From the Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556
Received for publication, July 31, 2000, and in revised form, October 2, 2000
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ABSTRACT |
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Vitamin D3 compounds are
currently in clinical trials for human breast cancer and offer an
alternative approach to anti-hormonal therapies for this disease.
1 1 To characterize the mechanisms of
1 Mitochondria play a central role in commitment of cells to apoptosis
via increased permeability of the outer mitochondrial membrane,
decreased transmembrane potential, release of cytochrome c
and apoptosis-inducing factor, and production of reactive oxygen species (ROS) (8, 9). Anti-apoptotic Bcl-2 family members, such as
Bcl-2 and Bcl-XL, can block these mitochondrial events, whereas pro-apoptotic Bcl-2 family members, including Bax, can trigger
these changes. For example, apoptotic signals induce
conformational changes in Bax, which lead to exposure of the
pro-apoptotic BH3 domain, and translocation to the mitochondria (10).
The effects of pro-apoptotic Bcl-2 family members are achieved by both
caspase-dependent and caspase-independent mechanisms (11,
12).
Although the role of caspases in apoptosis triggered by cell surface
death receptors such as TNFR1 has been well established, it is not
clear if apoptosis triggered by nuclear receptors such as the VDR is
mediated via similar caspase-dependent pathways. To probe
the mechanisms whereby vitamin D3 signaling modulates apoptosis in MCF-7 cells, we studied the effects of
1 Our results indicate that, although caspase inhibition can block some
of the late stages of 1 Cells and Cell Culture--
MCF-7 cells (originally obtained
from ATCC) were used to generate the vitamin D3-resistant
variant (MCF-7D3Res) that has been described previously
(13). Both cell lines were cultured in Clonogenicity Assay--
MCF-7 cells were incubated with
1 Subcellular Fractionation--
Cells were trypsinized, pooled
together with media and washes containing floating cells, and pelleted
by centrifugation at 500 × g for 3 min at 4 °C.
Pellets were resuspended with 3 volumes of Buffer A (20 mM
HEPES-KOH, pH 7.5, 10 mM KCl, 1.5 mM
MgCl2, 1 mM EDTA, 1 mM EGTA, 10 mM benzamidine, 1 mM dithiothreitol, 250 mM sucrose, plus protease and phosphatase inhibitors),
lysed with a Dounce homogenizer, and fractionated by differential
centrifugation (16). Briefly, homogenates were centrifuged twice at
500 × g for 5 min at 4 °C, and the nuclear pellet
was resuspended in Buffer A, sonicated 2 × 10 s, and stored
at Immunoblot Analysis--
Subcellular fractions isolated as
described above were solubilized in Laemmli sample buffer, separated by
SDS-PAGE, and transferred to nitrocellulose. Proteins derived from
mitochondria and/or S100 extracts were immunoblotted with Bax rabbit
polyclonal (13666E; PharMingen, San Diego, CA) or cytochrome
c mouse monoclonal antibodies (7H8.2C12; PharMingen) diluted
1:500 or 1:250, respectively, in PBS plus 5% skim milk. S100 extracts
were also probed with cytochrome oxidase subunit II mouse monoclonal
antibody (clone 12C4-F12; Molecular Probes, Eugene, OR) to exclude
mitochondrial contamination. Nuclear extracts were probed with PARP
mouse monoclonal antibody (C2.10, Enzyme Systems Products) diluted
1:1000. Specific antibody binding was detected by horseradish
peroxidase-conjugated secondary antibodies (Amersham Pharmacia Biotech)
diluted 1:5000 in PBS, 5% skim milk, 0.1% Tween 20 and
autoradiography with enhanced chemiluminescence (Pierce).
Immunocytochemistry--
MCF-7 cells grown on Lab-Tek II chamber
slides (Fisher Scientific) were treated with ethanol vehicle, 100 nM 1 Flow Cytometry--
For analysis of mitochondrial membrane
potential and reactive oxygen species, cells harvested by
trypsinization were pooled with media plus washes and pelleted by
centrifugation. Cell suspensions (1 × 106 cells) were
incubated with 1 µM tetramethylrhodamine ethyl ester (TMRE, Molecular Probes) in PBS containing 130 mM KCl to
abolish the plasma membrane potential. After incubation for 10 min at 37 °C, cells were washed once in PBS and then analyzed for TMRE red
fluorescence by flow cytometry. Live cells rapidly and reversibly take
up TMRE, and accumulation of the dye in mitochondria has been shown to
be potential driven (17). For analysis of ROS, cell suspensions (5 × 105 cells) were incubated with 4 µM
hydroethidine (HE, Molecular Probes) in PBS for 15 min at 37 °C, and
conversion of HE to ethidium by superoxide anion was analyzed by flow cytometry.
For analysis of DNA fragmentation, MCF-7 cells were harvested by
trypsinization, collected by centrifugation, fixed in 2% formaldehyde
in PBS, and permeabilized in 70% EtOH at
For detection of PS externalization, 1 × 106 cells
were incubated in the presence of 10 µg/ml annexin V-FITC
(BioWhittaker, Walkersville, MD) and 5 µg/ml PI in binding buffer (10 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 2.5 mM CaCl2) for 15 min at 37 °C. Cells were
washed twice in binding buffer, fixed in 2% formaldehyde in PBS for 15 min on ice, and then washed two more times in PBS plus 0.2% BSA.
