From the Departments of Radiation Oncology and
Pharmacology,
Department of Anatomy, and the ¶ Department
of Physiology and Biophysics, Case Western Reserve University,
Cleveland, Ohio 44106-4942
Received for publication, January 25, 2001, and in revised form, March 1, 2001
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ABSTRACT |
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Apoptosis is an evolutionarily conserved pathway of biochemical and
molecular events that underlie cell death processes involving the
stimulation of intracellular zymogens. The process is a genetically programmed form of cell death involved in development, normal turnover
of cells, and in cytotoxic responses to cellular insults. Once
apoptosis is initiated, biochemical and morphological changes occur in
the cell. These changes include: DNA fragmentation, chromatin condensation, cytoplasmic membrane blebbing, cleavage of apoptotic substrates (e.g. PARP, lamin B), and loss of mitochondrial
membrane potential with concomitant release of cytochrome c
into the cytoplasm (7-9). Apoptosis is a highly regulated, active
process that requires the participation of endogenous cellular enzymes
that systematically dismantle the cell. The most well characterized
proteases in apoptosis are caspases, aspartate-specific cysteine
proteases, that work through a cascade that can be initiated by
mitochondrial membrane depolarization leading to the release of
cytochrome c and Apaf-1 into the cytoplasm (10), that then
activates caspase 9 (11). Non-caspase-mediated pathways are less understood.
We previously showed that apoptosis following Ca2+ is recognized as an important regulator of apoptosis
(16-21). The cytoplasmic Ca2+ concentration is maintained
at ~100 nM in resting cells by relatively impermeable
cell membranes, active extrusion of Ca2+ from the cell by
plasma membrane Ca2+-ATPases, plasma membrane
Na+/Ca2+ exchangers, and active uptake of
cytosolic Ca2+ into the endoplasmic reticulum (ER) by
distinct Ca2+-ATPases. In contrast, the concentration of
Ca2+ in the extracellular milieu and in the ER is much
higher (in the millimolar range). Evidence for involvement of
Ca2+ influx into the cytosol as a triggering event for
apoptosis has come from studies with specific Ca2+ channel
blockers that abrogate apoptosis in regressing prostate following
testosterone withdrawal (22). Other support for the involvement of
Ca2+ in apoptosis comes from the observation that agents
that directly mobilize Ca2+ (e.g.
Ca2+ ionophores or the sarcoplasmic reticulum
Ca2+-ATPase pump inhibitor, thapsigargin, TG) can
trigger apoptosis in diverse cell types (23-27). Inhibition of the
sarcoplasmic reticulum Ca2+-ATPase pump by TG causes a
transient increase in cytoplasmic Ca2+ from ER
Ca2+ stores, and a later influx of Ca2+ from
the extracellular milieu, leading to the induction of apoptotic cell
death (24, 27, 28). Consequently, emptying of intracellular Ca2+ stores may trigger apoptosis by disrupting the
intracellular architecture and allowing key elements of the effector
machinery (e.g. Apaf-1) to gain access to their substrates
(e.g. caspase 9). Ca2+ has also been shown to be
necessary for apoptotic endonuclease activation, eliciting DNA cleavage
after many cellular insults (29-31). Buffering intracellular
Ca2+ released from stored Ca2+ pools
(e.g. ER) with BAPTA-AM, or removal of extracellular
Ca2+ with EGTA, can protect cells against apoptosis (32,
33). Therefore, increases in intracellular Ca2+ levels
appear to be important cell death signals in human cancer cells that
might be exploited for anti-tumor therapy. Finally, Ca2+
may act as a signal for apoptosis by directly activating key proapoptotic enzymes (e.g. calpain); however, these
proteolytic responses are poorly understood. The role of
Ca2+ in cell death processes involving caspase activation
has been examined in detail (28, 34-36). However, the role of
Ca2+ in non-caspase-dependent cell death
responses is relatively unexplored.
Recent studies have suggested that alterations in mitochondrial
homeostasis play an essential role in apoptotic signal transduction induced by cytotoxic agents (37, 38). Various apoptotic stimuli have
been shown to induce mitochondrial changes, resulting in release of
apoptogenic factors, apoptosis-inducing factor (39), and mitochondrial
cytochrome c (9) into the cytoplasm. These changes are
observed during the early phases of apoptosis in human epithelial
cells, and were linked to the initial cascade of events, sending the
cell to an irreversible suicide pathway. During high, sustained levels
of cytosolic Ca2+, mitochondrial Ca2+ uptake is
driven by mitochondrial membrane potential to maintain Ca2+
homeostasis in the cytosol. In de-energized mitochondria,
Ca2+ can be released by a reversal of this uptake pathway
(40). These data, therefore, linked changes in Ca2+
homeostasis and mitochondrial membrane potential to the initiation of
apoptosis. Li et al. (41) reported that We previously characterized the activation of a novel cysteine protease
in various breast cancer cell lines with properties similar to the
Ca2+-dependent cysteine protease, calpain,
after exposure to Reagents--
Cell Culture--
MCF-7:WS8 (MCF-7) human breast cancer cells
were obtained from Dr. V. Craig Jordan, (Northwestern University,
Chicago, IL). MDA-MB-468 cells were obtained from the American Type
Culture Collection and transfected with NQO1 cDNA in the pcDNA3
constitutive expression vector as described previously (5). Tissue
culture components were purchased from Life Technologies, Inc., unless otherwise stated. MCF-7 cells were grown in RPMI 1640 cell culture medium supplemented with 10% fetal bovine serum, in a 37 °C
humidified incubator with 5% CO2, 95% air atmosphere as
previously described (2, 5). For all experiments, log-phase breast
cancer cells were exposed to 5 µM TUNEL Assay--
Cells were seeded at 1 × 106
cells/10-cm Petri dish and allowed to grow for 24 h. Log-phase
cells were then pretreated for 30 min with 10 µM
BAPTA-AM, 3 mM EGTA, or 50 µM dicumarol
followed by a 4-h pulse of 5 µM Cell Growth Assays--
MCF-7 cells were seeded at 5 × 104 cells per well in a 12-well plate and allowed to attach
overnight. The following day, log-phase cells were pretreated for 30 min with 5 µM BAPTA-AM, followed by a 4-h pulse of
Confocal Microscopy--
MCF-7 cells were seeded at 2-3 × 105 cells per 35-mm glass bottom Petri dishes (MatTek
Corp., Ashland, MA) and allowed to attach overnight. Cells were rinsed
twice in a Ca2+/Mg2+ balanced salt solution
(BSS, 130 mM NaCl, 5 mM KCl, 1.5 mM
CaCl2, 1 mM MgCl2, 25 mM HEPES, pH 7.5, 5 mM glucose, 1 mg/ml bovine serum albumin) and loaded with the Ca2+-sensitive
fluorescent indicator, fluo-4-AM (5 µM), in BSS for ~20-30 min at 37 °C. Cells were rinsed twice in BSS and incubated for an additional 20 min at 37 °C to allow for hydrolysis of the AM-ester. Cells were imaged with a Zeiss 410 confocal microscope (Thornwood, NY) equipped with a ×63 N.A. 1.4 oil immersion
planapochromat objective at room temperature (the same results were
observed at room temperature and 37 °C). Confocal images of fluo-4
fluorescence were collected using a 488-nm excitation light from an
argon/krypton laser, a 560-nm dichroic mirror, and a 500-550 nm
band-pass barrier filter. Three basal images were collected before drug
addition (8 µM Mitochondrial Membrane Potential Determinations--
MCF-7 cells
were seeded at 2.5-3 × 105 cells per 6-well plate,
and allowed to grow for 24 h. Log-phase cells were pretreated for
30 min with 10 µM BAPTA-AM, 3 mM EGTA, or 50 µM dicumarol followed by a 4-h pulse of 5 µM ATP Measurements--
Cells were seeded at 2.5 × 105 cells per well in 6-well dishes and allowed to attach
for 24 h. Fresh medium was added to the cells along with
Ca2+ chelators or dicumarol 30 min prior to Western Blot Analyses--
Whole cell extracts from control or
Ca2+ Chelators Prevent
To examine whether BAPTA-AM could affect Ca2+ Chelators Do Not Block Apoptosis Induced by Other
Agents--
It was possible based on the data in Fig. 1 that calcium
chelators may block Exposure of NQO1-expressing MCF-7 Cells to
After exposure to 8 µM
Since the ER is a major store of Ca2+ in the cell, we
tested if the initial rise in intracellular Ca2+ levels
after exposure of MCF-7 cells to Loss of Mitochondrial Membrane Potential After Loss of ATP After
Loss of ATP following Ca2+ Chelators Prevent
A simple explanation for the aforementioned results could be that BAPTA
blocks bioactivation of
Mitochondrial membrane depolarization induced by
The dramatic loss of intracellular ATP in MCF-7 cells following
Dicumarol also abrogated DNA fragmentation induced by When homeostatic mechanisms for regulating cellular
Ca2+ are compromised, cells may die, either by necrosis or
apoptosis (20, 21, 36). We demonstrated that bioactivation of -Lapachone (
-Lap) triggers apoptosis
in a number of human breast and prostate cancer cell lines through a
unique apoptotic pathway that is dependent upon NQO1, a two-electron
reductase. Downstream signaling pathway(s) that initiate apoptosis
following treatment with
-Lap have not been elucidated. Since
calpain activation was suspected in
-Lap-mediated apoptosis, we
examined alterations in Ca2+ homeostasis using
NQO1-expressing MCF-7 cells.
