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Tumor Biology |
Lurie Cancer Center, Northwestern University Medical School [X-H. Y., A. D. T.] and Departments of Medicine [X-H. Y., X. L., C. J. F.] and Pathology [B. R. B., A. D. T.], Evanston Northwestern Healthcare, Evanston, Illinois 60201, and Department of Microbiology/Immunology, Finch University of Health Sciences/The Chicago Medical School, North Chicago, Illinois 60064 [T. L. S.]
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Caspase 3 (also known as cpp32, yama, and apopain) is a key caspase in
this signaling cascade (8, 9, 10, 11, 12)
. Caspase 3 activity has
been detected in apoptosis induced by a variety of apoptotic signals,
including death receptor activation (13)
, growth factor
deprivation (14)
, ionizing radiation (15)
,
and treatment with granzyme B (16)
or different
chemotherapeutic agents (17)
. Caspase 3 knockout mice
displayed abnormal brain tissue development due to lack of apoptosis
(18)
. A growing number of substrates cleaved by caspase 3
have been identified, such as
PARP3
(10)
, sterol-regulatory element-binding protein
(19)
, gelsolin (20)
, the U1-associated
Mr 70,000 protein
(21)
, D4-GDI (22)
, DFF (23)
,
DNA-dependent protein kinase 
and 
(24
, 25)
,
-fordrin (26)
, and huntingtin (27)
.
Caspase 3 is believed to play a pivotal role in apoptotic execution.
Alterations in apoptosis-associated genes are often observed in
cancers. The p53 tumor suppressor gene, a key
regulator in DNA damage-induced apoptosis, is frequently mutated in
human tumors (28)
. Overexpression of apoptotic inhibitors,
such as bcl-2 and bcl-xL (29
, 30)
,
and down-regulated apoptosis-promoting factors, such as Bax-
and Fas (31
, 32)
, has been detected in primary tumors and
tumor cell lines. These alterations have been linked to
chemotherapeutic resistance (31
, 33)
. Correction of these
alterations has resulted in sensitization of the defective cells to
chemotherapeutic agents (34)
.
Caspase 3 deficiency was recently detected in MCF-7 breast cancer cells. It is due to a deletion mutation in exon 3 of the gene (35) . Overexpression of caspase 3 in MCF-7 cells indicates that caspase 3 plays a critical role in both death receptor- and mitochondria-mediated apoptotic pathways (35, 36, 37, 38) . Given the important role of caspase 3 in apoptotic execution and the correlation between the alterations of other apoptotic regulators and chemotherapeutic resistance, we postulated that caspase 3 deficiency might also significantly contribute to chemotherapeutic resistance. Although caspase 3-like activity has been detected in the apoptosis induced by various chemotherapeutic drugs (17) , the specific role of caspase 3 in this process warrants further investigation due to the overlapping activities among effector caspases (18 , 39) . To evaluate the role of caspase 3 in chemotherapy-induced apoptosis, we reconstituted caspase 3 in MCF-7 cells and characterized the apoptotic responses of the MCF-7 cells to doxorubicin and etoposide in comparison with control cells. We found that reconstitution of caspase 3 significantly sensitized MCF-7 cells to doxorubicin- and etoposide-induced apoptosis.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Drug Treatment and Sample Collection.
For doxorubicin (Bedford Laboratories, Bedford, OH) and etoposide
(Bristol-Myers Squibb Co., Princeton, NJ) treatments in studies other
than the MTT assay (see below), 1 x 106 cells were seeded into 60-mm dishes 24 h
before drug treatment. Various doses were added to the dishes 18 h
before cell collection. Treated cells to be analyzed by flow cytometry
and DAPI staining were trypsinized. Cells to be analyzed by DEVD
(Asp-Glu-Val-Asp) cleavage assay and Western blot were scrapped off the
dish. In all cases, medium from individual dishes, which might contain
floating dead cells, was collected and mixed with the cell pellet from
the same dish.
MTT Survival Assay.
Three hundred cells were placed into each well of 96-well plates.
Twenty-four h later, the medium was replaced with new medium containing
defined doses of doxorubicin or etoposide. Six days after treatment,
the medium was changed with phenol red-free medium containing 500
µg/ml MTT (Sigma). Three h after incubation, MTT-containing medium
was removed. The incorporated dye was dissolved in 100 µl/well DMSO,
and the plates were read at the wavelength of 570 nm using an ELISA
reader. Absorbance in the treated cells was expressed as a percentage
of control. Eight parallel samples were treated in each concentration
point. Five separate experiments were performed.
