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Biochemistry and Biophysics |
Department of Pharmaceutics and Pharmacodynamics, Center for Pharmaceutical Biotechnology, University of Illinois at Chicago, Chicago, Illinois 60612 [S. M., R. Y., A-N. T. K.], and Department of Microbiology and Immunology, Baylor College of Medicine, Houston, Texas 77030 [T-H. Y.]
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ABSTRACT |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, leading to growth arrest of
ER-positive cells. In addition to its cytostatic effects mediated
through the ER, TAM has also been shown to be cytotoxic to both
ER-positive and ER-negative cells (4
, 5) , and such a
cytotoxic effect is believed to be mediated by the induction of
apoptosis (6
, 7)
. Although several signaling
intermediates, such as PKC (8
, 9)
, transforming growth
factor ß (10)
, and c-myc (11)
, have been
implicated, the precise molecular mechanism of TAM-induced apoptosis
remains unclear.
One of the key events in apoptosis is the activation of a cascade of
intracellular cysteine proteases known as caspases (12)
.
To date, at least 13 caspases have been identified in mammalian cells
(13)
. These have been classified into three families,
depending on their functions in the caspase cascade (14)
.
The caspases with a death effector domain comprise caspase-8 and -10.
They are associated with the cell membrane DRs of TNF-, Fas, or
TNF-related apoptosis-inducing ligand via their death domains. The
caspases with a caspase recruiting domain include caspase-1, -4, -5,
-2, and -9. Finally, the effector or downstream caspases include
caspases 3, 6, and 7. They are activated by death effector domain
caspases or by caspases with a caspase recruiting domain by
cleavage at specific tetrapeptide residues. Caspase-3
(15)
, a downstream caspase, has been shown to play a
pivotal role in the terminal, execution phase of apoptosis induced by
diverse stimuli. On proteolytic activation by upstream caspases,
caspase-3 is able to cleave a variety of substrates, including PARP
(15)
, DNA fragmentation factor (16)
, PKC-
(17)
, and others. The cleavage of various substrates
contributes to the typical morphological and biochemical features
observed in apoptosis. Because of the diversity of its substrates,
caspase-3 is thought to be a general mediator of physiological as well
as stress-induced apoptosis.
JNK1 is a member of the of mitogen-activated protein kinase family that has been shown to regulate a number of physiological and pathological processes, including chemical-induced cell transformation (18) and apoptosis (19 , 20) . JNK1 is strongly activated by anticancer (21, 22, 23) and chemopreventive (24 , 25) agents. JNK1 activation by cytotoxic agents was shown to be mediated by oxidative or mechanical stress in the form of ROS or microtubule perturbations, respectively (21 , 22) . The molecular ordering of JNK1 and caspase pathways seems to be stimulus dependent. Induction of caspase activity by the anticancer drugs etoposide and camptothecin has been shown to be mediated by JNK1 activation (26) , whereas in doxorubicin- or death ligand-induced apoptosis, caspase activation appears to be independent of the JNK pathway (27) . Thus, although a growing body of evidence suggests that both caspase and JNK are important mediators of apoptosis induced by various agents, the relationship between these two pathways remains elusive.
In this study, we investigated the role of caspase and JNK in TAM-induced apoptosis. Our results showed that TAM-induced apoptosis in breast cancer cell lines MDA-MB-231, BT-20, and MCF-7 was mediated by both caspase-3 and JNK1.
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MATERIALS AND METHODS |
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-tocopherol), and DAPI were obtained from Sigma (St.
Louis, MO). The fluorogenic tetrapeptide substrates of caspase-3
(Ac-DEVD-MCA), caspase-1 (Ac-YVAD-MCA), and caspase-6 (Ac-VEID-MCA)
were obtained from Peptides International (Louisville, KY), and the
substrates of caspase-8 (Acetyl-Ile-Glu-Thr-Asp-methylcoumaryl-7-amide
(Ac-IETD-MCA) and caspase-9
(Acetyl-Leu-Glu-His-Asp-trifluoromethylcoumaryl-7-amide
(Ac-LEHD-FCA) and caspase inhibitors Ac-DEVD-CHO and
z-VAD-FMK were purchased from Calbiochem (San Diego, CA). Before
TAM treatment, cells (at approximately 80% confluence) were cultured
in serum-free MEM. For pretreatment, the cells were incubated with the
indicated agents for 1 h before challenge with TAM.