Pellets were resuspended in PBS plus 0.2% BSA and analyzed by flow
cytometry. There were less than 1% PI+ cells in the
population, and they were therefore excluded from analysis.
All flow cytometric analyses were performed on an Epics XL Flow
Cytometer (Coulter Corp., Miami, FL) equipped with an argon laser. TMRE
and HE were analyzed on FL3 using a 620-nm band pass filter. For DNA
fragmentation analysis, FITC was analyzed on FL1 using a 520-nm band
pass filter and PI was analyzed on FL3 with no color compensation. For
PS externalization, annexin V-FITC was analyzed on FL1 and PI was
analyzed on FL2 (580-nm band pass filter) using software color
compensation. Data was modeled with the Multiplus AV software (Phoenix
Flow Systems).
Caspase Activity Assay--
Caspase activity was analyzed with
the ApoAlert CPP32/caspase-3 assay kit according to manufacturer's
protocol (CLONTECH, Palo Alto, CA). Briefly, after
harvesting by trypsinization, 2 × 106 cells were
pelleted and stored at Statistical Evaluation--
Data are expressed as mean ± S.E. One-way analysis of variance was used to assess statistical
significance between means. Differences between means were considered
significant if p values less than 0.05 were obtained with
the Bonferroni method using GraphPad Instat software (GraphPad
Software, San Diego, CA).
Disruption of Mitochondrial Function, as Determined by
Subcellular Localization of Bax and Cytochrome c, and ROS Generation,
by 1
Translocation of Bax to mitochondria has been associated with release
of cytochrome c, an event that is considered a commitment point for activation of apoptosis. As expected for viable cultures, no
cytochrome c was detected in cytosolic fractions from MCF-7 cells treated with ethanol vehicle for up to 120 h (Fig.
2A). In contrast,
redistribution of cytochrome c from mitochondria to cytosol
was detected within 48 h of 1
Long term exclusion of cytochrome c from the electron
transport chain can lead to impairment of proton flow and generation of
ROS due to incomplete reduction of molecular oxygen. Hence, mitochondrial generation of ROS in response to
1 1
Proteolytic activity associated with caspase activation was
analyzed by three distinct methods: cleavage of an endogenous caspase
substrate (PARP), and flow cytometric analysis of PS exposure and DNA
fragmentation, which others have shown are provoked by caspases (7). As
demonstrated in Fig. 4A, PARP
was cleaved after treatment of MCF-7 cells with either
1 Caspase Activity Induced by 1 Caspase Inhibition Does Not Block
1
To further probe mitochondrial function, the membrane
potential-sensitive probe TMRE was used to detect mitochondrial
membrane potential by flow cytometry.
1
Subcellular localization of cytochrome c protein was
examined by fluorescence microscopy to confirm the finding that
cytochrome c release can proceed independently of caspase
activation after 1
Since zVAD.fmk did not block cytochrome c release or
mitochondrial dysfunction induced by
1 Here we report for the first time that
1 In addition to Bax translocation, we report that treatment of MCF-7
cells with 1 Examination of events downstream of mitochondria indicated that
1 Although caspase inhibition did not block mitochondrial events induced
by 1 The data presented in this paper indicate that
1 In summary, 1,25-Dihydroxyvitamin D3
(1
,25(OH)2D3), the active form of vitamin
D3, induces apoptosis in breast cancer cells and tumors,
but the underlying mechanisms are poorly characterized. In these
studies, we focused on the role of caspase activation and mitochondrial
disruption in 1
,25(OH)2D3-mediated apoptosis in breast cancer cells (MCF-7) in vitro. The effect of
1
,25(OH)2D3 on MCF-7 cells was compared with
that of tumor necrosis factor
, which induces apoptosis via a
caspase-dependent pathway. Our major findings are that
1
,25(OH)2D3 induces apoptosis in MCF-7 cells
by disruption of mitochondrial function, which is associated with Bax
translocation to mitochondria, cytochrome c release, and
production of reactive oxygen species. Moreover, we show that Bax
translocation and mitochondrial disruption do not occur after 1
,25(OH)2D3 treatment of a MCF-7 cell clone
selected for resistance to
1
,25(OH)2D3-mediated apoptosis. These
mitochondrial effects of 1
,25(OH)2D3 do not
require caspase activation, since they are not blocked by the
cell-permeable caspase inhibitor z-Val-Ala-Asp-fluoromethylketone. Although caspase inhibition blocks
1
,25(OH)2D3-mediated events downstream of
mitochondria such as poly(ADP-ribose) polymerase cleavage, external
display of phosphatidylserine, and DNA fragmentation, MCF-7 cells still
execute apoptosis in the presence of z-Val-Ala-Asp-fluoromethylketone, indicating that the commitment to
1
,25(OH)2D3-mediated cell death is
caspase-independent.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,25-Dihydroxyvitamin D3
(1
,25(OH)2D3),1
the active form of vitamin D3, acts through the nuclear
vitamin D3 receptor (VDR) and is a potent negative growth
regulator of breast cancer cells both in vitro and in
vivo (1). A variety of synthetic vitamin D3 analogs
that induce mammary tumor regression in animals are now undergoing
clinical trials in human patients (2, 3). Our laboratory has shown that
1
,25(OH)2D3 induces morphological and
biochemical markers of apoptosis (chromatin and nuclear matrix condensation, and DNA fragmentation) in breast cancer cells (MCF-7) (4,
5); however, the precise mechanism by which
1
,25(OH)2D3 and the VDR mediate
apoptosis is poorly understood.