DEVD Cleavage Assay.
Drug-treated cells were washed with PBS and resuspended in lysis buffer
[50 mM Tris-HCl (pH 8.0), 130 mM KCl, 1
mM EDTA, 10 mM EGTA, and 10 µM
digitonin] at 320 µl/60-mm dish. After incubation at 37°C for 10
min, the samples were spun for 3 min (5000 rpm), and the supernatant
was collected. After adding 100 µl of lysate to each well of a
fluorometer plate, 100 µl of substrate solution, 2 µM
DEVD-AMC (PharMingen, San Diego, CA) in lysis buffer was added
right before the reading. Fluorescence was measured in a microplate
fluorometer (Cambridge Technology, Cambridge, MA) using an
excitation wavelength of 360 nm and an emission wavelength of
460 nm. Results are reported as the fluorogenic activity over 1 h
(T60 to
T0). Samples were prepared in
triplicate.
Western Blot.
PBS-washed cells were treated with lysis buffer (41)
on
ice for 30 min. Lysed cells were centrifuged at 14,000 rpm for 10 min
to remove cellular debris. Protein concentrations of the supernatant
were determined using BCA Protein Assay (Pierce, Rockford, IL). Fifty
µg of cell lysate were loaded onto each lane of a gel. Protein was
separated by either 10% or 15% SDS-PAGE and transferred to
nitrocellulose membranes. The membranes were blocked with TBS-T (5%
milk in Tris-buffered saline-Tween 20) washing buffer (41)
and probed with specific primary antibodies. Concentrations of the
primary antibodies used ranged from a 1:500 dilution to a 1:2,000
dilution. Antibodies against caspases 3 and 7 were purchased from
Transduction Laboratories (Lexington, KY). Antibodies against caspase 6
and DFF were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The
anti-lamin B antibody was from PharMingen. The anti-PARP antibody was
from Boehringer Mannheim (Indianapolis, IN). Washed membranes were then
probed with horseradish peroxide-labeled antimouse, antirabbit, or
antigoat secondary antibodies (Amersham Pharmacia, Arlington Heights,
IL), respectively. The membranes were washed again and treated with
enhanced chemiluminescence reagents (Amersham Pharmacia). The specific
protein bands were visualized by autoradiography (41)
.
Flow Cytometry.
Drug-treated cells were trypsinized and washed with PBS. The cells were
then fixed in 50 µl of 0.125% paraformaldehyde in PBS at 37°C for
5 min, followed by the addition of 450 µl of ice-cold methanol to
each sample. After being washed three times with PBS containing 0.1%
Triton X-100 and treated with RNase A (0.04 Kunitz units) for 30 min,
the cells were stained with 50 µg/ml propidium iodide. Cell analysis
was performed using a Coulter Epics 751 flow cytometer. The fraction of
the total cell population present in the G1, S
phase, G2-M phase, and hypodiploid peak was
obtained from DNA histograms by mathematical modeling using MPLUS
software (41)
.
Nuclear Staining.
Drug-treated cells were collected and washed with PBS followed by
fixation with 2% paraformaldehyde at 4°C for 30 min. The cells were
stained with 0.5 µg/ml DAPI for 30 min. Stained cells were then
washed and mounted on slides using a cytospinner. Nuclear morphology of
the cells was visualized using an Olympus fluorescence microscope.
IC50 Determination and Statistical Analysis.
For IC50 determination from the MTT assay,
nonlinear regression analysis was performed with Cricket Graph software
to generate curves for IC50 calculation.
Significance evaluation was performed by the paired t test.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, the protein levels of caspase 3 in MCF-7/pv, MCF-7/c3, and Jurkat cell
controls were detected by Western blot. The caspase 3-specific antibody
detected a strong protein band with a molecular weight of about 32,000
in MCF-7/c3 and Jurkat cells but not in MCF-7/pv cells. Caspase 3
levels reconstituted in MCF-7/c3 cells were comparable to those in
Jurkat cells that express high levels of caspase 3. The results below
demonstrate that the reconstituted caspase 3 was functional. We have
since maintained MCF-7/c3 cells in culture for over 1 year, and the
cells have shown stable expression of high levels of caspase 3.
