activation and concomitant drug-induced apoptosis in H460 cells do not
require the activity of FADD-containing receptor signaling complexes.
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, increased levels of
cytochrome c are clearly evident in the cytosolic fraction
48 h after treatment. This release preceded caspase activation
because exposure of the cells to the preferential caspase-8-inhibitor
IETD-fmk or the broad-spectrum caspase inhibitor zVAD-fmk did not block
this process (Fig. 4A)
. Next, cytochrome c
release was monitored in H460 cells stably expressing CrmA or the
antiapoptotic Bcl-2 or Bcl-xL proteins (Fig. 4B)
. CrmA did not affect cytochrome c release;
however, this release was almost completely blocked in Bcl-2- and
Bcl-xL-expressing cells in comparison with
untreated controls (Fig. 4C)
. As predicted from this
finding, cisplatin-induced apoptosis was strongly suppressed by both
Bcl-2 and Bcl-xL (Fig. 4D)
; in
Bcl-2-expressing cells, a complete block of apoptosis was detected
(P = 0.001), whereas
Bcl-xL overexpression resulted in an
approximately 85% reduction in cell death (P = 0.006). Moreover, Bcl-2 also blocked the processing of caspase-8
and IETD proteolytic activity (Fig. 4, E and F)
.
Similar results were obtained on the use of topotecan or gemcitabine in
these experiments (data not shown).
It has been reported that caspase-8 can be activated by caspases-9 in a
mitochondria-controlled manner (29)
. Although we showed
that caspase-9 is not involved in the apoptotic process in H460 cell
(Fig. 1)
, we wanted to further exclude this alternative. In H460 cells
that overexpressed caspase-9S, caspase-8 cleavage remained clearly
detectable after cisplatin treatment (Fig. 5)
. Taken together, these data provide
strong evidence for the existence of a mitochondria-dependent pathway
that is able to activate procaspase-8, independently from caspase-9,
during anticancer drug-induced apoptosis.
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, this inhibitor had no
effect on caspase-8 cleavage, although we could observe a 30–50%
decrease in apoptosis (result not shown). In addition, a more
downstream event, such as PARP cleavage, was prevented by
overexpression of CrmA and caspase-8-DN (Fig. 6B)
, likely
attributable to the suppression of DEVD-like activity triggered by
cisplatin exposure in these stable transfectants (Fig. 6C)
.
These results show that effector caspases are activated after the
cleavage of procaspase-8 in drug-induced apoptosis in H460 cells.
Drug-induced Apoptosis Is Inhibited by Caspase-8 Blockade in
Different NSCLC Cell Lines.
We further investigated whether the findings that indicated a central
role for caspase-8 in chemotherapy-induced apoptosis in H460 cells
would apply also to other NSCLC cell lines. Therefore, H460 cells,
together with the NSCLC cell lines H322 and SW1573, were exposed to
both cisplatin and the preferential caspase-8 inhibitor IETD-fmk. The
extent of blockade of drug-induced apoptosis was analyzed in comparison
with the effect obtained with the broad-spectrum caspase inhibitor
zVAD-fmk. Jurkat-T-leukemia cells were selected for use in these
experiments as a control, because the pattern of inhibition of
chemotherapy-induced apoptosis provided by IETD-fmk and zVAD-fmk has
been reported in these cells (31
, 45)
. As shown in Fig. 7A
, a similar pattern of
suppression of chemotherapy-induced apoptosis (50–60%) was provided
by IETD-fmk in all of the three NSCLC lines analyzed
(P = 0.025). Interestingly, the protection
provided by IETD-fmk was similar to the blockade obtained with the
pancaspase inhibitor zVAD-fmk, which again suggested a crucial role of
caspase-8 (Fig. 7A)
. In contrast, in Jurkat cells, IETD-fmk
provided no protection (P = 0.2), whereas
zVAD-fmk blocked at least 85% of drug-induced apoptosis
(P = 0.006). These results were reproducible
on using other drugs (topotecan or gemcitabine), concentrations
(IC50 or IC80), or time
point for analysis (data not shown). We also analyzed the cleavage of
IETD-AFC as a marker of caspase-8 activity in the NSCLC cell lines
H460, H322, and SW1573 during cisplatin- and Fas-induced apoptosis. The
findings were then compared with the results observed in Jurkat cells
under the same experimental conditions. Fas-induced death was used as a
marker for caspase-8 activity during apoptosis. As shown in Fig. 7B
, the amount of IETD-AFC cleavage generated during
cisplatin and Fas-induced apoptosis was similarly high in all of the
three NSCLC cell lines analyzed. In contrast, in Jurkat cells, Fas
exposure led to a substantial increase in IETD-AFC cleavage, whereas
exposure to cisplatin had hardly any effect on caspase-8 activity (Fig. 7B)
. These results are consistent with the findings observed
with the caspase-inhibitor IETD-fmk. We thus conclude that our findings
pointing to an important role for caspase-8 in chemotherapy-induced
apoptosis in H460 cells can be likely extrapolated to other NSCLC
cells.