,25(OH)2D3-mediated apoptosis in breast
cancer cells, we compared specific intracellular events in MCF-7 cells
after treatment with 1
,25(OH)2D3 or tumor necrosis factor
(TNF
). TNF
was chosen as a positive control since this cytokine induces apoptosis in MCF-7 cells by a well defined
pathway triggered by tumor necrosis factor receptor 1 (TNFR1), a cell
surface death receptor. Death receptors contain homologous cytoplasmic
regions termed "death domains," which transmit apoptotic signals
through recruitment of adaptor molecules that activate caspases, a
family of cysteine proteases involved in cell disassembly. The best
characterized death receptors (Fas, TNFR1) use Fas-associated death
domain and TNFR1-associated death domain adaptors to recruit and
activate caspase-8 (6). Cleavage of specific substrates by caspases
during apoptosis promotes the degradation of key structural proteins,
including poly(ADP-ribose) polymerase (PARP), and lead to external
display of phosphatidylserine (PS), DNA fragmentation, and cellular
condensation (7).
,25(OH)2D3 on mitochondrial function and
caspase activity using a cell-permeable inhibitor of caspase-related
proteases (z-Val-Ala-Asp-fluoromethylketone, zVAD.fmk). In addition,
the effects of 1
,25(OH)2D3 and TNF
on a
vitamin D3-resistant variant of MCF-7 cells
(MCF-7D3Res cells) were examined to identify events that
contribute to vitamin D3 resistance (13, 14). The
MCF-7D3Res cells do not undergo cell cycle arrest or
apoptosis in response to 1
,25(OH)2D3;
however, these cells retain sensitivity to other inducers of apoptosis
such as TNF
and anti-estrogens (13).
,25(OH)2D3-mediated
apoptosis in MCF-7 cells, the commitment to cell death is
caspase-independent. These data implicate Bax distribution and
mitochondrial disruption as critical caspase-independent events in
1
,25(OH)2D3-mediated apoptosis of breast
cancer cells.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-minimal essential medium
(Life Technologies, Inc.) containing 25 mM HEPES and 5%
fetal bovine serum (Life Technologies, Inc.). Cells were routinely
plated at 5000 cells/cm2 and passaged every 3-4 days.
Stock cultures of MCF-7D3Res cells were routinely grown in
medium containing 100 nM
1
,25(OH)2D3 (kindly provided by LEO
Pharmaceuticals, Ballerup, Denmark). For experiments, MCF-7 and
MCF-7D3Res cells were plated in
-minimal essential
medium containing 5% fetal bovine serum plus antibiotics, and treated
with 1
,25(OH)2D3 or ethanol vehicle 2 days
after plating. Parallel cultures were treated with TNF
(Sigma) 2-3
days before scheduled harvest of 1
,25(OH)2D3-treated dishes. Caspase
inhibitors, zVAD.fmk or zDEVD.fmk (Enzyme Systems Products, Livermore,
CA) were added at the same time (1
,25(OH)2D3
or TNF
) or 2 days after (1
,25(OH)2D3)
initial treatment. For cell growth assays, cells were seeded at 1000 cells/well in 24-well plates, treated for the indicated times, and
analyzed by crystal violet assay. Briefly, cells were fixed with 1%
glutaraldehyde for 15 min, incubated with 0.1% crystal violet (Fisher
Scientific, Pittsburgh, PA) for 30 min, destained with H2O,
and solubilized with 0.2% Triton X-100. Absorbance at 562 nm (minus
background at 630 nm) was determined on a microtiter plate reader.
,25(OH)2D3 for 6 days or TNF
for 1 day
in the presence or absence of 25 µM zVAD.fmk. For 1
,25(OH)2D3 treatment, medium was replaced
every 2 days. After treatments, cells were trypsinized, media and
washes were pooled, and cells were pelleted by centrifugation and
resuspended in fresh medium. The cells were seeded in 96-well plates at
5, 50, 500, and 2500 cells/well in 24 replicates. After 14 days, the
cells were fixed and stained with crystal violet as described above, and clonogenic potential was estimated by counting positive wells (15).
80 °C in multiple aliquots. The supernatants were combined and
further centrifuged at 10,000 × g for 30 min at
4 °C, and the resultant mitochondrial pellets were resuspended in
Buffer A, sonicated, and stored at
80 °C in multiple aliquots. The
supernatant from the 10,000 × g spin was further
centrifuged at 100,000 × g for 1 h at 4 °C.
The resulting supernatant was designated S100 (containing cytosol) and
stored at
80 °C in multiple aliquots. Protein concentrations were
determined by the Micro BCA protein assay (Pierce).
,25(OH)2D3, or 2.5 ng/ml
TNF
for 96 h (ethanol, 1
,25(OH)2D3)
or 48 h (TNF
) in the presence or absence of 25 µM
zVAD.fmk. The cells were fixed in 4% formaldehyde in PBS for 5 min at
room temperature, permeabilized in methanol at
20 °C for 5 min,
and blocked overnight with PBS plus 1% BSA containing 0.02% sodium
azide. The slides were then incubated with cytochrome c
mouse monoclonal antibody (6H2.B4; PharMingen), diluted 1:100 in
blocking buffer, for 3 h at 37 °C in a humidified chamber.