-Lap-exposed MCF-7 cells exhibited an
early increase in intracellular cytosolic Ca2+, from
endoplasmic reticulum Ca2+ stores, comparable to
thapsigargin exposures.
1,2-Bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester, an intracellular Ca2+ chelator,
blocked early increases in Ca2+ levels and inhibited
-Lap-mediated mitochondrial membrane depolarization, intracellular
ATP depletion, specific and unique substrate proteolysis, and
apoptosis. The extracellular Ca2+ chelator, EGTA, inhibited
later apoptotic end points (observed >8 h, e.g. substrate
proteolysis and DNA fragmentation), suggesting that later execution
events were triggered by Ca2+ influxes from the
extracellular milieu. Collectively, these data suggest a critical, but
not sole, role for Ca2+ in the NQO1-dependent
cell death pathway initiated by
-Lap. Use of
-Lap to trigger an
apparently novel, calpain-like-mediated apoptotic cell death could be
useful for breast and prostate cancer therapy.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Lap1 is a naturally
occurring compound present in the bark of the South American Lapacho
tree. It has antitumor activity against a variety of human cancers,
including colon, prostrate, promyelocytic leukemia, and breast (1-3).
-Lap was an effective agent (alone and in combination with taxol)
against human ovarian and prostate xenografts in mice, with little host
toxicity (4). We recently demonstrated that
-Lap kills human breast
and prostate cancer cells by apoptosis, a cytotoxic response
significantly enhanced by NAD(P)H:quinone oxidoreductase (NQO1, E.C.
1.6.99.2) enzymatic activity
(5).2
-Lap cytotoxicity
was prevented by co-treatment with dicumarol (an NQO1 inhibitor) in
NQO1-expressing breast and prostate cancer cells (5).2 NQO1
is a cytosolic enzyme elevated in breast cancers (6) that catalyzes a
two-electron reduction of quinones (e.g.
-Lap, menadione), utilizing either NADH or NADPH as electron donors. Reduction of
-Lap by NQO1 presumably leads to a futile cycling of
the compound, wherein the quinone and hydroquinone form a redox cycle
with a net concomitant loss of reduced NAD(P)H (5).
-Lap administration
was unique, in that an ~60-kDa PARP cleavage fragment, as well as
distinct intracellular proteolytic cleavage of p53, were observed in
NQO1-expressing breast or prostate cancer cells (5).2 These
cleavage events were distinct from those observed when caspases were
activated by topoisomerase I poisons, staurosporine, or administration
of granzyme B (5, 12, 13). Furthermore,
-Lap-mediated cleavage
events were blocked by administration of global cysteine protease
inhibitors, as well as extracellular Ca2+ chelators (12).
Based on these data, we concluded that
-Lap exposure of
NQO1-expressing breast and prostate cancer cells caused the activation
of a Ca2+-dependent protease with properties
similar to calpain; in particular, the p53 cleavage pattern of
-Lap-exposed cells was remarkably similar to the pattern observed
after calpain activation (14, 15).
-Lap caused a
decrease in mitochondrial membrane potential with release of cytochrome c into the cytoplasm in a number of human carcinoma cell
lines, shortly after drug addition. Other alterations in metabolism
(e.g. ATP depletion) have not been examined in
-Lap-treated cells.
-Lap (12). Using NQO1-expressing breast cancer
cells, we show that
-Lap elicits a rise in intracellular
Ca2+ levels shortly after drug administration that
eventually leads to apoptosis. This paper suggests a critical, but not
sufficient, role for Ca2+ in the cell death pathway
initiated by NQO1-dependent bioactivation of
-Lap.
Possible combinatorial effects (e.g. NAD(P)H depletion as
well as intracellular calcium alterations) that initiate
-Lap-mediated apoptosis in NQO1-expressing breast cancer cells will
be discussed.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Lapachone
(3,4-dihydro-2,2-dimethyl-2H-naphtho[1,2b]pyran-5,6-dione)
was synthesized by Dr. William G. Bornmann (Memorial Sloan
Kettering, New York), dissolved in dimethyl sulfoxide at 10 mM, and the concentration verified by spectrophotometric
analysis (2, 5). EGTA, Hoescht 33258, and thapsigargin were obtained from Sigma. BAPTA-AM
(1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra-(acetoxymethyl ester)) was obtained from Calbiochem (La
Jolla, CA). JC-1
(5,5'6,6'-tetrachloro-1,1',3,3'-tetraethyl-benzimidazolylcarbocyanine iodide) and Fluo-4-AM were obtained from Molecular Probes, Inc. (Eugene, OR).
-Lap for 4 h
(unless otherwise indicated), after which fresh medium was added and
cells were harvested at various times post-treatment.
-Lap, as described
above, or 24 h treatment of 10 µM ionomycin or 1 µM staurosporine. Medium was collected from experimental
as well as control conditions 24 h later, and attached along with
floating cells were monitored for apoptosis using TUNEL 3'-biotinylated
DNA end labeling via the APO-DIRECT kit (Pharmingen, San Diego, CA) as
described (5). Apoptotic cells were analyzed and quantified using an
EPICS XL-MCL flow cytometer that contained an air-cooled argon laser at
488 nm, 15 mW (Beckman Coulter Electronics; Miami, Fl), and XL-MCL
acquisition software provided with the instrument.