MTS Assay for Cell Viability.
Cells were plated at a density of 104
cells/well
into 96-well plates and cultured overnight before challenge with a
series of concentrations of TAM for 24 h. Cell viability was
determined by CellTiter96 Non-Radioactive Cell
Proliferation Assay Kit (Promega, Madison, WI) using MTS as substrate.
Nuclear Staining Assay.
After treatment, detached cells were collected in the medium, and the
attached cells were harvested by trypsinization and combined with the
detached fraction. Cells were centrifuged at 1000 x g for 5 min, washed once with PBS, and fixed in a solution
of methanol:acetic acid (3:1) for 30 min. The fixed cells were placed
on slides and stained with 2 µg/ml DAPI for 15 min. Excess dye was
washed off with PBS. Nuclear morphology was observed under a
fluorescence microscope.
Internucleosomal DNA Fragmentation Assay.
After treatment, cells were harvested as described above. The
cell pellet was resuspended in a lysis buffer containing 10
mM Tris (pH 7.4), 150 mM NaCl, 5 mM
EDTA, and 0.5% Triton X-100. Cell lysate was left on ice for 30 min.
DNA was extracted by adding an equal volume of neutral
phenol:chloroform:isoamyl alcohol mixture (pH 8.0; Fisher Scientific)
and precipitated with 0.1 volume of 5 M sodium chloride and
2 volumes of 100% ethanol at -20°C overnight. The DNA sample was
dissolved in TE buffer [10 mM Tris (pH 8.0) and 1
mM EDTA] and treated with 1 µg/ml RNase at 37°C for
2 h. DNA fragments were resolved by electrophoresis in a 1.5%
agarose gel and visualized by ethidium bromide staining.
Caspase Activity Assay.
After treatment, cells were washed twice with ice-cold PBS and
harvested as described previously (28)
. The catalytic
activity of caspases was measured with their fluorogenic substrate
(28)
. Briefly, 10 µg of total protein, as determined by
the Bio-Rad (Richmond, VA) protein assay, was incubated with 200
µM fluorogenic peptide substrates Ac-DEVD-MCA,
Ac-YVAD-MCA, Ac-VEID-MCA, Ac-IETD-MCA, or Ac-LEHD-FCA in
a 50-µl assay buffer at 37°C for 2 h. The release of AMC was
measured with a spectrofluorometer (PerSeptive Biosystems, Inc.,
Framingham, MA) at an excitation wavelength of 360 nm and an emission
wavelength of 460 nm. The release of 7-amido-4-trifluoromethylcoumarin
was monitored at 360/530 nm.
Western Blotting Analysis of PARP Cleavage.
Cells were harvested in a lysis buffer as described previously
(28)
. Fifty µg of total cytosolic protein, as determined
by Bio-Rad protein assay, were resolved in 10% SDS-PAGE and
transferred to polyvinylidene difluoride membrane (Millipore) using a
semidry transfer system (Fisher Scientific). The membrane was
blocked with 5% nonfat dry milk in TBS for 1 h at room
temperature and then incubated with 2 µg/ml primary polyclonal
antihuman PARP antibody (Upstate Biotechnology Inc.) in TBS (containing
3% nonfat milk) at 4°C overnight. The membrane was washed three
times with TBS and blotted with a horseradish peroxidase-conjugated
secondary antibody (1:2000 dilution; Santa Cruz Biotechnology) at room
temperature for 1 h and then washed in TBS three times. The
protein was visualized using enhanced chemiluminescence system
(Amersham).
In Vitro Immunocomplex Kinase Assay for JNK1
Activity.
The JNK1 assay was performed as described previously
(24)
. Briefly, after cell harvesting, equal amounts of
protein were incubated with rabbit anti-JNK1 antiserum (Ab101) for
2 h at 4°C in the presence of protein A-Sepharose 4B conjugate.
The immunocomplexes were precipitated by centrifugation at high speed
for 1 min and washed twice with lysis buffer and twice with kinase
buffer. Kinase reactions were initiated by adding 30 µl of kinase
assay buffer containing 10 µg of GSH
S-transferase-c-Jun1–79(1–79) fusion protein, 2 µCi of
[
-32P]ATP, and 20 µM
ATP. After incubation for 30 min at 30°C, the reactions were
terminated by adding 10 µl of 4x Laemmli buffer and by heating at
94°C for 5 min. The phosphorylation products were resolved in 10%
SDS-PAGE and visualized by autoradiography.