Slides were washed three times for 5 min each time with PBS, followed
by incubation for 1 h at room temperature with anti-mouse
secondary antibody conjugated to Alexa-488 (a photostable dye with
spectral properties similar to fluorescein; Molecular Probes) diluted
1:50 in blocking buffer. Slides were washed three times for 5 min with
PBS, incubated for 15 min at room temperature with 1 µg/ml Hoechst
33258 (Sigma), washed five times for 5 min with PBS, rinsed with
distilled H2O, and coverslips were applied with an
antifade reagent. Fluorescence was detected using an Olympus AX70
microscope equipped with a Spot RT digital camera.
20 °C. DNA strand breaks
in cells undergoing apoptosis were indirectly labeled with
bromodeoxyuridine by terminal transferase (Roche Molecular
Biochemicals) and detected by FITC-conjugated monoclonal antibody to
bromodeoxyuridine using the APO-BRDU kit according to manufacturer's
protocol (Phoenix Flow Systems, San Diego, CA). Cells were
counterstained with 5 µg/ml propidium iodide (PI; Sigma) containing
RNase A (Roche Molecular Biochemicals) for detection of total DNA, and
two-color analysis of DNA strand breaks and cell cycle was achieved by
flow cytometry.
20 °C. For analysis, cell pellets were
lysed, re-pelleted to remove cell debris, and supernatants were
incubated with 50 µM DEVD-AFC for 1 h at 37 °C.
The samples were analyzed using a fluorescence spectrophotometer with
excitation = 380 nm and emission = 508 nm.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,25(OH)2D3--
To identify specific
intracellular events involved in
1
,25(OH)2D3-mediated apoptosis, we examined
the signaling pathway downstream of the VDR in MCF-7 cells. Since
disruption of mitochondrial function is a primary event in apoptosis
that can be triggered by translocation of Bax to mitochondrial outer
membrane, we first examined the subcellular distribution of Bax after
1
,25(OH)2D3 treatment of MCF-7 cells. As
demonstrated in Fig. 1, Bax
redistribution from the cytosolic to the mitochondrial fraction
occurred after treatment with 1
,25(OH)2D3 or
TNF
in MCF-7 cells (Fig. 1, top). Not only was Bax
translocated to mitochondria, but both
1
,25(OH)2D3- and TNF
-treated cells
exhibited cleavage of Bax from the intact 21-kDa protein to an 18-kDa
fragment, an observation that is consistent with reports of Bax
cleavage during drug-induced apoptosis (18). In both
1
,25(OH)2D3- and TNF
-treated cells, the
Bax cleavage product was detected in mitochondrial, but not cytosolic,
fractions, and others have proposed that Bax cleavage enhances
homodimerization and its pro-apoptotic properties (19). These are the
first data to implicate translocation and cleavage of Bax during
1
,25(OH)2D3-induced apoptosis. To determine
the relationship between Bax translocation and sensitivity to
1
,25(OH)2D3-induced apoptosis, we examined the subcellular distribution of Bax in a vitamin
D3-resistant variant of MCF-7 cells, which does not undergo
apoptosis after treatment with 1
,25(OH)2D3
but retains sensitivity to other triggers, including
TNF
.2 In the
MCF-7D3Res cells, 1
,25(OH)2D3
did not induce translocation or cleavage of Bax (Fig. 1,
bottom). However, in these cells, Bax translocation and
cleavage was triggered by TNF
, indicating that Bax functions appropriately in MCF-7D3Res cells during apoptosis induced
by agents other than 1
,25(OH)2D3. The
inability of Bax to redistribute to mitochondria in response to
1
,25(OH)2D3 in the vitamin
D3-resistant variant suggests that Bax translocation may be
a critical initiating event in
1
,25(OH)2D3-mediated apoptosis of MCF-7
cells.
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Fig. 1.
Subcellular distribution of Bax after
treatment with
1 ,25(OH)2D3 or
TNF
in parental MCF-7 or
MCF-7D3Res cells. MCF-7 or MCF-7D3Res
cells were plated at a density of 2 × 105
cells/150-mm dish. Two days after plating, cells were treated with
vehicle control (ethanol) or 100 nM
1
,25(OH)2D3 for 96 h, or with 2.5 ng/ml
TNF
for 48 h. Mitochondria and S100 isolated as described under
"Experimental Procedures" were separated on SDS-PAGE, transferred
to nitrocellulose, and immunoblotted with polyclonal antibody to Bax.
The results are representative of at least three independent
experiments.
,25(OH)2D3
treatment in MCF-7 cells, before any morphological apoptotic features
were detected. The absence of cytochrome oxidase in cytosolic fractions confirmed that extracts were free of mitochondrial contamination (data
not shown). In MCF-7D3Res cells,
1
,25(OH)2D3 did not trigger release of
cytochrome c; however, cytochrome c was detected
in cytosolic fractions after TNF
treatment of both MCF-7 and
MCF-7D3Res cell lines (Fig. 2B; see also Fig.