-Lap (0-5 µM). Drugs were removed and fresh medium
added. Cells were allowed to grow for an additional 6 days. DNA content
(a measure of cell growth) was determined by fluorescence using Hoechst
dye 33258 as described (5) and changes in growth were monitored using a
PerkinElmer HTS 7000 Plus Bio Assay Plate Reader (Norwalk, CT) with 360 and 465 nm excitation and emission filters, respectively. Data
were expressed as relative growth, T/C (treated/control), using
experiments performed at least twice.
-Lap, ± 50 µM dicumarol
or 200 nM TG). The mean pixel intensity was set to equal
one for analyses of fold-increase in fluo-4 fluorescence intensity.
Subsequently, images were collected after the indicated treatments at
90-s intervals. BAPTA-AM (20 µM) was co-loaded with fluo-4-AM where indicated. Mean pixels were determined in regions of interest for individual cells at each time point.
-Lap, unless otherwise indicated. Cells were
trypsinized and resuspended in phenol red-minus RPMI medium for
analyses. Cells were maintained at 37 °C for the duration of the
experiment, including during analyses. Prior to analyses, cells were
loaded with 10 µg/ml JC-1 for 9-14 min and samples were analyzed
using a Beckman Coulter EPICS Elite ESP (Miami, FL) flow cytometer.
JC-1 monomer and aggregate emissions were excited at 488 nM
and quantified using Elite acquisition software after signal collection
through 525- and 590-nm band pass filters, respectfully. Shifts in
emission spectra were plotted on bivariant dot plots, on a cell-by-cell
basis, to determine relative mitochondrial membrane potential of
treated and control cells.
-Lap
exposure (4 h unless otherwise indicated). Floating cells were
collected, pelleted, and lysed in 1.67 M perchloric acid.
Attached cells were lysed directly in 1.67 M perchloric
acid. Following a 20-min incubation at room temperature, attached cells
were scraped and transferred to corresponding microcentrifuge tube,
cooled on ice for several minutes, and spun to pellet protein
precipitates. Deproteinized samples were neutralized with 3.5 M KOH and HEPES/KOH (25 mM HEPES, 15 mM KOH, pH 8), and incubated on ice for 15 min.
Precipitates were removed by centrifugation and samples stored at
20 °C. Cell extracts were analyzed for ATP and ADP levels using a
luciferase-based bioluminescent assay and rephosphorylation protocols,
as described (42).
-Lap-exposed MCF-7 cells were prepared and analyzed by
SDS-polyacrylamide gel electrophoresis/Western blot analyses as
previously described (2, 5, 12). Loading equivalence and transfer
efficiency were monitored by Western blot analyses of proteins that are
known to be unaltered by experimental treatments (2), and using Ponceau
S staining of the membrane, respectively. Probed membranes were then
exposed to x-ray film for an appropriate time and developed. Dilutions
of 1:10,000 for the C-2-10 anti-PARP antibody (Enzyme Systems
Products, Livermore, CA), and 1:2000 for anti-p53 DO-1 and anti-lamin B
(Santa Cruz Biotechnology, Santa Cruz, CA) antibodies were used as
described (2, 12).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Lap-induced Apoptotic DNA
Fragmentation and Protect against Cell Death--
Log-phase MCF-7
cells were treated for 4 h with 5 µM
-Lap, fresh
medium was then applied, and cells were harvested 24 h later and
analyzed for DNA fragmentation (i.e. apoptotic cells
staining positive in a TUNEL assay). Treatment of MCF-7 cells with
-Lap resulted in >90% apoptotic cells (Fig.
1, A and B).
However, MCF-7 cells exposed to a 30-min pretreatment with 10 µM BAPTA-AM or 3 mM EGTA, followed by a 4-h
pulse of 5 µM
-Lap, exhibited only 20 or 39%
apoptotic cells, respectively, in 24 h.
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Fig. 1.
-Lap-mediated apoptosis and
relative cell growth is Ca2+-dependent.
DNA fragmentation was assessed using the TUNEL assay. Log phase MCF-7
cells were treated with the indicated Ca2+ chelator for 30 min prior to a 4-h pulse of 5 µM
-Lap. TUNEL assays
were performed to monitor apoptosis 24 h after
-Lap addition
(A and B). A, shown are the results of
any one experiment from studies performed at least three times. The
number in the upper right corner represents
percent cells staining positive in the TUNEL assay. Results are
graphically summarized in B as the average of at three
independent experiments, mean ± S.E. Student's t test
for paired samples, experimental group compared with MCF-7 cells
treated with
-Lap alone are indicated (*
p < 0.01). C, cells were exposed to a 4-h
pulse of various concentrations of
-Lap either alone
(closed), or after a 30-min pretreatment with 5 µM BAPTA-AM (open). Relative DNA per well was
determined by Hoescht 33258 fluorescence, and graphed as relative
growth (treated/control DNA); mean relative DNA per well, ± S.E. Shown
are representative results of experiments performed at least twice.
Student's t test for paired samples, experimental group
compared with MCF-7 cells treated with
-Lap alone are indicated (*,
p < 0.05; and **, p < 0.005).
-Lap lethality, we measured
relative growth of MCF-7 cells with or without exposure to
-Lap, and
in the presence or absence of BAPTA-AM. MCF-7 cells were treated for 30 min with 5 µM BAPTA-AM, subsequently exposed to a 4-h
pulse of
-Lap (1.5
5 µM), and relative cell growth
was measured 6 days later (Fig. 1C). The LD50
dose of
-Lap in MCF-7 cells was ~2.5 µM in colony
forming assays, which correlated well with IC50 relative
growth inhibition, as measured by DNA content (2, 5). At 1.5 µM
-Lap, cells exhibited little or no toxicity. At
-Lap doses of 3 or 5 µM, cells exhibited considerable
toxicity, >90% growth inhibition, as previously reported (2, 5).
Toxicity was significantly prevented by 5 µM BAPTA-AM
pretreatment. BAPTA-AM pretreated cells exhibit only 44 and 73% growth
inhibition after 3 or 5 µM
-Lap treatments,
respectively (Fig. 1C). BAPTA alone did not affect MCF-7
cell growth compared with untreated controls.
-Lap-mediated apoptosis by sequestering calcium required for the activation of apoptotic endonucleases. We, therefore, examined both intra- and extracellular Ca2+ chelators for
their ability to prevent apoptosis in NQO1-transfected MDA-468
(MDA-468-NQ3) cells induced by
-Lap, ionomycin (which induces
Ca2+-mediated cell death (36)), and staurosporine (STS,
which inhibits protein kinase C and works via a caspase-mediated cell
death pathway (43, 44)). We used MDA-468-NQ3 cells to assay for
caspase-mediated endonuclease activation and DNA fragmentation since
they express the endonuclease-activating caspase 3, unlike MCF-7 cells
(45). We previously demonstrated that MDA-468-NQ3 cells responded
similarly to
-Lap as MCF-7 cells (Fig.
