 |
INTRODUCTION |
It is now well established that anti-cancer drugs kill tumor cells
by activating essential components of the apoptotic pathway (1-7). In
this regard the pivotal role of intracellular caspases (8) and
mitochondrial-derived pro-apoptotic factors, such as cytochrome
c and apoptosis-inducing factor (9), has been highlighted in
recent communications (6, 10-15). As a result, the current model
supports a cross-talk between caspases and mitochondria during the
commitment and execution of the apoptotic signal (16-25); however, the
early events responsible for triggering the recruitment and activation
of caspases and mitochondrial factors are still sketchy. An important
observation reported with CD95- and drug-induced apoptosis is the
marked acidification of the intracellular milieu of cells that exhibit
morphological and biochemical changes consistent with apoptosis
(26-30). Prevention of acidification blocks subsequent apoptosis in
some systems (31), and, conversely, generation of a rapid intracellular
pH decrease induces caspase activation and apoptosis (32, 33). However,
the mechanism of intracellular acidification and its temporal
relationship to caspase activation and mitochondrial dysfunction during
apoptosis are debatable. Contradictory findings have been reported with
the general caspase inhibitor
benzyloxycarbonyl-valinyl-alaninyl-aspartyl-(O-methyl)-fluoromethylketone as to whether intracellular acidification induces caspases or is a
downstream consequence of caspase activation (32, 34). Similarly, there
is a fair amount of controversy with respect to the role of the
mitochondrial permeability transition
(MPT)1 pore in cytosolic
acidification during chemically induced apoptosis (35, 36). In a recent
report, we demonstrated that hydrogen peroxide
(H2O2)-mediated apoptosis was accompanied by
intracellular acidification (37). These findings, together with the
fact that an increase in intracellular H2O2 is
observed in tumor cells upon exposure to anticancer agents (38-40),
stimulated our interest in investigating the temporal relationship
between drug-induced H2O2 production,
intracellular acidification, and caspase activation.
We have recently demonstrated that two novel anticancer agents
generated upon photo-oxidation of merocyanine 540 (MC540) triggered cytosolic translocation of cytochrome c in tumor cells (6, 41). Despite efficient cytochrome c release and induction of MPT, one of the agents, namely C5, was ineffective in triggering apoptosis due to insufficient caspase activation (41). We concluded that the mere release of cytochrome c in the absence of
caspase activation was not sufficient for effective drug-induced apoptosis.
In the present report, the role of intracellular acidification in the
apoptotic activity of the two photoproducts was investigated. We show
that HL60 cells acidify upon exposure to C2, and that the intracellular
acidification and caspase activation is dependent upon
H2O2 production. We also provide evidence to
support that oxidant-dependent release of cytochrome
c from purified mitochondria occurs independent of the MPT,
but could be inhibited by scavengers of H2O2.
These findings provide a mechanistic explanation for the differential
response of tumor cells to two drugs that activate cytochrome
c translocation but differ in their abilities to trigger caspase activation and effective apoptosis, and highlight the critical
role of H2O2 in acidifying the intracellular
milieu, thus creating a permissive environment for caspase activation and apoptosis.
 |
EXPERIMENTAL PROCEDURES |
Cells and Culture Conditions--
The human promyelocytic
leukemia cell line HL60 was obtained from ATCC (Rockville, MD) and
maintained in culture in a 37 °C incubator with 5% CO2
in RPMI 1640 supplemented with 10% fetal bovine serum (Life
Technologies, Inc.).
Purification of Photo-oxidation Products C2 and
C5--
Photoactivation of MC540 (Sigma) and purification of
photoproducts C2 and C5 were performed as described recently (6). Purified compounds were resuspended in Me2SO at 100 mg/ml
and stored at
20 °C.