Transient Transfection Cell Death Assay.
Cells were plated in 6-well plates 12 h before transfection at a
density of 1.5 x 105 cells/well.
Cells were cotransfected with the indicated plasmids by using calcium
phosphate precipitation method. After removing the transfection medium,
cells were incubated in culture medium for 12 h and then treated
with 2.5 µM TAM for 24 h. Cells were then washed
with PBS and fixed in 1% paraformaldehyde for 5 min on ice. The fixed
cells were washed once in PBS and stained overnight in a solution
containing 1 mM MgCl2, 10
mM K4Fe(CN)6,
10 mM
K3Fe(CN)6, and 1
mM X-gal in PBS. The ß-galactosidase-expressing
cells (blue-stained cells) were counted under a light microscope
from five randomly chosen fields. Cell survival was determined as
follows: (percentage of blue-stained cells in treated group/percentage
of blue-stained cells in untreated group) x 100% (25)
.
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RESULTS |
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). Because the clinically relevant concentrations of TAM are close to 5
µM (29)
, we used the concentration of 5
µM for all subsequent treatments.
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, indicating that TAM-induced cell death
was due to apoptosis in ER-negative MDA-MB-231 cells. This was
further confirmed by an independent method, the DNA fragmentation assay
(Fig. 2C)
. Similar results were obtained when ER-negative
BT-20 cells were treated with TAM (data not shown).
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, TAM at the apoptosis-inducing concentration (5
µM) strongly stimulated caspase-3-like activity
in MDA-MB-231 cells. Under similar conditions, TAM also activated
caspase-8, -9, and-6, although to a lesser extent. Furthermore, the
activation of these caspases preceded the onset of apoptosis as
determined by DAPI and trypan blue staining. Similar results were
obtained in BT-20 cells (data not shown). However, in MCF-7 cells, we
did not detect any increased activity of caspase-3 and -6 after TAM
treatment, although caspase-8 and -9 were activated to a level similar
to that seen in MDA-MB-231 and BT-20 cells (data not shown). In
contrast to a large peak of caspase-3 activity, little caspase-1
activity was observed throughout the experiment in either of these cell
lines.
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. The cleavage of PARP
was inhibited by pretreatment of cells with Ac-DEVD-CHO, a
caspase-3-specific inhibitor, or with z-VAD-FMK, a general inhibitor of
caspases. The lipid-soluble antioxidant V-E also inhibited TAM-induced
PARP cleavage (Fig. 3B)
.
Caspase Inhibitors Ac-DEVD-CHO and z-VAD-fmk Block the Induction of
Apoptosis by TAM.
The above-mentioned results indicate that caspases are activated
in TAM-treated cells. To define the role of such caspase activation in
TAM-induced apoptosis, we treated MDA-MB-231 cells with caspase
inhibitors before challenge with TAM. The broad specificity caspase
inhibitor, z-VAD-fmk (25 µM), abrogated the induction of
apoptosis by TAM as measured by the number of fragmented nuclei and
trypan blue-positive cells (Fig. 4A)
. Similar results were obtained in both MCF-7 and BT-20
cells (data not shown). The caspase-3-specific inhibitor, Ac-DEVD-CHO
(50 µM), also attenuated TAM-induced apoptosis
in MDA-MB-231 cells (Fig. 4A)
. The caspase inhibitors also
abrogated caspase-3 activity in MDA-MB-231 cells (Fig. 4B)
.
These results demonstrate that TAM induces apoptosis in breast cancer
cells in a caspase-dependent manner.
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. Thus, the activation of JNK1 by TAM is dose dependent.
JNK1 activation by TAM is also time dependent, with maximum activation
being achieved 1 h after treatment with 5
µM TAM (Fig. 5B)
. This earlier
kinetics of JNK1 activation as compared with caspase activation
suggests that JNK1 is either upstream or independent of caspases.
Consistent with this notion, pretreatment of cells with caspase
inhibitor z-VAD-fmk or Ac-DEVD-CHO had no significant effect on JNK1
activation by TAM (Fig. 5C)
.
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, overexpression of DN JNK1(APF) prevented cell death
induced by TAM. The JNK1 activity in DN JNK1-transfected cells was not
induced in response to TAM treatment (Fig. 6B)
. This result
suggests that JNK1 participates in the regulation of TAM-induced
apoptosis.