7).
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Fig. 2.
Cytosolic localization of cytochrome
c after treatment with
1 ,25(OH)2D3 or
TNF
. A, time course of cytosolic
cytochrome c after treatment with vehicle control (ethanol)
or 100 nM 1
,25(OH)2D3 in MCF-7
cells. Cells were plated at a density of 1 × 105
cells/150-mm dish. Two days after plating, the cells were treated with
ethanol or 1
,25(OH)2D3 and re-fed 2 days
later. S100 fractions prepared at the indicated time points as
described under "Experimental Procedures" were separated on
SDS-PAGE, transferred to nitrocellulose, and immunoblotted with
cytochrome c (7H8.2C12) antibody. B, cytosolic
cytochrome c in MCF-7D3Res cells. Cells were
plated and treated with ethanol or
1
,25(OH)2D3 as described above, and 2.5 ng/ml TNF
was added 3 days before harvest. All the dishes were
harvested on day 5 of treatment, and S100 fractions were prepared and
immunoblotted as described above. The results are representative of at
least three independent experiments.
,25(OH)2D3 and TNF
was examined by flow
cytometry. Production of superoxide anion was indirectly assessed as
oxidation of hydroethidine to ethidium, which fluoresces red upon DNA
intercalation. As presented in Fig. 3,
ROS production was enhanced by 1
,25(OH)2D3
in MCF-7, but not MCF-7D3Rescells, whereas TNF
increased
ROS production comparably in both cell lines. Time-course studies have
demonstrated that ROS production is enhanced within 72 h of
1
,25(OH)2D3 treatment in MCF-7 cells (data
not shown).
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Fig. 3.
ROS production after treatment with
1 ,25(OH)2D3 or
TNF
in parental MCF-7 or
MCF-7D3Res cells. Cells were plated at a density of
1 × 105 cells/150-mm dish. Two days after plating,
the cells were treated with vehicle control (ethanol), 100 nM 1
,25(OH)2D3, or media only,
re-fed 2 days later, and 2.5 ng/ml TNF
was added to dishes
containing media only. All dishes were harvested on day 5 when ROS
generation was assessed by flow cytometry as described under
"Experimental Procedures." Data are expressed as the percentage of
cells positive for ROS after negative subtraction of data derived from
vehicle-treated MCF-7D3Res cells. The results are
representative of at least three independent experiments.
,25(OH)2D3 Mediates PARP Cleavage, PS
Externalization, and DNA Fragmentation in a
Caspase-dependent Manner--
Cytochrome c
released into the cytosol is thought to trigger caspase activation
downstream of mitochondria through binding to Apaf-1 and autoactivation
of procaspase-9. Activated caspase-9 can activate additional effector
caspases responsible for cell disassembly and events such as PS
externalization, PARP cleavage, and DNA fragmentation. To determine the
involvement of caspase-dependent proteolysis in
1
,25(OH)2D3-mediated apoptosis, we examined
whether a broad spectrum cell-permeable caspase inhibitor (zVAD.fmk)
could abrogate the effects of 1
,25(OH)2D3 in
MCF-7 cells.
,25(OH)2D3 or TNF
, and in both cases,
cleavage was blocked by zVAD.fmk. Furthermore, both
1
,25OH)2D3 and TNF
induced PS
externalization, which was also completely blocked by zVAD.fmk (Fig.
4B). Finally, the effects of
1
,25(OH)2D3 and TNF
on DNA fragmentation
was assessed as terminal transferase-mediated incorporation of
bromodeoxyuridine, detected by FITC-conjugated anti-bromodeoxyuridine
antibody by flow cytometry (Fig. 5).
1
,25(OH)2D3 treatment of MCF-7 cells induced
DNA fragmentation primarily in the G1 phase of the cell cycle, with only a small population of cells (5%) accumulating in
sub-G1. TNF
treatment induced extensive DNA
fragmentation in the G1 phase of the cell cycle, with
accumulation of 29% of the population in sub-G1. Despite
the differences in the magnitude and profiles of DNA fragmentation
between 1
,25(OH)2D3- and TNF
-treated cells, zVAD.fmk completely blocked DNA fragmentation induced by both
agents.
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Fig. 4.
Effects of caspase inhibitor on PARP cleavage
and PS exposure after treatment with
1 ,25(OH)2D3 or
TNF
in MCF-7 cells. A, PARP
cleavage. Cells were plated and treated as described in Fig. 3 in the
presence or absence of 25 µM zVAD.fmk. All the dishes
were harvested on day 5. Nuclear extracts prepared as described under
"Experimental Procedures" were separated on SDS-PAGE and
immunoblotted with mouse monoclonal antibody to PARP. B, PS
externalization. Cells plated and treated as described above were
incubated with annexin V-FITC and PI as described under "Experimental
Procedures." Data are expressed as the percentage of annexin
V-FITC-positive cells after negative subtraction of data generated with
vehicle treated cells. The results are representative of at least three
independent experiments.
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Fig. 5.
Effect of caspase inhibitor on DNA
fragmentation after treatment with
1 ,25(OH)2D3 or
TNF
in MCF-7 cells. Cells were plated and
treated as described in Fig. 4, and DNA fragmentation was determined by
flow cytometry as described under "Experimental Procedures." The
results are representative of at least three independent
experiments.