Measurement of Intracellular pH with
2',7'-Bis(2-carboxyethyl)-5,6-carboxyfluorescein
(BCECF)--
Intracellular pH (pHi) was
measured by loading cells with membrane-impermeant dye BCECF (Sigma) as
described elsewhere (42). Briefly, HL60 cells (1 × 106) before or after exposure (2-12 h at 37 °C) to C2
(50 µg/ml), C5 (150 µg/ml), or H2O2
(35-138 µM) were washed once with HBSS, resuspended in
0.1 ml of HBSS, and loaded with 10 µl of 1 mM BCECF at
37 °C for 30 min in the dark. Cells were resuspended in 0.5 ml of
HBSS and analyzed using a Coulter Epics Elite ESP (Coulter, Hialeah,
FL) flow cytometer with the excitation set at 488 nm. A minimum of
10,000 events was analyzed, and the ratio of BCECF fluorescence at 525 and 610 was used to obtain intracellular pH from a pH calibration
curve. In order to generate a pH calibration curve, HL60 cells were
loaded with BCECF as above, washed once with HBSS, and then resuspended
in high K+ buffer (135 mM
KH2PO4, 20 mM NaCl, and 110 mM KH2PO4, and 20 mM
NaCl with a range of pH between 6.0 and 8.0). Immediately before flow
cytometry, cells were loaded with 20 µM nigericin (1 mM stock in absolute alcohol; Sigma), and fluorescence
ratio measurements (525 nm/610 nm) of cells in nigericin-containing
buffers of a range of pH were then used to relate histogram channel
numbers to pHi. To evaluate the effect of
scavenging intracellular H2O2 on
pHi, cells were incubated with either catalase (1000 units/ml) or the synthetic non-protein catalase/SOD mimetic EUK-8
(30 µM) for 1 h before treatment with C2 or C5.
Where indicated, pHi was clamped by incubating
cells in medium at the required pH in the presence of 1 µg/ml
nigericin. 1 µg/ml nigericin did not affect cell viability for up to
8 h.
Flow Cytometric Analysis of Intracellular
H2O2 Concentration--
Intracellular
H2O2 was determined by staining with
2',7'-dichlorofluorescein diacetate (Molecular Probes, Eugene, OR),
which is oxidized to dichlorofluorescein by
H2O2 (43). HL60 cells were exposed to 50 µg/ml C2 or 150 µg/ml C5 for 2-8 h, loaded with 20 µM 2',7'-dichlorofluorescein diacetate at 37 °C for 30 min, and analyzed by flow cytometry (Coulter Epics Elite ESP;
excitation 488 nm). Catalase (1000 units/ml) was used to
scavenge intracellular H2O2 before addition of
the drugs and flow cytometric analysis of H2O2.
To assess, if C2-induced H2O2 production was caspase-dependent, cells were incubated for 1 h with
caspase inhibitors DEVD, IETD, or LEHD (100 µM) prior to
the addition of C2 (50 µg/ml) for 4 h, followed by flow
cytometry for H2O2.
Determination of Caspases 3, 8, and 9 Activities--
Caspases
3, 8, and 9 activities were assayed by using AFC-conjugated substrates
supplied by Bio-Rad. HL60 cells (1 × 106 cells/ml)
were incubated with C2 (50-100µg/ml) or C5 (150µg/ml) for 8 h, washed twice with 1× phosphate-buffered saline, resuspended in
50µl of chilled cell lysis buffer (provided by the supplier), and
incubated on ice for 10 min. 50 µl of 2× reaction buffer (10 mM HEPES, 2 mM EDTA, 10 mM KCl, 1.5 mM MgCl2, 10 mM dithiothreitol) and
6 µl of the fluorogenic caspase-specific substrate (DEVD-AFC for
caspase 3, IETD-AFC for caspase 8, and LEHD-AFC for caspase 9) were
added to each sample and incubated at 37 °C for 30 min. Protease
activity was determined by the relative fluorescence intensity at 505 nm following excitation at 400 nm using a spectrofluorimeter (Luminescence Spectrometer LS50B, PerkinElmer Life Sciences,
Buckinghamshire, United Kingdom). Where indicated, cell lysates were
analyzed for poly(ADP-ribose) polymerase (PARP) cleavage by SDS-PAGE
and Western blotting with a monoclonal anti-PARP antibody (clone C2-10
from PharMingen, San Diego, CA). Detection of the 116-kDa and/or the cleaved 85-kDa bands was carried out using horseradish
peroxidase-conjugated anti-mouse IgG. In addition, the effect of
H2O2 on caspase 3 activity was assessed by
exposing HL60 cells to increasing concentrations of
H2O2 (35-276 µM) for 8 h.