2 and Ref. 5). EGTA significantly protected MDA-468-NQ3 cells against ionomycin-induced apoptosis, but
not against STS-induced apoptosis (Fig. 2). MDA-468-NQ3 cells treated
for 24 h with 10 µM ionomycin exhibited 49%
apoptotic cells, whereas, MDA-468-NQ3 cells pretreated for 30 min with
3 mM EGTA followed by a 24-h exposure to ionomycin
exhibited only 4% apoptotic cells. Cells treated for 24 h with 1 µM STS in the absence or presence of 3 mM
EGTA exhibited 56 and 46% apoptosis, respectively. BAPTA-AM (10 µM) did not significantly block apoptosis induced by
ionomycin. BAPTA-AM pretreatment of STS-exposed MDA-468-NQ3 cells did
not significantly decrease apoptosis (p < 0.4)
compared with cells exposed to STS alone; the modest effect of BAPTA-AM on STS-induced apoptosis may reflect the Ca2+ dependence of
the apoptotic endonucleases involved in this response. Neither BAPTA-AM
nor EGTA alone elicited apoptotic responses at the doses used in the
aforementioned experiments (Figs. 1B and 2). Furthermore,
preliminary data suggest that DFF45 (ICAD) was cleaved in
NQO1-expressing MCF-7 or MDA-468-NQ3 cells at 8 h after
-Lap
treatment, in a temporal manner corresponding to the induction of
apoptosis (data not shown). Cleavage of DFF45, an endogenous inhibitor
of the magnesium-dependent and Ca2+-independent
apoptotic endonuclease, DFF40 (CAD), suggests that DFF40 is activated
following treatment with
-Lap. Taken together with results in Fig.
1, these data strongly suggest that a rise in intracellular
Ca2+ levels is part of a critical signaling pathway for the
induction of apoptosis in NQO1-expressing human breast cancer cells
following
-Lap exposure.
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Fig. 2.
Ca2+ chelators did not block
Ca2+-activated endonuclease activation after
-Lap. NQO1-expressing MDA-468-NQ3 cells
(generated from non-expressing human breast cancer cells (5)) were
treated with either 3 mM EGTA or 30 µM
BAPTA-AM for 30 min prior to drug addition; either a 4-h pulse of 8 µM
-Lap, or 24 h continuous treatment of 10 µM ionomycin or 1 µM STS. Cells were then
analyzed using the TUNEL assay for DNA fragmentation. Shown are
mean ± S.E. of at least two independent experiments. Student's
t test for paired samples, experimental group compared with
cells treated with drug alone are indicated (*, p < 0.05).
-Lap Results in
Increased Intracellular Ca2+--
We next directly
examined whether intracellular Ca2+ levels were increased
in log-phase MCF-7 cells after
-Lap treatment using the
cell-permeant intracellular Ca2+ indicator dye, fluo-4.
Cells were loaded with 5 µM fluo-4-AM, and where
indicated, 20 µM BAPTA-AM, incubated for ~25 min to allow for the dye to permeate cells, rinsed, and then incubated for an
additional ~20 min for hydrolysis of the AM-ester. Following drug
addition, images were collected every 90 s for ~60 min using confocal microscopy. Three basal images were recorded before drug addition and average pixels per cell were determined (indicative of
fluo-4 fluorescence and, therefore, basal intracellular
Ca2+ levels) and used for analyses over time. The
fluorescence of basal images were averaged and set to equal one; fold
increases were determined from changes in fluo-4 fluorescence over control.
-Lap, MCF-7 cells exhibited an
~2-fold increase in fluo-4 fluorescence from 4 to 9 min, after which time Ca2+ levels returned to basal levels in a majority of
cells examined (43 of 50, 86%) (Fig.
3A). The rise in intracellular
Ca2+ levels in MCF-7 cells following
-Lap exposure was
prevented by preloading cells with BAPTA-AM (20 µM) (Fig.
3B). Interestingly, not all
-Lap-exposed MCF-7 cells were
affected by pretreatment with BAPTA-AM; 3 of 26 cells (12%) exhibited
a rise in intracellular Ca2+ levels after exposure to
-Lap despite the presence of this Ca2+ chelator.
However, BAPTA-AM pretreated MCF-7 cells that did exhibit a rise in
intracellular Ca2+ levels following
-Lap treatment
exhibited a similar, but delayed Ca2+ increase (10-20
min), as compared with
-Lap-exposed MCF-7 cells in the absence of
BAPTA-AM (4-9 min). This may be due to a saturation of the chelator or
heterogeneity of the tumor cell population. These results are
consistent with previous reports that the buffering capacity of
BAPTA-AM may be overwhelmed with time (34, 46). Higher doses of
BAPTA-AM were not used due to toxicity caused by the drug alone (data
not shown).
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Fig. 3.
Intracellular Ca2+ changes
after -Lap. Intracellular
Ca2+ levels were measured in live cells via confocal
microscopy using the Ca2+ indicator dye, fluo-4-AM. MCF-7
cells were loaded with either fluo-4-AM alone (A, C, and
D) or fluo-4-AM and 20 µM BAPTA-AM
(B).
-Lap (8 µM) was added to cells after
basal images were recorded. Images were collected every 90 s for
45-75 min, as indicated. The number in the upper
right corner of each Ca2+ image represents the time
(min) after
-Lap addition. A, representative cells before
and after
-Lap treatments are shown as pseudocolored images. These
results are also displayed in graph form showing
fold change (as compared with basal levels) in fluo-4 fluorescence in
cells after
-Lap treatment over time, with or without co-loading of
BAPTA-AM (A and B). C, TG (200 nM) was added to MCF-7 cells after basal images were
recorded. Once fluo-4 fluorescence returned to basal levels, cells were
subsequently exposed to
-Lap. D,
-Lap was added to
MCF-7 cells after basal images were recorded. After fluo-4 fluorescence
returned to basal levels, TG was subsequently added to the cells. Each
line represents the change in fluo-4 fluorescent emission of
an individual cell over time; each graph is representative of one of at
least three independent experiments.
-Lap was due to release of
Ca2+ from this organelle. If
-Lap exposure led to
release of Ca2+ stored in the ER, then TG (a sarcoplasmic
reticulum Ca2+-ATPase pump inhibitor) administration should
not cause additional Ca2+ release. Similarly, if the
sequence of drug administration were reversed, additional
Ca2+ release would also not be observed. When
-Lap was
added after TG-induced depletion of ER Ca2+ stores, no
measurable rise in intracellular Ca2+ levels occurred in 25 of 27 (93%) cells analyzed (Fig. 3C). Similarly, when TG
was added to cells after
-Lap, only 1 of 18 (6%) cells that
initially responded to
-Lap exhibited a rise in intracellular Ca2+ levels following subsequent TG administration (Fig.
3D). At the end of the experiment, all cells analyzed
remained responsive to ionomycin. Thus, cells exposed to
-Lap and/or
TG were still capable of altering Ca2+ levels, and the
Ca2+ indicator dye was not saturated. We noted that the
increase in fluo-4 fluorescence (2-3-fold over basal levels, Fig.
3A) in MCF-7 cells observed after exposure to
-Lap was
comparable to that elicited by TG (1.5-2.5-fold over basal levels,
Fig. 3C), further suggesting that the two agents mobilized
the same ER pool of Ca2+. All cells analyzed
started with comparable basal levels of Ca2+ and appeared
to load equal amounts of the indicator dye, as determined by basal
fluorescence (measured by pixels per cell) at the beginning of each
analysis; relative basal fluo-4 fluorescence for each experiment in
Fig. 3 were: A, 56 ± 7; B, 52 ± 7; C, 78 ± 8; D, 79 ± 8 S.E. Untreated or BAPTA-AM-loaded MCF-7 cells did not show any fluctuations in basal Ca2+ levels during the time
course of the experiment, nor did any of the drugs interfere with the
Ca2+ indicator dye (data not shown).