,25(OH)2D3
Is Not DEVDase--
DEVDase cleavage activity was measured with the
fluorogenic substrate DEVD-AFC, which can detect activity of caspases-3
and -7. Since MCF-7 cells do not express functional caspase-3 (data not
shown, Ref. 20), any DEVDase activity detected in these cells would
most likely correspond to caspase-7. For these experiments, MCF-7 cells
were pretreated with 1
,25(OH)2D3 for 2 days
before cytosolic extracts were collected for DEVDase activity assays at
the indicated time points. As demonstrated in Fig.
6, TNF
, but not
1
,25(OH)2D3, induced DEVDase cleavage
activity in MCF-7 cells. Even with extended treatment times in
additional studies, no DEVDase cleavage activity could be detected
after 1
,25(OH)2D3 treatment of MCF-7 cells
(data not shown). This observation indicates that other known or as yet
unidentified effector caspases may be activated by
1
,25(OH)2D3, which mediate PARP cleavage, PS exposure, or DNA fragmentation.
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Fig. 6.
Time course of DEVDase cleavage activity
after treatment with
1 ,25(OH)2D3 or
TNF
in MCF-7 cells. Cells were plated at
a density of 2 × 105 cells/150-mm dish. Two days
after plating, the cells were pretreated with ethanol, 100 nM 1
,25(OH)2D3, or media only.
Two days later, medium was replaced with ethanol (
), 100 nM 1
,25(OH)2D3 (
), or 10 ng/ml TNF
(
). Cytosolic extracts of cells harvested at the
indicated time points were incubated with DEVD-AFC for 1 h at
37 °C and analyzed on a fluorescence spectrophotometer. Data
represent mean ± S.E. of two independent experiments performed in
duplicate.
,25(OH)2D3-mediated Cytochrome c Release,
Mitochondrial Dysfunction, or Cell Death--
Since zVAD.fmk blocked
1
,25(OH)2D3-mediated
caspase-dependent events downstream of mitochondria, we
examined the effects of the caspase inhibitor on cytochrome
c release and mitochondrial function. As shown in Fig.
7, zVAD.fmk did not abrogate
1
,25(OH)2D3-mediated cytochrome c
release or ROS production under the same conditions where PS exposure,
PARP cleavage, and DNA fragmentation were blocked. In contrast, the
caspase inhibitor effectively blocked cytochrome c release
and ROS generation triggered by TNF
(Fig. 7, A and C).
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Fig. 7.
Effects of caspase inhibitor on cytochrome
c release, mitochondrial membrane potential, and ROS
production after treatment with
1 ,25(OH)2D3 or
TNF
in MCF-7 cells. A, cytochrome
c release. S100 extracts prepared from cells treated as
described in Fig. 4 were separated on SDS-PAGE and immunoblotted with
cytochrome c (7H8.2C12) mouse monoclonal antibody.
B, mitochondrial membrane potential. Cells plated and
treated as described above were incubated with 1 µM TMRE
as described under "Experimental Procedures" and analyzed by flow
cytometry. Data are expressed as the percentage of cells with reduced
mitochondrial membrane potential. C, ROS production. Cells
treated as described above were incubated with 4 µM HE as
described under "Experimental Procedures" and analyzed by flow
cytometry. The data represent the percentage of cells positive for ROS.
Each bar represents the mean ± S.E. of two to four
independent experiments. **, p < 0.001; *,
p < 0.01; treated versus ethanol control as
evaluated by analysis of variance. The results (A and
B) are representative of at least three independent
experiments.
,25(OH)2D3 treatment significantly enhanced
the percentage of cells with reduced mitochondrial membrane potential,
and zVAD.fmk did not block the decrease in mitochondrial membrane
potential induced by 1
,25(OH)2D3. TNF
treatment also enhanced the percentage of cells with decreased
mitochondrial membrane potential; however, in contrast to
1
,25(OH)2D3, the effect of TNF
was
completely blocked by zVAD.fmk (Fig. 7B).
,25(OH)2D3 treatment. In
Fig. 8, cytochrome c
fluorescence (middle panels) is presented
alongside phase contrast (top panels) and Hoechst
nuclear staining (bottom panels) to compare
cytochrome c localization in individual viable and apoptotic
cells. In vehicle-treated control cells, apoptotic morphology was
not present, and cytochrome c staining was restricted
to punctate cytoplasmic regions, consistent with mitochondrial
localization (Fig. 8). After treatment with 1
,25(OH)2D3 or TNF
, apoptotic cells
identified by phase contrast and Hoechst nuclear staining exhibited
chromatin condensation, nuclear fragmentation, and cytosolic
vacuolization. In these apoptotic cells, diffuse cytoplasmic
cytochrome c staining was detected throughout the cell,
which obscured the nuclei, consistent with redistribution of cytochrome
c from mitochondria to cytoplasm (21). Consistent with the
immunoblotting data (Fig. 7A), treatment with zVAD.fmk
failed to prevent 1
,25(OH)2D3-mediated
cytochrome c release, as demonstrated by persistence of
diffuse cytoplasmic cytochrome c staining in
1
,25(OH)2D3- plus zVAD.fmk-treated cells. However, zVAD.fmk did prevent the morphological signs of
apoptosis, including chromatin condensation and nuclear
fragmentation, consistent with its ability to block PS redistribution,
PARP cleavage, and DNA fragmentation induced by
1
,25(OH)2D3.