In order to evaluate the effect of H2O2 on
caspase 3 activation induced by C2 or C5, HL60 cells were incubated (1 h) with catalase (1000 units/ml) or EUK-8 (30 µM) prior
to the addition of 50 µg/ml C2 or 150 µg/ml C5.
Propidium Iodide Staining for DNA Fragmentation and
Apoptosis--
Cell death was assessed by propidium iodide (PI)
staining for DNA fragmentation as described elsewhere (5). Briefly,
HL60 cells (1 × 106 cells/ml) were treated with 50 µg/ml C2 or 150 µg/ml C5 for 12-18 h, fixed with 70% ethanol, and
stained with PI for DNA content analysis. At least 10,000 events were
analyzed by flow cytometry with the excitation set at 488 nm and
emission at 610 nm. Data are shown as percentage of cells with
sub-diploid DNA and are mean ± S.D. of three experiments.
Detection of Cytosolic Cytochrome c--
For determination of
cytosolic cytochrome c, HL60 cells (30 × 106) were treated with 50 µg/ml C2 or 150 µg/ml C5 for
12 h, cytosolic fractions were obtained and analyzed by Western
blotting for cytochrome c as described previously (6). In a
separate experiment, M14 cells (2 × 103) were grown
on coverslips, exposed to C2 or C5, fixed with methanol:acetone (1:1,
v/v), and incubated for 2 h at 37 °C with 1:150 dilution (in
3% bovine serum albumin) of anti-cytochrome c antibody
(clone 7H8.2C12; PharMingen) as described previously (6). Following three washes with 1× phosphate-buffered saline + 1% fetal bovine serum, cells were exposed to 1:20 dilution of anti-mouse fluorescein isothiocyanate-conjugated IgG (Pharmingen, San Diego, CA) for 1 h,
washed twice, and analyzed by confocal microscopy (NUMI Core Facility,
National University of Singapore, Singapore).
Detection of Cytochrome c in Mitochondrial
Supernatants--
Mitochondria were isolated from mouse liver (BALB/c)
as described elsewhere (44). 0.5 mg of mitochondria were incubated with
C2 (100 µg) or C5 (150 µg) for 1 h at 30 °C in the presence and absence of 1000 units/ml catalase or 30 µM EUK-8,
followed by centrifugation at 4000 × g for 5 min.
Fifty µg of the supernatants were subjected to 12% SDS-PAGE, and
transferred to polyvinylidene difluoride membranes using a Trans-blot
SD semi-dry system (Bio-Rad). Membranes were then blocked overnight
with 5% dry milk in TBST (50 mM Tris/HCl, pH 7.4, 150 mM NaCl, 0.1% Tween 20). Incubation with anti-cytochrome
c antibody (1:5000) was performed at room temperature for
1 h in Tris-buffered saline + 0.05% Tween 20 and 1% bovine serum
albumin. Following three washes with TBST, the membranes were exposed
to goat anti-mouse IgG-horseradish peroxidase conjugate (1:5000)
supplied as 0.8 mg/ml (Pierce) for 1 h and washed thrice with
TBST. Chemiluminescence was detected by the SuperSignal substrate
Western blotting kit (Pierce).
Intracellular O2
Measurement--
A lucigenin-based chemiluminescence assay (45) was
used for measuring intracellular O2
as
described previously (46). Chemiluminescence was monitored for 20, 40, and 60 s in a TD-20/20 luminometer (Turner Designs, Sunnyvale,
CA). Data are shown as relative light units (RLU) per 2 × 106 cells ± S.D. of three to six independent
measurements. For detection of mitochondrial
O2
, purified mitochondria (0.5 mg)
were exposed to 50 µg of C2 or 150 µg of C5 for 0-20 min and
O2
was measured immediately as
described above.