-Lap Is
Attenuated by Intracellular, but Not Extracellular, Ca2+
Chelation--
Mitochondrial membrane potential was previously shown
to drop from a hyperpolarized state to a depolarized state after
treatment of various human cancer cells with
-Lap (41). A drop in
mitochondrial membrane potential in
-Lap-treated cells was
accompanied by a concomitant release of cytochrome c into
the cytosol (41). To explore whether early changes in intracellular
Ca2+ levels were upstream of mitochondrial changes in
NQO1-expressing breast cancer cells, log phase MCF-7 cells were
pretreated for 30 min with either 10 µM BAPTA-AM or 3 mM EGTA and then exposed to 5 µM
-Lap for
4 h. Prior to analyses, cells were loaded with JC-1, a cationic
dye commonly used to monitor alterations in mitochondrial membrane
potential (47, 48). Mitochondrial depolarization measurements using
JC-1 were indicated by a decrease in the red/green fluorescence
intensity ratio (a movement of events from upper left to
lower right, Fig. 4), as seen
following a 10-min treatment with the potassium ionophore, valinomycin
(100 nM), which causes a collapse of mitochondrial membrane
potential by uncoupling mitochondrial respiration (Fig. 4e)
(49); cells in the upper left-hand quadrant exhibited high
mitochondrial membrane potential, whereas, cells in the lower
right-hand quadrant have low mitochondrial membrane potential and
are depolarized. Cells in the upper right-hand quadrant exhibited intermediate membrane potential. Mitochondrial membrane potential decreased in MCF-7 cells in a time- and
dose-dependent manner following exposure to
-Lap (Figs.
4, a-d, and data not shown). By 4 h, the majority of
-Lap-treated MCF-7 cells exhibited low mitochondrial membrane
potential (53%), while the majority of control cells maintained high
mitochondrial membrane potential (51%) (Fig. 4, b, a and
g, f, respectively). This drop in mitochondrial membrane
potential observed 4 h after treatment with
-Lap (low, 53%)
was abrogated by pretreatment with BAPTA-AM (low, 23%), but not by
EGTA (low, 48%) (Fig. 4, g-i, respectively). Pretreatment with 10 µM BAPTA-AM prevented the decrease in
mitochondrial membrane potential (low, 23%); however, BAPTA-AM did not
maintain
-Lap-exposed cells in a high-potential state (high, 28%)
as observed in control untreated cells (high, 51%). Approximately half
of the BAPTA-AM-exposed cells were in an intermediate membrane
potential state (45%) (Fig. 4h). We noted, however, that
BAPTA-AM or EGTA exposures alone caused depolarization of the
mitochondria, with a majority of the cells residing in the same
intermediate energized state as observed following BAPTA-AM and
-Lap
(Fig. 4, j-k). Therefore, BAPTA-AM prevented mitochondrial
depolarization induced by
-Lap to the same extent as in cells
treated with BAPTA-AM alone. Pretreatment with 3 mM EGTA
did not affect the loss of mitochondrial membrane potential caused by
-Lap (low 48%), implying that an early rise in intracellular
Ca2+ levels from intracellular stores was sufficient to
cause a drop in mitochondrial membrane potential, and that
extracellular calcium was not needed for these effects in
-Lap-treated cells (Fig. 4, h-i).
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Fig. 4.
-Lap-induced loss of
mitochondrial membrane potential is mediated by alterations in
Ca2+ homeostasis. Mitochondrial membrane potential was
measured in control or drug-treated MCF-7 cells with the JC-1 dye.
A, cells were treated with 5 µM
-Lap and
assayed for changes in mitochondrial membrane potential at 1, 2, and
4 h post-treatment. Exposure of MCF-7 cells to 100 nM
valinomycin for 15 min served as a positive control as described (49).
Cells in the upper left-hand quadrant exhibit high
mitochondrial membrane potential, while cells in the lower
right-hand quadrant exhibit low mitochondrial membrane potential.
B, cells were treated for 30 min with either 10 µM BAPTA-AM or 3 mM EGTA prior to a 4-h
treatment with 5 µM
-Lap. At 4 h, cells were
harvested for analyses of changes in mitochondrial membrane potential
using JC-1 as described above. Shown are representative experiments
performed at least three times, and numbers in each quadrant
represent the average of cells in that quadrant of at least three
independent experiments. S.E. for any single number was not more than
11%.
-Lap Is Attenuated by Intracellular
Ca2+ Chelation--
The bioactivation of
-Lap by NQO1
is thought to lead to a futile cycling between quinone and hydroquinone
forms of the compound, presumably due to the instability of the
hydroquinone form of
-Lap (5). This futile cycling led to depletion
of NADH and NADPH, electron donors for NQO1 in in vitro
assays (5). Exhaustion of reduced enzyme co-factors may be a critical
event for the activation of the apoptotic pathway in NQO1-expressing
cells following
-Lap exposure. We, therefore, measured intracellular
ATP and ADP in log-phase MCF-7 cells after various doses and times of
-Lap (using a luciferase-based bioluminescent assay (42)).
Intracellular ATP levels were reduced in MCF-7 cells after treatment
with
-Lap in a dose- and time-dependent manner (Fig.
5A). At all doses of
-Lap
above the LD50 of the drug (~2.5 µM) in
MCF-7 cells (2), intracellular ATP levels were reduced by >85% at
4 h, the time at which drug was removed (Fig. 5A,
left); the loss of ATP correlated well with
-Lap-induced cell
death in MCF-7 cells (Fig. 1C). ADP levels remained
relatively unchanged after various doses of
-Lap, however, the
[ATP]/[ADP][Pi] ratio decreased dramatically.
Intracellular ATP levels began to drop to 70% of control levels 2 h after 5 µM
-Lap exposure, the time at which
-Lap
began to elicit mitochondrial membrane depolarization (Figs. 5,
A, right, and 4, c). ATP levels continued to drop
to 8% of control levels by 4 h after drug exposure (Fig.
5A, right). In contrast, ADP levels remained relatively unchanged during the course of the experiment, with an increase at 30 min (172% control levels) that returned to control levels by 1 h
post-treatment. Cellular ATP levels in
-Lap-treated cells did not
appear to recover to normal levels within the 6-24-h interval after
drug removal (data not shown).
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Fig. 5.
ATP depletion after
-Lap treatment is Ca2+ dependent.
Intracellular ATP and ADP levels were measured using a luciferase-based
bioluminescent assay. A, cells were treated with the
indicated dose of
-Lap for 4 h or were treated with 5 µM
-Lap for the time indicated, and harvested for ATP
analyses. ATP levels were expressed as nanomoles of ATP per
106 cells. Purified ATP was used as a standard to determine
intracellular ATP concentrations. B, cells were either
pretreated or untreated with the indicated Ca2+ chelators
for 30 min prior to drug addition, and
-Lap (5 µM) was
then added for 4 h. Cells were harvested for analyses following
-Lap exposure. Results represent the average of at least three
independent experiments, ± S.E. Student's t test for
paired samples, experimental group compared with drug alone are
indicated (*, p < 0.05; **, p < 0.01).