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. The results indicated that MCF-7/c3 cells were significantly
sensitized to both drugs (P < 0.01 for
doxorubicin; P < 0.05 for etoposide). This
suggests that caspase 3 reconstitution sensitized MCF-7 cells to
doxorubicin and etoposide treatments.
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-treated group or 400 µM etoposide
(Fig. 2B)
-treated group, MCF-7/c3 cells displayed diffused
apoptosis as compared with MCF-7/pv cells, which showed only sporadic
islands of cell death.
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, DEVD cleavage activity in drug-treated MCF-7/pv cells was very
limited, even in the cells treated with 5 µM doxorubicin
or 200 µM etoposide. However, DEVD cleavage activity in
MCF-7/c3 cells increased over 10–20-fold when the cells were treated
with 200 µM etoposide or 2.5 µM
doxorubicin. The strong caspase 3-like activity in drug-treated
MCF-7/c3 cells indicates that caspase 3 expressed in MCF-7/c3 cells was
functional and that activation of reconstituted caspase 3 contributed
to the sensitization.
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. Although Western blotting was less
sensitive than DEVD cleavage assay, the results obtained using either
method were consistent with each other. Because caspases 6 and 7 are
commonly activated in different apoptosis, we compared the extent and
pattern of their activation between MCF-7/pv and MCF-7/c3 cells. In
drug-treated MCF-7/pv cells, which were deficient of caspase 3, caspase
7 processing/activation was minimal (Fig. 4, A and B)
. In contrast, activation of caspase 7 in MCF-7/c3 cells
was remarkably increased when the cells were treated with 10 and 50
µM doxorubicin or 200 and 400
mM etoposide, as indicated by the formation of
p32 and p20 fragments. These results indicate that caspase 7 activation
in doxorubicin- and etoposide-treated cells was primarily caspase 3
dependent. These observations were consistent with our reported finding
of granzyme B-induced apoptosis (16)
.
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, a p32
band product was identified in doxorubicin-treated MCF-7/pv cells but
not in MCF-7/c3 cells, consistent with caspase 6 activation at low
levels in the absence of caspase 3. Reconstitution of caspase 3,
however, significantly enhanced caspase 6 activation at both 10 and 50
µM doxorubicin. Because the combined size of
pLarge and pSmall subunits of caspase 6 is about
Mr 32,000 (42)
, the
appearance of a p32 band in MCF-7/pv cells suggests that caspase 6 was
processed by a caspase other than caspase 3 between the propeptide and
pLarge subunit. The disappearance of the p32 band and an increase in
the pLarge subunit (p20) in treated MCF-7/c3 cells suggests that
caspase 3 processes caspase 6 between the pLarge and the pSmall
subunits. In etoposide-treated MCF-7/c3 cells, the extent of caspase 6
activation was not as great as that observed in doxorubicin-treated
MCF-7/c3 cells (Fig. 4B)
. However, a cleavage product with a
size around Mr 30,000 appeared
specifically in etoposide-treated MCF-7/c3 cells. Disappearance of the
p32 band in MCF-7/c3 cells treated with 800 µM
etoposide also suggests the cleavage between pLarge and pSmall
subunits. Taken together, these results support that activation of
caspase 3, as well as the subsequent activation of caspases 6 and 7,
contributed to the sensitization in MCF-7/c3 cells.
Cleavage of Cellular Death Substrates in MCF-7/c3 Cells.
Because proteolytic cleavage of cellular death substrates by activated
caspases is responsible for the cellular dysfunction and structural
destruction of apoptosis (6)
, we studied the cleavage of
PARP, lamin B, and DFF as representative substrates in the control and
reconstituted cells. As shown in Fig. 5
, there was only limited cleavage of all three substrates in MCF-7/pv
cells treated with either drug (even when the doxorubicin concentration
was as high as 50 µM, and the etoposide concentration was
as high as 400 µM). In contrast, all three substrates
were significantly or even completely cleaved in the drug-treated
MCF-7/c3 cells. These results are further evidence supporting a pivotal
role for caspase 3 in chemotherapy-induced apoptosis.