Determination of Mitochondrial Matrix Swelling--
Large
amplitude mitochondrial swelling was determined spectroscopically by
the loss of absorbance at 540 nm as described elsewhere (47). Where
indicated, 10 µM cyclosporin A (CsA; Sigma), 1000 units
of catalase, or 30 µM EUK-8 were added to the
mitochondria prior to the addition of C5 (150 µg).
 |
RESULTS |
Intracellular Acidification Is Required for Efficient Caspase 3 Activation in HL60 Cells--
We have recently shown that the release
of cytochrome c in the absence of efficient caspase
activation was not sufficient for effective drug-induced apoptosis in
HL60 cells (41). Although both C2 and C5, recently described
photoproducts of MC540, induced cytosolic translocation of cytochrome
c as shown by Western blotting of HL60 cell cytosols and
immunofluorescence analyses in an adherent cell line M14 (Fig.
1 (a and b) and
Ref. 41), only C2 triggered efficient processing of caspase 3 and PARP
cleavage (Fig. 1, c and d). Intrigued by these
results, we hypothesized that the intracellular environment may not be
conducive for effective caspase activation in C5-treated cells, unlike
C2. Therefore, we wondered if the ability of C2 to effectively trigger
caspase activation and apoptosis could be explained by the induction of
intracellular acidification. Our results showed that, within 4 h
of treatment with C2 (50 µg/ml), the pHi of
HL60 cells dropped from 7.4 to 7.0 (Fig.
2a). Interestingly, kinetic
analysis of caspase 3 activity clearly showed that a significant
increase in caspase 3 activity occurred between 4 and 8 h of
exposure to C2 (Fig. 2b), suggesting that caspase 3 activation was downstream of intracellular acidification. Similar
treatment with C5 (150 µg/ml) for 4-18 h had no effect on
pHi or caspase 3 activation (Fig. 2,
a and c). We next asked if the activity of C5
could be enhanced by acidification of the intracellular milieu. To do
so, we first incubated the cells for 12 h with C5 to allow
intracellular events triggered by C5, and then clamped
pHi by culturing cells for the next 6 h in
pH 7.0 medium in the presence of 1 µg/ml nigericin (Fig.
2a). Whereas clamping pHi at 7.0 for
6 h in cells cultured in medium alone or by exposure to C5 for 12 or 18 h had no effect on caspase 3 activation, exposure to C5
followed by clamping pHi at 7.0 resulted in a
significant increase in caspase 3 activation (Fig. 2c). This
was further confirmed by Western blot analysis of cleavage of caspase 3 substrate PARP (48). PARP was not cleaved by exposure to either C5 (150 µg/ml for 18 h) or nigericin (1 µg/ml for 8 h), but C5
followed by intracellular acidification with nigericin (pH 7.0)
efficiently induced PARP cleavage as shown in Fig. 2d.
Furthermore, the percentage of cells with sub-diploid DNA (indicative
of apoptotic fraction) significantly increased (>50% as compared with
10% with C5 alone) upon reducing the pHi to 7.0 for 6 h following C5 exposure (Fig. 2e).

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 1.
Cytosolic translocation of cytochrome
c induced upon incubation of tumor cells with C2 or
C5. a, HL60 cells (30 × 106) were
exposed to 50 µg/ml C2 or 150 µg/ml C5 for 12 h and cytosolic
fractions were subjected to SDS-PAGE and Western blotting for
cytochrome c as described under "Experimental
Procedures." b, M14 cells (1 × 103) were
grown on coverslips, treated with C2 or C5 for 12 h, and
cytochrome c localization was determined by confocal
microscopy using anti-cytochrome c as described under
"Experimental Procedures." c, HL60 (1 × 106) cells were treated with C2 or C5 for 12 h
followed by flow cytometric analysis of caspase 3 processing using a
PE-conjugated anti-active caspase 3 that recognizes the processed form
of caspase 3 (p17). c, lysates obtained from C2- and
C5-treated HL60 cells (18 h) were also analyzed for cleavage of PARP by
Western blotting using a monoclonal anti-PARP antibody (clone
C2-10).
|
|

View larger version (38K):
[in this window]
[in a new window]
|
Fig. 2.