Blocking Caspase-8 Favors Clonogenic Survival in H460 Cells on Drug
Treatment.
Finally, to further examine the relevance of caspase-8 in
drug-induced cytotoxicity in H460 cells, we questioned whether the
inhibition of this caspase would simply delay cell death or whether it
would be able to rescue cells, favoring clonogenic survival. Clonogenic
growth assays were performed in which control vector-transfected cells,
together with CrmA and caspase-8-DN overexpressing cells, were exposed
to IC50 and IC80
concentrations of anticancer drugs. CrmA- and
caspase-8-DN-expressing cells showed a significant increase in
clonogenic potential when compared with cells transfected with the
empty vector or the parental line (Table 2
; P = 0.015
and 0.025, respectively). Similar observations were made in H460 cells
overexpressing Bcl-2 or Bcl-xL
(P = 0.006). In contrast, stable expression
of the caspase-9 inhibitors, caspase-9S and XIAP, did not increase
clonogenic survival (Table 2)
, being consistent with their inability to
suppress drug-induced apoptosis.
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DISCUSSION |
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Different lines of experimentation clearly indicate the importance and
apical role of caspase-8 and the lack of function of caspase-9 in
chemotherapy-induced apoptosis in H460 cells. Firstly, stable
overexpression of the caspase-9 inhibitors caspase-9S and XIAP in the
NSCLC cell line H460 failed to suppress apoptosis that was induced by
various chemotherapeutic drugs, which correlated with the observed lack
of caspase-9 activity in these cells (Fig. 1)
. Secondly, drug-induced
apoptosis was blocked in H460 transfectants that expressed known
inhibitors of caspase-8, e.g., CrmA and caspase-8 DN
(Fig. 2)
. These results are in line with the observation that the
preferential caspase-8 peptide-inhibitor IETD-fmk blocked
druginduced apoptosis in NSCLC cells (Fig. 7A)
. The
similar blockade of drug-induced apoptosis induced by IETD-fmk and the
substantial amount of cleavage of IETD-AFC generated by exposure to
chemotherapy in three distinct NSCLC cell lines suggest that our
results may be characteristic of NSCLC cells, rather than being cell
type-specific phenomena related to H460 cells. In addition, these
findings are in contrast to the results obtained with drug-induced
apoptosis in Jurkat cells, which we included as control in our studies
(Figs. 1
and 7)
. These findings in Jurkat cells are in line
with a previous report in HL-60 cells (31)
. Our findings
in H460 cells expressing CrmA are at odds with the reported lack of
suppression of drug-dependent apoptosis by CrmA in lymphoid cells
(32
, 47) but are in line with another report showing a
blockage of drug-induced by CrmA in leukemia cells (48)
. A
possible explanation for this discrepancy may be an effect of CrmA on
other caspases. In fact, when analyzing our results, we have to take
into consideration that both CrmA and chemical caspase inhibitors like
IETD-fmk have been shown to inhibit caspases other than caspase-8 in
cell-free systems (44)
. However, the confirmation of our
findings in H460 cells overexpressing caspase-8-DN gives weight to our
data and point to a crucial role of caspase-8 in the process of
drug-induced apoptosis in the NSCLC cell line H460.
Some investigators have reported the activation of procaspase 8
in drug-induced apoptosis. However, in these studies, the activation of
caspase-8 by chemotherapy appeared to be a mere bystander effect and
not a key event in the execution of the apoptotic program
(29, 30, 31
, 49)
. An alternative pathway for
chemotherapy-dependent activation of caspase-8 has been proposed in
leukemia, neuroblastoma, and colon cancer cells and would be based on
Fas/FasL signaling (50, 51, 52)
. However, we and others have
been unable to substantiate this model (30
, 38 , 45
, 53, 54, 55)
; and, moreover, our current finding that drug-induced
apoptosis in H460 cells is not inhibited by FADD-DN overexpression
further demonstrates that death receptors are not involved in mediating
this response (Fig. 3)
. In H460 cells, caspase-8 activation appears to
be an apical event in the execution phase, because we conclude
from our finding that this activation precedes the
chemotherapy-mediated increase in caspase-3-like activity and the PARP
cleavage that take place independently from caspase-9 activation (Figs. 1
and 6)
.
Caspase-8 Activation in H460 Cells Is Mitochondria-dependent.
In analogy with the mechanisms that can activate caspase-9, we studied
the role of mitochondria in drug-dependent caspase-8 activation in H460
cells. On the basis of the following observations, we conclude that
this activation requires a mitochondrial step (see Fig. 4
):
(a) overexpression of Bcl-2 and
Bcl-xL blocks caspase-8 cleavage as well as
IETD-AFC proteolytic activity; (b) overexpression of the caspase-8
inhibitors CrmA and caspase-8-DN fails to block cytochrome c
release from mitochondria after exposure to chemotherapy; and
(c) IETD-fmk and zVAD-fmk do not block cytochrome
c release.