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-tocopherol) prevented TAM-induced activation of
caspase-3-like proteases, and JNK1 and also abolished TAM-induced
apoptosis (Fig. 7
, A—C). In contrast, water-soluble antioxidants
NAC or GSH had little effect. Similar results were obtained in MCF-7
and BT-20 cells (data not shown). These data suggest that oxidative
stress generated in the cell membrane is responsible for the initiation
of JNK1 and caspase pathways that ultimately lead to apoptosis in
TAM-treated breast cancer cells.
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DISCUSSION |
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, or
anticancer drugs like camptothecin and etoposide that act independent
of the ER and are known to induce apoptosis via the caspase cascade,
failed to activate the caspase-3 in MCF-7 cells (data not shown). One
explanation of the observed phenomenon is provided in two recent
reports (32
, 33)
that document the absence of caspase-3
activity in MCF-7 cells due to a mutation in exon 3 of the
CASP-3 gene. This may also explain the absence of
genome digestion in MCF-7 cells. Thus, other caspases may be operative
in TAM-induced apoptosis in MCF-7 cells. This was supported by our
results that the general caspase inhibitor z-VAD-fmk completely blocked
TAM-induced apoptosis in MCF-7 cells (data not shown). The activation
of caspase-8 by TAM in breast cancer cell lines is intriguing,
suggesting that TAM, like other anticancer drugs, may induce the
expression of DRs or apoptotic ligands such as TNF-, Fas ligand, or
TNF-related apoptosis-inducing ligand or signal through death
domain-containing adapter proteins (27
, 34)
. Caspase-9 can
be activated by caspase-8 or activated independently by apoptotic
protease-activating factor 1 on binding of cytochrome c
released from the mitochondria (Ref. 35
and the references
therein). Indeed, the involvement of mitochondria in the proapoptotic
action of TAM was implicated by a time-dependent reversal of both the
calcium-induced mitochondrial membrane depolarization and calcium
release in TAM-treated rat liver microsomes, suggesting that the effect
was on the permeability transition pore (36)
. The
activation by TAM of the effector caspases, caspase-3 and -6, could
then be explained by proteolytic cleavage by these activated upstream
caspases. Caspase-1, however, did not seem to play a role in
TAM-induced apoptosis because no activation was detected under
experimental conditions. Thus, DR- or mitochondria-mediated activation
of the caspase cascade may be a potential mechanism underlying
TAM-induced apoptosis in breast cancer. TAM also activated JNK1 under conditions that induced apoptosis. Kinetically, the JNK1 activation was a much earlier event as compared with caspase activation and apoptosis. Furthermore, neither of the caspase inhibitors significantly blocked JNK1 activation, although DN JNK1, at least partially inhibited TAM-induced apoptosis. This suggests that JNK1 is upstream of the caspase cascade. Thus, our results indicate that both the caspase and JNK pathways are required in TAM-induced apoptosis in breast cancer cells because interfering with either pathway blocked apoptosis. Importantly, the lipid-soluble antioxidant V-E also blocked both JNK1 and caspase-3 activation and completely abolished TAM-induced cell death, implicating the involvement of oxidative stress. Two possibilities exist for generation of oxidative stress by TAM: either autoxidation of the phenolic moiety, producing superoxides and hydrogen peroxide through redox cycling, or local effects in the membrane due to TAM partitioning into lipid bilayer. Ferlini et al. (31) demonstrated the generation of ROS in Jurkat and A2780 ovarian cancer cells treated with TAM. In the current study, the water-soluble antioxidants NAC and GSH failed to block caspase or JNK activation and cell death. Thus, generation of ROS alone may not be sufficient to activate the signaling pathways leading to apoptosis. V-E, on the other hand, inhibits cell membrane oxidation (37) . Hence, membrane oxidation by TAM seems to play an important role in caspase and JNK signaling and apoptosis. In this regard, the actions of TAM may be similar in part to UV radiation. Therefore, it will be interesting to test whether TAM-induced ceramid accumulation (38) , PKC translocation (39) , or other second messengers contribute to the activation of JNKs and caspases by TAM. A recent study demonstrating protective effect of V-E against TAM-induced lysis of erythrocytes suggests that disruption of structural characteristics of biomembranes and release of peripheral membrane-, cytoskeleton-, or membrane-bound cytosol proteins by TAM contributes to its anticancer action (42) .