View larger version (69K):
[in a new window]
Fig. 8.
Effects of
1 ,25(OH)2D3 or
TNF
on morphology and cytochrome c
release in MCF-7 cells. Cells grown on Lab-Tek II
chamber slides were treated with ethanol, 100 nM
1
,25(OH)2D3 ± 25 µM zVAD.fmk,
or 2.5 ng/ml TNF
, fixed after 96 h (ethanol,
1
,25(OH)2D3) or 48 h (TNF
),
immunostained with cytochrome c (6H2.B4) mouse monoclonal
antibody, and visualized with Alexa-488-conjugated secondary antibody.
Nuclei were counterstained with Hoechst 33258. The images were taken
with an Olympus AX70 fluorescence microscope (original magnification,
×400). Top, phase contrast; middle, cytochrome
c (green); bottom, Hoechst
(blue). The results are representative of at least two
independent experiments.
,25(OH)2D3, but did protect MCF-7 cells
from morphological signs of apoptosis, including DNA fragmentation, we
examined whether zVAD.fmk-treated cells actually remained viable and/or
maintained clonogenic potential. As shown in Fig.
9, both zVAD.fmk and zDEVD.fmk caspase
inhibitors rescued MCF-7 cells from TNF
-mediated cell death, as
demonstrated by total cell numbers, with zVAD.fmk offering the greater
protection. However, neither zVAD.fmk nor zDEVD.fmk caspase inhibitors
could protect MCF-7 cells from
1
,25(OH)2D3-mediated apoptosis, since the
reduction in total cell number was not abrogated by either inhibitor
(Fig. 9). Finally, the clonogenic potential was determined after
treatment of cells with 1
,25(OH)2D3 or
TNF
in the presence or absence of zVAD.fmk followed by re-plating at
limiting dilutions in fresh medium. In vehicle control-treated
cultures, at least 1 out of 5 cells had the ability to produce clones.
In TNF
-treated cultures, clonogenicity was less than 1 out of 500 cells (f < 0.002) but in the presence of zVAD.fmk, the
frequency of cells with clonogenic potential was significantly
increased (f
0.2). In
1
,25(OH)2D3 treated cultures, clonogenicity
was less than 1 out of 50 cells (f < 0.02), and this
was not enhanced in the presence of zVAD.fmk.
View larger version (15K):
[in a new window]
Fig. 9.
Effect of caspase inhibitors on MCF-7 cell
number after treatment with
1 ,25(OH)2D3 or
TNF
. Cells were seeded at a density of
1000 cells/well in 24-well plates. Two days after plating, cells were
treated with ethanol, 100 nM
1
,25(OH)2D3, or 10 ng/ml TNF
for 5 days ± 25 µM zVAD.fmk (broad spectrum,
bottom) or zDEVD.fmk (caspase-3/7-specific, top).
Total cell number was determined by crystal violet assay as described
under "Experimental Procedures." Data represent mean ± S.E.
of four replicate determinations. The results are representative of at
least two independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
,25(OH)2D3 induces apoptosis in MCF-7 cells
by disruption of mitochondrial function, which is accomplished by
translocation of Bax to mitochondria, and increased permeability of the
outer mitochondrial membrane. Of particular interest, neither Bax
translocation nor the mitochondrial disruption is induced by
1
,25(OH)2D3 in a variant line of MCF-7 cells
selected for resistance to
1
,25(OH)2D3-mediated apoptosis (13).
Collectively, these data implicate an essential role for mitochondrial
signaling in the induction of apoptosis by
1
,25(OH)2D3 and identify the pro-apoptotic
protein Bax as an important downstream target of the VDR in MCF-7 cells.
,25(OH)2D3 induces cytochrome
c release and ROS generation, events that have been observed
in cells induced to undergo apoptosis by overexpression of Bax (22,
23). These data further support the concept that
1
,25(OH)2D3-mediated apoptosis may be driven
by Bax translocation. A role for the pro-apoptotic protein Bax in
1
,25(OH)2D3-mediated apoptosis is consistent
with previous studies that support a role for Bcl-2, the anti-apoptotic antagonistic partner of Bax, in mediating the effects of
1
,25(OH)2D3 on both breast and prostate
cancer cells. Thus, 1
,25(OH)2D3
down-regulates Bcl-2 (24, 25) and overexpression of Bcl-2 blocks
1
,25(OH)2D3-induced apoptosis (26, 27).
Since Bcl-2 and Bax act antagonistically in the regulation of
apoptosis, these data suggest that down-regulation of Bcl-2 in
conjunction with translocation of Bax may be necessary for
1
,25(OH)2D3-mediated apoptosis. Further
studies will be necessary to identify the signals generated by
1
,25(OH)2D3 that induce Bax translocation to
mitochondria. Since events upstream of Bax translocation to
mitochondria in response to 1
,25(OH)2D3 are abrogated in the vitamin D3-resistant MCF-7 variant,
comparison of early events in VDR signaling in these cells will be an
important subject for future studies.