-Lap was prevented by a 30-min pretreatment
with an intracellular Ca2+ chelator, but not an
extracellular Ca2+ chelator (Fig. 5B). At 4 h, pretreatment with 10 or 30 µM BAPTA-AM elicited only
58 and 43% ATP loss, respectively, compared with
-Lap alone (92%
loss). The extracellular Ca2+ chelator, EGTA, did not
significantly affect the loss of ATP, nor
[ATP]/[ADP][Pi] ratio observed in MCF-7 cells after
-Lap treatment (Fig. 5B). Exposure of MCF-7 cells to TG
(200 nM) did not elicit decreases in ATP or ADP levels
4 h after drug exposure, compared with untreated control cells.
-Lap-induced
Proteolysis--
We previously showed that apoptosis in various breast
cancer cell lines induced by
-Lap was unique, causing a pattern of PARP and p53 intracellular cleavage events distinct from those induced
by caspase activating agents (12). After
-Lap treatment, we observed
an ~60-kDa PARP cleavage fragment and specific cleavage of p53 in
NQO1-expressing breast cancer cells. Furthermore, we showed that this
proteolysis in
-Lap-treated cells was the result of activation of a
Ca2+-dependent protease with properties similar
to µ-calpain (12). PARP and p53 proteolysis in
-Lap-exposed,
NQO1-expressing cells was prevented by pretreatment with the
extracellular Ca2+ chelators, EGTA and EDTA, in a
dose-dependent manner (at 8 and 24 h) (Ref. 12, and
data not shown). Additionally, PARP, p53, and lamin B proteolysis
induced at 24 h in MCF-7 cells following
-Lap treatment were
abrogated by pretreatment with 10 or 30 µM BAPTA-AM (Fig.
6). These data strongly suggest that a
Ca2+-dependent pathway and potentially a
Ca2+-dependent protease are operative in
-Lap-mediated apoptosis.
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Fig. 6.
Intracellular Ca2+ chelators
prevent apoptotic proteolysis after -Lap
treatment. Apoptotic proteolysis was measured in MCF-7 cells
exposed to a 4-h pulse of 5 µM
-Lap, with or without a
30-min pretreatment of the indicated dose of BAPTA-AM. Whole cell
extracts were prepared 24 h after drug addition, and analyzed
using standard Western blotting techniques with antibodies to PARP,
p53, and lamin B. Shown is a representative Western blot of whole cell
extracts from experiments performed at least three times.
-Lap by NQO1 in a manner similar to that of
dicumarol (5). However, BAPTA (free acid) did not affect the enzymatic
activities of NQO1 using standard enzymatic assays (data not shown)
(5). The free acid (active) form of BAPTA, instead of its
AM ester
form, was used in these assays since intracellular accumulation of this
Ca2+ chelator was not necessary and was physiologically
relevant in the in vitro enzyme assay. Using
-Lap as a
substrate, NQO1 enzymatic activity in the presence of 10 mM
BAPTA (a dose of the free acid form of BAPTA that was >1000-fold
higher than that used in the experiments of Figs. 1-6) was reduced by
<20%. Thus, BAPTA-AM did not affect the activity of NQO1, a
two-electron reductase required for
-Lap cytotoxicity (5). We
conclude that BAPTA-AM prevents
-Lap-induced apoptosis by blocking
Ca2+-mediated signaling events via chelating intracellular
Ca2+.
-Lap Bioactivation by NQO1 Is Critical for
Ca2+-mediated Signaling--
We previously reported that
cells expressing NQO1 are more sensitive to the cytotoxic effects of
-Lap (5).2 NQO1 is inhibited by dicumarol, which
competes with NADH or NADPH for binding to the oxidized form of the
enzyme. Dicumarol thereby prevents reduction of quinones (50, 51). We
demonstrated that dicumarol attenuates
-Lap-mediated proteolysis of
apoptotic substrates (e.g. PARP and p53), apoptosis, and
survival in NQO1-expressing cells (5).2 As expected,
increases in intracellular Ca2+ levels in NQO1-expressing
human cancer cells elicited by
-Lap were abrogated by co-treatment
with 50 µM dicumarol in 26 of 27 cells (96%) examined
(Fig. 7A, lower panel). The
ability of dicumarol to inhibit increases in intracellular
Ca2+ levels was greater than that observed with BAPTA-AM,
where intracellular Ca2+ level increases were prevented in
only 89% of cells examined (Fig. 3B). Thus, NQO1 was
critical for the rise in intracellular Ca2+ levels observed
in MCF-7 cells after
-Lap exposure.
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Fig. 7.
NQO1-dependent activation of
-Lap is critical for Ca2+
signaling. A, intracellular Ca2+ was
measured on live cells using the Ca2+ indicator dye,
fluo-4-AM, and confocal microscopy as described in the legend to Fig.
3. Three basal images were recorded before drug treatments.
-Lap (8 µM) was then added to MCF-7 cells, either alone
(upper panel) or in combination with 50 µM
dicumarol (lower panel). Images were collected every 90 s for 50-60 min. Shown are representative graphs displaying changes in
fluo-4 fluorescence for the duration of the experiment. Each
line represents the fold change in fluo-4 fluorescent
emission (as compared with basal levels) of an individual cell from one
experiment, and the graph is representative of experiments performed at
least three times. B, mitochondrial membrane potential was
measured using the JC-1 dye as described in the legend to Fig. 4. MCF-7
cells were treated with 50 µM dicumarol 30 min prior to
-Lap exposure. Four hours later, cells were harvested for analyses
of mitochondrial membrane potential. Shown are mean ± S.E. of the
percentage of cells with low mitochondrial membrane potential of at
least two independent experiments. C, ATP and ADP levels
were assayed as described in the legend to Fig. 5. Cells were
pretreated with dicumarol for 30 min prior to drug addition, 5 µM
-Lap was added for 4 h, and cells were
harvested immediately thereafter for analyses. Results represent the
mean of at least three independent experiments ± S.E. Student's
t test for paired samples, experimental groups compared with
drug alone are indicated (* p < 0.05). D,
apoptosis, using the TUNEL assay, was assessed as per Fig. 1. MCF-7
cells were treated with 50 µM dicumarol 30 min prior to a
4-h exposure of 5 µM
-Lap. Cells were then harvested
for TUNEL analyses at 24 h post-treatment. Shown are mean ± S.E. of at least three independent experiments.
Student's t test for paired samples, experimental groups
compared with
-Lap exposure alone are indicated (*,
p < 0.005). DC, 50 µM
dicumarol.
-Lap was also
abrogated by pretreatment with dicumarol (Fig. 7B). By
4 h, the majority of
-Lap-treated cells exhibited low
mitochondrial membrane potential (58%), while very few control cells
were depolarized (9%) (Fig. 7B). Pretreatment with
dicumarol attenuated this response to
-Lap, with only 34% being
depolarized. The inability of dicumarol to prevent mitochondrial
depolarization in 34% of
-Lap-treated cells was probably due to the
high background of control cells (20%) that were depolarized after
exposure to dicumarol alone. In comparison with intracellular
Ca2+ buffering, BAPTA-AM elicited only a minor
depolarization of the mitochondria on its own (low, 14%) and thus was
able to elicit a greater protective effect (Fig. 4B); only
23% of cells exposed to BAPTA-AM and
-Lap exhibited low
mitochondrial membrane potential as compared with
-Lap exposed cells
in the presence of dicumarol (34%).
-Lap
exposure was inhibited by a 30-min pretreatment with 50 µM dicumarol (Fig. 7C).