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-induced apoptosis
(35)
. To examine the effect of caspase 3 reconstitution on
DNA fragmentation and nuclear morphology in doxorubicin- and
etoposide-induced apoptosis, we analyzed these changes in drug-treated
MCF-7/c3 cells and control MCF-7/pv cells. Flow cytometry analysis
detected significant DNA fragmentation (the hypodiploid peak) only in
drug-treated (doxorubicin or etoposide) MCF-7/c3 cells (Fig. 6, A and B)
. Nuclear morphology corresponding to
DNA fragmentation was verified using DAPI staining of the treated
cells. In contrast to drug-treated MCF-7/pv cells, which only displayed
nuclear condensation in apoptotic cells, apoptotic MCF-7/c3 cells
showed typical nuclear fragmentation (Fig. 6C)
. These
results suggest that caspase 3 was also required for DNA and nuclear
fragmentation in chemotherapy-induced apoptosis.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Doxorubicin and etoposide are active chemotherapeutic agents used in clinical oncology. Doxorubicin is a key adjuvant drug for breast cancer treatment. It triggers apoptosis through several mechanisms. As with many chemotherapeutic agents, it induces DNA damage by interacting with topoisomerase II, leading to DNA breakage (43) . It can also induce membrane alterations and the generation of ceramide at higher concentrations (44) . Recently, it has been reported that up-regulation of the Fas/Fas ligand system may also be involved in doxorubicin-mediated killing (45) . For etoposide-induced apoptosis, DNA damage secondary to topoisomerase II inhibition appears to be a major mechanism (46) . Despite the variance in the chemotherapeutic initiation process, the resulting release of cytochrome c from mitochondria followed by activation of caspase 9 and the effector caspases is believed to be the final common pathway in chemotherapy-induced cell death (38 , 47 , 48) . Microinjection of cytochrome c induced apoptosis in 293 cells with functional caspase 3 or caspase 3-transfected MCF-7 cells but not in caspase 3-deficient MCF-7 cells (36) , indicating that caspase 3 was required for cytochrome c-mediated apoptosis. Abrogation of mitochondrial cytochrome c release and caspase 3 activation have been associated with acquired multidrug resistance (49) . As shown in this presentation, caspase 3 reconstitution restored the integrity of the doxorubicin- and etoposide-induced killing mechanism. This direct evidence links caspase 3 deficiency and chemotherapeutic efficacy, suggesting caspase 3 defects as one mechanism for chemoresistance.
Activation of caspase 3 in chemotherapy-induced apoptosis has been
reported by many groups (17
, 38
, 47
, 50
, 51)
. Involvement
of caspase 3 in this process was shown either by detecting its
activation as a representative of effector caspases (38
, 50
, 52)
or by using synthetic inhibitors, such as DEVD-CHO
(51
, 53)
, to block caspase 3-like activities.
Nevertheless, little work has been done to differentiate the role of
caspase 3 from that of other effector caspases in this process. In our
experiments, comparison between caspase 3-deficient and -reconstituted
cell lines more specifically defined the specific role of caspase 3 in
doxorubicin- or etoposide-induced apoptosis and in the activation of
other effector caspases. Although caspases 3, 6, and 7 are all
categorized as effector caspases (4
, 5)
, our results
demonstrated an additional apical-like nature of caspase 3. These data,
derived from a whole cell system (in contrast to a cell-free system),
show that activation of caspase 6 and especially caspase 7 was largely
dependent on caspase 3 activation (Fig. 4)
. Although caspase 6
activation was detected in caspase 3-deficient cells, efficient
activation of caspase 6 required caspase 3 activity (Fig. 4)
. As a
result, by direct cleavage and amplification through the activation of
other effector caspases, caspase 3 reconstitution led to a striking
increase in death substrate cleavage and DNA fragmentation (Figs. 5
and 6)
. Our preliminary results showed that reconstituted caspase 3 also
had feedback effects on its upstream
factors.4
Although our experiments were based on an in vitro cell line model, our data are consistent with a recent report that was based on an in vivo model. Using a rat AH130 liver tumor model, Yamabe et al. (54) found that transduction of human caspase 3 in combination with etoposide administration induced extensive apoptosis and significantly reduced tumor volume, as compared with the group with caspase 3 transduction or etoposide treatment alone. Although our focus was on reconstitution of caspase 3, and theirs was on caspase 3 overexpression-mediated therapy, both reports demonstrate that caspase 3 is critical in chemotherapy-induced apoptosis and that caspase 3 reconstitution/overexpression and chemotherapy have synergetic effects.