,25(OH)2D3 induced features of apoptosis
associated with caspase activation, such as PARP cleavage, PS exposure,
and DNA fragmentation. To determine whether caspase activation was
required for 1
,25(OH)2D3-mediated apoptosis,
we used the broad spectrum, cell-permeable caspase inhibitor zVAD.fmk.
We observed that 1
,25(OH)2D3 signaling on
mitochondria does not require caspase activation, since zVAD.fmk was
unable to block 1
,25(OH)2D3-induced
cytochrome c release, decrease in mitochondrial membrane
potential, or ROS production. Again, this is consistent with apoptosis
driven by Bax translocation, which promotes cytochrome c
release via caspase-independent pathways (28-31). Our data also
complement that of Mathiasen et al. (27), who reported that
inhibition of caspase activity by overexpression of CrmA, a
cowpox-derived caspase inhibitor, or caspase inhibitory peptides
(Ac-DEVD-CHO, Ac-IETD-CHO, and zVAD.fmk) did not block vitamin
D3-mediated growth arrest or apoptosis.
,25(OH)2D3, zVAD.fmk did block events
downstream of mitochondria such as PARP cleavage, external display of
PS, and DNA fragmentation. These findings are similar to reports of Bax-induced apoptosis, where caspase inhibitors had no effect on
Bax-induced cytochrome c release or mitochondrial
disruption, but prevented cleavage of nuclear and cytosolic substrates
and DNA degradation (28-31). However, our data conflict with that of Mathiasen et al. (27), who observed that zVAD.fmk did not
block 1
,25(OH)2D3-mediated DNA fragmentation
in MCF-7 cells. This discrepancy may reflect differences in doses (1 µM versus 25 µM) or experimental design between the two studies. The lower dose of zVAD.fmk (1 µM) used by Mathiasen et al. may have been
insufficient to block mitochondrial-initiated caspases (caspase-9)
(32).
,25(OH)2D3 triggers both
caspase-independent and caspase-dependent pathways in MCF-7
cells, and suggest that 1
,25(OH)2D3 can
activate downstream effector caspases. Since cytochrome c
release has been associated with autoactivation of procaspase-9,
1
,25(OH)2D3 may activate caspase-dependent pathways via cytochrome c
release. However, no DEVDase activity was detected in
1
,25(OH)2D3-treated cytoplasmic extracts,
suggesting that other, possibly unidentified, effector caspases may be
activated by 1
,25(OH)2D3, or that
caspase-dependent events occur at later stages in the
apoptotic program. Although blocking caspase activation prevented some
of the morphological aspects of
1
,25(OH)2D3-mediated apoptosis, MCF-7 cells
were not rescued from death by zVAD.fmk. This finding is consistent
with reports that many cell types eventually die by a slower,
non-apoptotic cell death if caspases are inactivated (8). These data
support the concept that mitochondrial damage represents a cell death commitment step in the course of apoptosis induced by many stimuli (33), including 1
,25(OH)2D3.
,25(OH)2D3 mediates apoptosis
of MCF-7 cells through mitochondrial signaling, which involves ROS
generation, and is regulated by the Bcl-2 family of apoptotic
regulators. Caspases act solely as executioners to facilitate
1
,25(OH)2D3-mediated apoptosis, and caspase
activation is not required for induction of cell death by
1
,25(OH)2D3. Our data suggest distinct
differences in the mechanisms of apoptosis induced by
1
,25(OH)2D3 and TNF
, since inhibition of
caspases was able to rescue MCF-7 cells from TNF
-mediated, but not
1
,25(OH)2D3-mediated, cell death. Although caspase inhibition blocked biochemical changes associated with caspase activation downstream of mitochondrial perturbations and loss
of cytochrome c, the commitment of MCF-7 cells to
1
,25(OH)2D3-mediated apoptosis is clearly
caspase-independent.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Thomas Waterfall for technical assistance.
![]() |
FOOTNOTES |
---|
* This work was supported by Army Breast Cancer Research Program Grant DAMD17-97-1-7183 and NCI National Institutes of Health Grant CA69700.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Biological
Sciences, University of Notre Dame, Notre Dame, IN 46556. Tel.:
219-631-3371; Fax: 219-631-7413; E-mail: jwelsh3@nd.edu.
Published, JBC Papers in Press, October 26, 2000, DOI 10.1074/jbc.M006876200
2 C. J. Narvaez and J. Welsh, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
1, 25(OH)2D3, 1
,25-dihydroxyvitamin
D3;
z, benzyloxycarbonyl;
Ac, acetyl;
VAD, Val-Ala-Asp;
DEVD, Asp-Glu-Val-Asp;
IETD, Ile-Glu-Thr-Asp;
fmk, fluoromethylketone;
CHO, aldehyde;
AFC, 7-amino-4-trifluoromethylcoumarin;
DEVDase, DEVD-cleavage specific caspase;
TNF
, tumor necrosis factor
;
TNFR1, tumor necrosis factor receptor 1;
VDR, vitamin D3
receptor;
PAGE, polyacrylamide gel electrophoresis;
PARP, poly(ADP-ribose) polymerase;
ROS, reactive oxygen species;
PS, phosphatidylserine;
PI, propidium iodide;
PBS, phosphate-buffered
saline;
TMRE, tetramethylrhodamine ethyl ester;
HE, hydroethidine;
FITC, fluorescein isothiocyanate;
BSA, bovine serum albumin.
![]() |
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