We are aware of one earlier report (56)
describing
mitochondria-controlled caspase-8 cleavage in neuroblastoma cells in
which, on exposure to betulinic acid, caspase-8 was activated
by apoptosis-inducing factor. Furthermore, in analogy with the
mechanism of drug-dependent caspase-9 activation, a possibility would
be that Apaf-1, together with cytochrome c and caspase-8
could form an alternative apoptosomal complex. This possibility is not
unprecedented, inasmuch as caspase-8 has been found to be able to
interact with Apaf-1, although these findings are under dispute
(29
, 57)
. In addition, caspase-8 has been shown to be able
to cleave and, thus, activate all known caspases in vitro
(58)
, which may be taken as an additional evidence for its
candidacy as an initiator caspase in death-receptor-independent types
of apoptosis (7)
. We are presently further exploring this
potential mechanism of caspase-8 activation in NSCLC cells. Another
question to be addressed is why NSCLC cells use a caspase-8-dependent
pathway instead of the more commonly used caspase-9 route. When we take
into consideration that caspase-9 and Apaf-1 are expressed at normal
levels in H460 cells (Fig. 1D)
, our findings suggest that
caspase-9 itself or some component of the complex that is responsible
for its activation may be constitutionally inhibited or nonfunctional
in H460 cells. To the best of our knowledge, the present report
provides the first indication for the existence of a constitutional
mechanism of inhibition at this level in cancer cells. Possible means
that render caspase-9 nonfunctional in these cells are currently under
investigation in our laboratory.
Implications of caspase-8-mediated Apoptosis in the Treatment of
NSCLC.
An important issue that is disputed in cancer research concerns the
correlation between chemotherapy-induced apoptosis and clonogenic
survival (59)
. Controversial data have been generated not
only about upstream members of the death machinery—such as p53, p21,
and c-myc (9
, 60, 61, 62)
—but also about caspases. The use of
peptide inhibitors of caspases was shown to abrogate the morphological
hallmarks of apoptosis; however, peptide inhibitors failed to
interfere with the final commitment of cells to die (63)
.
In contrast, a correlation between apoptosis and clonogenic survival
has been reported in cells overexpressing CrmA (32)
. In
this study, we also found a close correlation between the suppressive
effect of overexpression of the caspase-8 inhibitors, caspase-8-DN, and
CrmA, on drug-induced cell death and the clonogenic outgrowth of H460
cells. Taken into a broader perspective, our results suggest that
constitutional mechanisms of caspase inhibition may have a considerable
impact on the resistance of cancer cells to chemotherapy, because, upon
caspase blockade, cells were allowed to proliferate and were not doomed
to die by an alternative or delayed mechanism.
In conclusion, our findings in H460 cells provide a novel picture of the events involved in the apoptosis induced by anticancer agents that challenges the assumption that chemotherapy-induced apoptosis is preferentially mediated in a caspase-9-dependent manner. We demonstrate that after mitochondria activation by anticancer drugs, caspase-8 is activated in a caspase-9-independent manner, and, in turn, mediates the activation of effector caspases leading to apoptosis. In fact, because of differences from the model reported in other cell types, our data suggest that the caspase pathway activated might vary not only according to the sort of stimuli, but also depending on the cellular context, as proposed previously (9) . These findings may help to explain the difference in chemosensitivity between NSCLC cells and leukemia cell lines cells, and it would be particularly interesting if these observations can be confirmed in other solid tumor lines.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 C. G. F. is supported by a personal grant from
Capes (Fundação Coordenação de
Aperfeiçoamento de Pessoal de Nível Superior)
Brasília, Brazil. 
2 To whom requests for reprints should be
addressed, at the Department of Medical Oncology, University Hospital
Vrije Universiteit, De Boelelaan 1117, 1081HV Amsterdam, the
Netherlands. Phone: 31-20-444-4300; Fax: 31-20-444-4079; E-mail: g.giaccone{at}azvu.nl
3 The abbreviations used are:
NSCLC, non-small cell lung cancer; TNF, tumor necrosis factor; TRAIL,
TNF-related apoptosis-inducing ligand; FADD, Fas-associated death
domain; IAP, apoptosis-inhibiting protein; XIAP, X-linked IAP; CrmA,
cytokine response modifier A; AFC, 7-amino-4-trifluoromethyl coumarin;
DEVD-AFC, benzyloxycarbonyl-Asp-Glu-Val-APC; IETD-AFC,
benzyloxycarbonyl-Ileu-Glu-Thr-Asp-AFC; zVAD-fmk,
carbobenzoxy-Val-Ala-Asp-fluoromethyl ketone; IETD-fmk,
Z-lle-Glu(Ome)-Thr-Asp(Ome)-fluoromethyl ketone; Apaf-1, apoptotic
protease-activating factor 1; NCI-H460, H460; DN, dominant negative;
WT, wild type; PI, propidium iodide; 7-AAD, 7-amino-actinomycin D; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; mAb.
monoclonal antibody; FasL, Fas ligand; PARP, poly(ADP-ribose)
polymerase; caspase-9S, caspase-9 short form.
Received 4/18/00. Accepted 10/30/00.
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