-Lap-treated MCF-7
cells pretreated with dicumarol exhibited only 34% loss of
intracellular ATP, compared with 92% loss after
-Lap treatment
alone (Fig. 7C). ADP levels were not altered by any of the
treatments used, however, the [ATP]/[ADP][Pi] ratio
decreased dramatically in
-Lap-treated cells, and was only partially
decreased with dicumarol pretreatment alone, as compared with control
untreated cells.
-Lap in MCF-7
cells. MCF-7 cells exhibited 94% apoptosis following
-Lap exposure
that was prevented by a 30-min pretreatment with 50 µM
dicumarol; only 6% of the cells staining positive in a TUNEL assay at
24 h post-treatment (Fig. 7D). These data are
consistent with prior results (5), and correlate well with the survival protection afforded by dicumarol to
-Lap-treated cells. Dicumarol did not induce DNA fragmentation on its own. These data are consistent with the protection from apoptosis observed with either intra- and
extracellular Ca2+ chelators. BAPTA-AM or EGTA protected
-Lap exposed MCF-7 cells from apoptosis (Fig. 1, A and
B). Collectively, these data implicate the bioactivation of
-Lap by NQO1 as a critical step in the rise of intracellular
Ca2+ levels following
-Lap exposure, and thus
-Lap-mediated downstream apoptotic events.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Lap
by NQO1 induced cell death in a manner that was dependent upon
Ca2+ signaling (Figs. 1-6).
-Lap can be reduced by NQO1
and may undergo futile cycling between quinone and hydroquinone forms
(
-Lap-Q and
-Lap-HQ, Fig. 8),
presumably depleting NADH and/or NADPH in the cell (5). We theorize
that depletion of NAD(P)H, along with a rise in intracellular
Ca2+ levels in response to
-Lap, activate a novel
caspase-independent apoptotic pathway, as described in this paper and
previously (2, 5, 12). The rise in intracellular Ca2+
appears to be dependent upon the bioactivation of
-Lap by NQO1, suggesting a critical and necessary signaling role for Ca2+
in the downstream apoptotic pathway induced by this drug. Dicumarol completely abrogated intracellular Ca2+ changes (Fig. 7),
as well as apoptosis and survival, following
-Lap exposure of
NQO1-expressing cells (5).2 When increases in intracellular
Ca2+ levels were directly prevented by pretreatment with
BAPTA-AM, downstream apoptotic responses, as well as lethality, caused
by
-Lap were prevented; when corrected for BAPTA-AM affects alone,
-Lap-induced apoptosis, proteolysis, and lethality were essentially blocked by preventing early Ca2+ release from ER stores.
Thus, correcting for the BAPTA-AM affects alone, the role of
Ca2+ in
-Lap-mediated apoptosis may be more significant
that that revealed by the data shown. These data strongly suggest that
DNA fragmentation, mitochondrial membrane depolarization, ATP loss, and
apoptotic proteolysis were a consequence of the increase in intracellular Ca2+ levels (Figs. 1-6 and 8).
Interestingly, the cell death pathway induced by
-Lap was quite
distinct from that observed after exposure to TG, an agent known to
specifically cause release of Ca2+ from ER stores and
mediate caspase-dependent apoptosis (24, 28, 33, 52). Thus,
Ca2+ release was necessary for
-Lap-induced
cytotoxicity, but apparently not sufficient for the unique apoptotic
responses induced by
-Lap.
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Fig. 8.
Proposed model for
-lapachone-mediated apoptosis in NQO1-expressing
cells. In cells that express NQO1,
-Lap is reduced from the
quinone (
-lap-Q) to the hydroquinone
(
-lap-HQ) form in a futile cycle that results in dramatic
losses of NAD(P)H (5). During the metabolism of
-Lap by NQO1,
Ca2+ is subsequently released from the ER causing a rise in
cytosolic Ca2+ levels by an as yet unknown mechanism. To
maintain low cytoplasmic Ca2+ levels, we theorize that
mitochondria sequester Ca2+ and numerous cellular ATPases
probably function to pump Ca2+ out of the cytosol. This
leads to mitochondrial membrane depolarization and ATP hydrolysis,
respectively (Figs. 4 and 5). Sustained depolarization of the
mitochondrial membrane leads to further loss of ATP and prevents ATP
synthesis by inhibiting respiration. The loss of ATP disrupts ionic
homeostasis within the cell and thereby allows extracellular
Ca2+ to enter the cell down its concentration gradient (see
"Discussion"). The secondary rise in cytosolic Ca2+
levels leads to protease (presumably activation of calpain or a
calpain-like protease) and, thus, endonuclease (DFF40) activation,
ultimately resulting in apoptosis.
-Lap and TG-induced Similar Ca2+ Responses, but
Different Patterns of Apoptosis--
-Lap elicited an early rise in
intracellular Ca2+ levels from the same ER store as
released by TG, however, subsequent cell death processes were
remarkably different between the two compounds. TG is known to cause
transient increases in intracellular Ca2+ levels, however,
these were insufficient to induce apoptosis. Much like
-Lap,
Ca2+ was needed from the extracellular milieu, along with a
sustained increase in intracellular Ca2+ levels, for
TG-induced apoptosis (23) in MCF-7 cells (27). Depolarization of the
mitochondrial membrane potential and loss of intracellular ATP in cells
exposed to
-Lap, may have prevented plasma membrane Ca2+
pumps and ER Ca2+ pumps from functioning and maintaining
Ca2+ homeostasis. This, in turn, may have facilitated
Ca2+ leakage down its concentration gradient into the
cytosol, providing a secondary and sustained elevation of
Ca2+ that initiated a protease cascade(s) and ultimately
caused apoptosis after exposure to
-Lap. This is consistent with
what we observed in NQO1-expressing cells after
-Lap treatment and
co-administration of Ca2+ chelators. Buffering
intracellular Ca2+ with BAPTA-AM partially abrogated all of
the downstream events induced in MCF-7 cells by
-Lap (and thus
prevented secondary Ca2+ entry by buffering the initial
rise in cytosolic Ca2+). In contrast, extracellular
chelation by EGTA only prevented those events initiated by secondary
Ca2+ entry (e.g. protease activation and DNA
fragmentation). Thus, a secondary rise in intracellular
Ca2+ levels after exposure to
-Lap seems probable, and
necessary, for protease activation and DNA fragmentation as was
observed for TG-induced caspase-mediated apoptosis (23, 27). However, a
secondary influx of Ca2+ does not appear to be necessary
for reduction in mitochondrial membrane potential or loss of
intracellular ATP after
-Lap exposure, since EGTA did not prevent
these responses.
Although MCF-7 cells treated with -Lap had similar calcium
responses, as do TG-exposed cells,
-Lap-exposed cells exhibited a
very different pattern of apoptosis than TG-treated cells.
-Lap-exposed cells exhibit loss of intracellular ATP and a decrease
in the [ATP]/[ADP][Pi] ratio. In contrast, TG-exposed
cells did not exhibit loss of ATP (Fig. 5, and as reported by Ref. 53).
Our data suggest that in contrast to TG where ATP-dependent
caspase activation results in cell death (28, 33, 34, 54), an
ATP-independent protease is activated after exposure to
-Lap.