In our experiments, two drug treatment conditions were used. For IC50 determination using MTT assays, the cells were exposed to the drugs for 6 days. Significant sensitization was detected when drug concentrations were between 0.5 and 37 nM for doxorubicin and 0.05 and 1 µM for etoposide, respectively. This compares favorably with the plasma concentrations of the two drugs in clinical application, which could reach up to 2 µM for doxorubicin (55) and 170 µM (100 µg/ml) for etoposide (56) , respectively. To evaluate early biochemical changes, the cells were also treated for shorter periods (18 h) at much higher concentrations, although this very high dose and short duration approach is not currently clinically feasible.
One noteworthy finding was that when MCF-7/c3 cells were treated with
doxorubicin for 18 h, caspase activation and death substrate
cleavage displayed a sharp increase when the drug concentration was
increased from 2 to 10 µM (Figs. 4
and 5)
. This suggests
that there may be a concentration threshold for doxorubicin to induce
maximal caspase 3-mediated apoptosis under a given treatment condition.
This is consistent with the clinical benefit observed with dose
intensification of doxorubicin, as shown for node-positive breast
cancer patients (57)
and topical administration of
doxorubicin for ovarian cancer (58)
. Because significant
sensitization to doxorubicin was also detected in MTT assays, we agree
with Han et al. (59)
on the action model of
doxorubicin. Doxorubicin appears to induce two types of cellular
response, i.e., slow cell death at low concentrations and
rapid cell death at high concentrations. This may be due to an
increased number of activated mechanisms at higher drug concentrations.
Distinct differences in apoptotic activities between MCF-7/pv and
MCF-7/c3 cell lines in response to doxorubicin or etoposide treatment
underscore the possible significance of caspase 3 deficiency in cancer
resistance. Caspase 3 reconstitution sensitized MCF-7 breast cancer
cells to commonly applied chemotherapeutic agents, suggesting that
caspase 3 deficiency may contribute to chemotherapeutic resistance.
Caspase 3 reconstitution also sensitized MCF-7 cells to radiotherapy
and granzyme B (16)
.4
Reconstitution
of caspase 3 in MCF-7 cells may also enhance apoptosis in response to
Fas ligand and tumor necrosis factor treatment, as shown by others
(35
, 37)
. Therefore, it appears that caspase 3 deficiency
may have a broad clinical relevance, including both chemo- and
radiotherapeutic resistance and immune-associated antitumor mechanisms.
Our preliminary results show down-regulation or deficiency of caspase 3
in many breast cancer specimens,4
which
supports the non-breast cancer findings of others (60)
. We
therefore speculate that caspase alterations may be linked to poorer
prognosis and therapeutic resistance in human breast cancer.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by Career Development Award
DAMD17-99-1-9180 from the Department of Defense (to X. H. Y.),
National Cancer Institute Grant P30CA60553 (to A. D. T., Lurie Cancer
Center), The Carol Gollob Foundation, and Marvin and Lori Gollob. 
2 To whom requests for reprints should be
addressed, at Department of Pathology (A. D. T.) or Medicine
(X. H. Y.), Evanston Northwestern Healthcare, Northwestern University
Medical School, 2650 Ridge Avenue, Evanston, IL 60201. Phone:
(847) 570-2484; Fax: (847) 570-1135; E-mail: a-thor{at}nwu.edu
3 The abbreviations used are: PARP,
poly(ADP-ribose) polymerase; DFF, DNA fragmentation factor; MTT,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DAPI,
4',6-diamidino-2-phenylindole.
Received 11/ 3/99. Accepted 10/ 4/00.
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A. S. Levenson, K. M. Svoboda, K. M. Pease, S. A. Kaiser, B. Chen, L. A. Simons, B. D. Jovanovic, P. A. Dyck, and V. C. Jordan Gene Expression Profiles with Activation of the Estrogen Receptor {alpha}-Selective Estrogen Receptor Modulator Complex in Breast Cancer Cells Expressing Wild-Type Estrogen Receptor Cancer Res., August 1, 2002; 62(15): 4419 - 4426. [Abstract] [Full Text] [PDF]
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T. J. Kottke, A. L. Blajeski, X. W. Meng, P. A. Svingen, S. Ruchaud, P. W. Mesner Jr., S. A. Boerner, K. Samejima, N. V. Henriquez, T. J. Chilcote, et al. Lack of Correlation between Caspase Activation and Caspase Activity Assays in Paclitaxel-treated MCF-7 Breast Cancer Cells J. Biol. Chem., January 4, 2002; 277(1): 804 - 815. [Abstract] [Full Text]
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