Drug-induced intracellular acidification
triggers caspase activation and apoptosis in HL60 cells.
a, 1 × 106 cells were incubated with C2
(50 µg/ml) for 4 h or C5 (150 µg/ml) for 4-18 h, loaded with
pH-sensitive probe BCECF, and the fluorescence ratio (525/610 nm) was
used to measure pHi as described under
"Experimental Procedures." In a separate experiment, cells were
first incubated with C5 for 12 h followed by clamping of
pHi at 7.0 with 1 µg/ml nigericin for 6 h. Data shown are mean ± S.D. of six independent experiments.
b, HL60 cells were exposed to 50 µg/ml C2 for 4-16 h,
followed by fluorimetric detection of caspase 3 activity as described
under "Experimental Procedures." Data are mean ± S.D. of four
independent experiments and are shown as -fold increase of caspase 3 activity (× increase) compared with the untreated cells (1×).
c, lysates obtained from cells treated with C5 (12 or
18 h) or C5 (12 h) + nigericin (6 h) were analyzed for caspase 3 activity as described under "Experimental Procedures." Data are
mean ± S.D. of four independent experiments and are shown as
-fold increase of caspase 3 activity (× increase) compared with the
untreated cells (1×). d, PARP cleavage was analyzed by
Western blotting of lysates from cells treated with C5 (18 h),
nigericin (8 h), or C5+nigericin using anti-PARP antibody (clone
C2-10). e, DNA fragmentation induced upon exposure to C2,
C5, or C5 + nigericin, for 18 h was assessed by PI staining and is
expressed as subdiploid DNA (%).
|
|
C2-induced Acidification Is Accompanied by
H2O2 Production--
In our recent report, we
demonstrated that H2O2-triggered apoptosis in
M14 melanoma cells was accompanied by acidification of intracellular
milieu (37). Similar results were obtained by incubating HL60 cells
with 138 µM H2O2. HL60 cells
exposed to H2O2 acidified within 4 h of
treatment, which was inhibited by prior incubation of cells with
H2O2 scavenger catalase (Fig. 3a). In addition, efficient
caspase 3 activation, PARP cleavage, and DNA fragmentation were induced
by exposure to 138 µM H2O2, which
was inhibited by preincubation with catalase (Fig. 3,
b-d).

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 3.
H2O2 induces
intracellular acidification and caspase 3 activation in HL60
cells. a, HL60 cells (1 × 106/ml)
were incubated with 138 µM H2O2
for 4 h in the presence or absence of catalase (1000 units/ml),
loaded with BCECF, and the fluorescence ratio (525/625 nm) obtained was
used to determine pHi from a pH standard curve
as described under "Experimental Procedures." b, Caspase
3 activity was assayed in lysates of HL60 cells incubated with 138 µM H2O2 for 8 h in the
presence or absence of catalase using a fluorimetric assay described
under "Experimental Procedures." Data are shown as -fold (×)
increase of activity over the untreated control cells (1×).
c, HL60 cells were incubated with increasing concentrations
of H2O2 for 12 h, and lysates were
analyzed for PARP cleavage by Western blotting. d, DNA
fragmentation induced upon exposure to 138 µM
H2O2 (18 h) in the presence and absence of
catalase was assessed by PI staining and is expressed as subdiploid DNA
(%).
|
|
Having shown that C2 induced intracellular acidification in HL60
cells, we next asked if the acidification was dependent upon intracellular production of H2O2. Indeed, HL60
cells exposed to C2 (50 µg/ml) for 4 h showed an increase in
dichlorofluorescein fluorescence by flow cytometry (Fig.