Ca2+ may regulate apoptosis by activating
Ca2+-dependent protein kinases and/or
phosphatases leading to alterations in gene transcription. However,
with the rapid loss of intracellular ATP after exposure to
-Lap
(2-4 h, Fig. 5),
-Lap-mediated cell death unlikely involves
stimulated kinases or phosphatases or new protein synthesis. Instead,
indirect kinase inhibition, due to ATP depletion, along with continued
phosphatase activity is likely. Consistent with this notion, we found
dramatic de-phosphorylation of pRb in cells exposed to
-Lap at
3 h (2), a time consistent with loss of ATP following exposure to
this drug. Furthermore, loss of ATP at 2 h may also be responsible
for inhibition of NF-
B activation induced by tumor necrosis
factor-
in
-Lap pre-exposed cells (55), since significant loss of
ATP would prevent proteosome-mediated I
B degradation. Thus,
Ca2+-dependent loss of ATP in NQO1-expressing
cells following
-Lap treatment may explain the reported pleiotropic
effects of this agent.
-Lap-exposed cells also exhibited a very different pattern of
substrate proteolysis compared with that observed after TG (2, 12, 28).
We previously showed that
-Lap elicited a unique cleavage of PARP
(~60-kDa fragment), compared with the classical caspase-3-mediated
fragmentation of the protein (~89 kDa) observed after TG exposure
(data not shown and Ref. 28). In a variety of NQO1-expressing cells
exposed to
-Lap, atypical PARP cleavage was inhibited by the global
cysteine protease inhibitors, iodoacetamide and
N-ethylmaleimide, as well as the extracellular Ca2+ chelators, EGTA and EDTA (12). In addition,
-Lap-mediated apoptotic responses were insensitive to inhibitors of
caspases, granzyme B, cathepsins B and L, trypsin, and
chymotrypsin-like proteases (12). In contrast, classic caspase
inhibitors blocked TG-induced caspase activation and apoptosis (28).
Caspase activation, as measured by pro-caspase cleavage via Western
blot analyses, does not occur following
-Lap
exposures.3 Thus, protease
activation after
-Lap treatment appears to be Ca2+-dependent, or alternatively, is activated
by another protease or event that is
Ca2+-dependent (Figs. 1-6 and Ref. 12).
Loss of Reducing Equivalents Is Also Necessary for -Lap-mediated
Apoptosis, Similar to Menadione-mediated Apoptosis--
Menadione is a
quinone that can be detoxified by NQO1 two-electron reduction. However,
menadione can also be reduced through two, one-electron reductions via
other cellular reductases (56), thus eliciting menadione's toxic
effects. Menadione toxicity, elicited via two, one-electron reductions,
exhibited many similarities to
-Lap-mediated,
NQO1-dependent, toxicity (5). These included: (a) elevations in cytosolic Ca2+ (57, 58);
(b) NAD(P)H depletion (5, 59, 60); (c) ATP depletion (<0.1% control)3 (61-63); and (d)
mitochondrial membrane potential depolarization3 (64). We
previously demonstrated that menadione caused similar substrate
proteolysis (p53 and atypical PARP cleavage) in NQO1-deficient cells,
or at high doses in cells that express NQO1 where detoxification processes were over-ridden (5).3 The semiquinone form
of menadione can undergo spontaneous oxidation to the parent quinone
(59, 63, 65, 66); a pattern similar to the futile cycling observed
after
-Lap bioactivation by NQO1 (5). Loss of reducing equivalents,
such as NADH, due to the futile cycling of menadione may cause
inactivation of the electron transport chain with the concomitant loss
of mitochondrial membrane potential, and thus, loss of ATP (67, 68).
These responses were also observed in MCF-7 cells exposed to
-Lap
(Figs. 4 and 5). Extensive mitochondrial Ca2+ accumulation
can also mediate mitochondrial depolarization (69, 70). Thus,
Ca2+ sequestration may elicit mitochondrial membrane
depolarization and consequent ATP depletion in cells exposed to
lap. These data further suggest that Ca2+ is necessary
for
-Lap-mediated cell death, but other factors are apparently
needed for the initiation of the novel execution apoptotic pathway
observed in cells treated with this compound.
The rise in intracellular Ca2+ appears to be dependent on
the bioactivation of -Lap by NQO1, suggesting a critical and
necessary signaling role for Ca2+ in the downstream
apoptotic pathway induced by this drug. These data suggest that DNA
fragmentation, mitochondrial membrane depolarization, ATP loss, and
apoptotic proteolysis were a consequence of the increase in
intracellular Ca2+ levels. Work in our laboratory is
focused on elucidating the signaling response(s) that elicits ER
Ca2+ release following
-Lap bioactivation by NQO1. The
cell death pathway induced by
-Lap is quite distinct from that
observed after exposure to TG, and
-Lap-mediated apoptosis exhibited
many similarities to menadione-mediated apoptosis. These observations further suggest that early release of Ca2+ from ER stores,
as well as influx of Ca2+ from the extracellular milieu are
necessary, but not sufficient for the novel apoptotic execution pathway
induced by
-Lap. Thus, changes in Ca2+ homeostasis in
conjunction with the presumed loss of reducing equivalents are both
necessary and sufficient for
-Lap-mediated apoptosis. We propose
that development of
-Lap for treatment of human cancers that have
elevated NQO1 levels (e.g. breast and lung) is warranted
(6). Since most clinical agents used to date kill cells by
caspase-dependent and p53-dependent pathways, and many cancers evade death by altering these pathways, development of
agents that kill by specific targets (NQO1-mediated) and in p53- and
caspase-independent manners are needed.
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ACKNOWLEDGEMENTS |
---|
We thank Sara Simmers and Rich Tarin for all
their technical help, as well as R. Michael Sramkoski,
MT-(ASCP)H. We are grateful to Dr. William G. Bornmann for
synthesizing -lapachone, and Edmunds Z. Reineks and Philip A. Verhoef for critical review of this manuscript. We are also indebted to
Sarah Hildebrand for her enduring support of our research.
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FOOTNOTES |
---|
* This work was supported by United States Army Medical Research and Materiel Command Breast Cancer Initiative Grant DAMD17-98-1-8260 (to D. A. B.), Predoctoral Fellowship DAMD17-00-1-0194 (to C. T.), and Postdoctoral Fellowship DAMD-17-97-1-7221 (to J. J. P.).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.
§ Partial fulfillment of the requirements for the Ph.D. degree, Case Western Reserve University, Dept. of Pharmacology.
** To whom correspondence should be addressed: Dept. of Radiation Oncology (BRB-326 East), Case Western Reserve University, 10900 Euclid Ave., Cleveland, OH 44106-4942. Tel.: 216-368-0840; Fax: 216-368-1142; E-mail: dab30@po.cwru.edu.
Published, JBC Papers in Press, March 2, 2001, DOI 10.1074/jbc.M100730200
2 S. M. Planchon, C. Tagliarino, J. J. Pink, W. G. Bornmann, M. E. Varnes, and D. A. Boothman. Exp. Cell Res., in press.
3 C. Tagliarino, J. J. Pink, and D. A. Boothman, unpublished results.
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ABBREVIATIONS |
---|
The abbreviations used are:
-Lap,
-lapachone;
MCP, MCF-7:WS8;
NQO1, NAD(P)H:quinone oxidoreductase,
DT-diaphorase (E.C. 1.6.99.2);
PARP, poly(ADP-ribose) polymerase;
TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end
labeling;
ER, endoplasmic reticulum;
TG, thapsigargin;
STS, staurosporine;
BAPTA-AM, 1,2-bis-(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid-acetoxymethyl ester.
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REFERENCES |
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