In summary, it is tempting to speculate that TAM, by virtue of its high lipophilicity and partitioning in the cell membrane, generates a transmembrane signal transduction cascade leading to the early activation of JNK1 and subsequent activation of caspases. The caspase-dependent apoptotic effect of TAM may be of clinical relevance in determining sensitivity versus resistance of human breast cancer to TAM.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by the NIH Grants R01-ES06887
and R01-CA73647 (to A-N. T. K.). 
2 Present address: DuPont Pharmaceuticals Company,
1094 Elkton Road, P. O. Box 30, Newark, DE 19711.
3 To whom requests for reprints should be
addressed, at Department of Pharmaceutics and Pharmacodynamics, Center
for Pharmaceutical Biotechnology, M/C 865, College of Pharmacy,
University of Illinois at Chicago, 833 South Wood Street, Chicago, IL
60612. Phone: (312) 413-9646; Fax: (312) 413-9303; E-mail: KongT{at}uic.edu
4 The abbreviations used are: TAM, tamoxifen; ER,
estrogen receptor; PKC, protein kinase C; JNK, c-Jun
NH2-terminal kinase; PARP, poly(ADP-ribose) polymerase;
DAPI, diamidinophenylindole; AMC, 7-amino-4-methyl coumarin; V-E,
vitamin E; NAC, N-acetyl L-cysteine;
Ac-DEVD-CHO, acetyl-Asp-Glu-Val-Asp-aldehyde; z-VAD-fmk,
benzyl-Val-Ala-Asp-fluoromethyl ketone; DR, death receptor; TNF, tumor
necrosis factor; Ac-DEVD-MCA,
Acetyl-Asp-Glu-Val-Asp-methylcoumaryl-7-amide; Ac-YVAD-MCA,
Acetyl-Tyr-Val-Ala-Asp-methylcoumaryl-7-amide; Ac-VEID-MCA,
Acetyl-Val-Glu-Ile-Asp-methylcoumaryl-7-amide; MTS,
3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium;
TBS, Tris-buffered saline [20 mM Tris-HCl (pH 7.4) and 8
grams/liter NaCl]DN, dominant negative; GSH, glutathione; ROS,
reactive oxygen species; X-gal,
5-bromo-4-chloro-3-indoyl-ß-O-galactoside.
Received 4/ 5/00. Accepted 9/ 1/00.
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M. Obrero, D. V. Yu, and D. J. Shapiro Estrogen Receptor-dependent and Estrogen Receptor-independent Pathways for Tamoxifen and 4-Hydroxytamoxifen-induced Programmed Cell Death J. Biol. Chem., November 15, 2002; 277(47): 45695 - 45703. [Abstract] [Full Text] [PDF]
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Z.-Z. Pan, W. Bruening, B. I. Giasson, V. M.-Y. Lee, and A. K. Godwin gamma -Synuclein Promotes Cancer Cell Survival and Inhibits Stress- and Chemotherapy Drug-induced Apoptosis by Modulating MAPK Pathways J. Biol. Chem., September 13, 2002; 277(38): 35050 - 35060. [Abstract] [Full Text] [PDF]
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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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 D. G. Stupack and D. A. Cheresh Get a ligand, get a life: integrins, signaling and cell survival J. Cell Sci., January 10, 2002; 115(19): 3729 - 3738. [Abstract] [Full Text] [PDF]
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K. Hisamoto, M. Ohmichi, Y. Kanda, K. Adachi, Y. Nishio, J. Hayakawa, S. Mabuchi, K. Takahashi, K. Tasaka, Y. Miyamoto, et al. Induction of Endothelial Nitric-oxide Synthase Phosphorylation by the Raloxifene Analog LY117018 Is Differentially Mediated by Akt and Extracellular Signal-regulated Protein Kinase in Vascular Endothelial Cells J. Biol. Chem., December 7, 2001; 276(50): 47642 - 47649. [Abstract] [Full Text] [PDF]
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 E. R. Levin Genome and Hormones: Gender Differences in Physiology: Invited Review: Cell localization, physiology, and nongenomic actions of estrogen receptors J Appl Physiol, October 1, 2001; 91(4): 1860 - 1867. [Abstract] [Full Text] [PDF]
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