4a), indicative of
intracellular H2O2 production. Pre-incubation
of cells with catalase (1000 units/ml) completely inhibited the
production of H2O2 (Fig. 4b) and the intracellular acidification induced by C2 (Fig. 4c). These
findings were confirmed by using a cell permeable catalase mimetic
EUK-8 (30 µM) (49) as shown in Fig. 4c. On the
contrary, similar to its effect on pHi,
treatment of cells with 150 µg/ml C5 for 4 or 8 h had no effect
on intracellular H2O2 production (Fig.
5a). However, addition of
H2O2 (69 µM) for 4 h
following 12 h incubation with C5 resulted in a drop in
pHi to 7.0 (data not shown), significant
activation of caspases 9 and 3, and increase in percentage of DNA
fragmentation as compared with C5 (18 h) or
H2O2 (4 h) alone (Fig. 5b).

View larger version (38K):
[in this window]
[in a new window]
|
Fig. 4.
Acidification induced by C2 is due to
intracellular generation of H2O2.
Intracellular H2O2 was detected by flow
cytometric analysis of DCHF-DA-stained HL60 cells following exposure to
C2 (50-100 µg/ml) for 4 h (a) or catalase (1000 units/ml) + C2 (50 µg/ml) for 4 h (b). At least
10,000 events were analyzed by WinMDI software. c, HL60
cells were treated with C2 (50 µg/ml) in the presence or absence of
H2O2 scavengers catalase (1000 units/ml) or
EUK-8 (30 µM) and pHi was
determined as described under "Experimental Procedures." Data shown
are the mean ± S.D. of four experiments.
|
|

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 5.
C5 is ineffective as an apoptotic trigger due
to lack of H2O2 production. a,
HL60 cells (1 × 106/ml) were incubated with C5 (150 µg/ml) for 4-8 h, loaded with DCHF-DA, and analyzed by flow
cytometry for H2O2 production. b,
HL60 cells were exposed to C5 (150 µg/ml) for 18 h or C5 for
12 h followed by H2O2 (69 µM) for 6 h or H2O2 (69 µM) for 6 h, and caspases 3 and 9 activities were
determined as described under "Experimental Procedures." Data are
shown as RLU, following excitation at 410 nm and emission at 505 nm.
Cell death was determined by PI staining for DNA fragmentation.
|
|
The drop in intracellular pH induced by H2O2
has been shown to involve activation of the nuclear repair enzyme PARP,
and inhibition of the ATP-dependent
Na+/H+ antiporter (50). In order to assess if
acidification induced by C2 was dependent upon PARP activation and the
subsequent inhibition of the Na+/H+ antiporter,
cells were preincubated with the PARP inhibitor 3-aminobenzamide, which
does not itself alter pHi. Subsequent addition of either C2 (50 µg/ml) for 4 h or H2O2
(138 µM) for 30 min completely inhibited the drop in pH
induced by either C2 or H2O2 (Fig.
6a). Similar results were
obtained with another inhibitor phenanthridinone (51), suggesting the
involvement of PARP activation in the acidifying activity of C2. Next
we questioned if the drop in intracellular pH could be due, in part, to
inhibition of the Na+/H+ antiporter. In order
to accomplish that the effect of a known inhibitor of the
Na+/H+ exchanger, amiloride, on C2 and
H2O2 induced acidification was investigated.
Following preincubation with C2 (50 µg/ml) or
H2O2 (138 µM), cells were
incubated with 10 µM amiloride for 30 min. Results showed
no subsequent effect of amiloride on intracellular pH following prior
incubation with either C2 or H2O2 (Fig.
6b). Conversely, prior inhibition of the
Na+/H+ exchanger with amiloride blocked the
subsequent effect of C2 or H2O2 on
pHi (data not shown). Thus, the absence of a
significant
pHi upon incubation with C2
following amiloride treatment, or vice versa, suggests the
presence of a common intracellular target. These results support
earlier findings that H2O2-mediated
intracellular acidification is due, at least in part, to inhibition of
the Na+/H+ exchanger (50).

View larger version (51K):
[in this window]
[in a new window]
|
Fig. 6.
Acidification induced by C2 is
PARP-dependent and involves inhibition of
Na+/H+ antiport. a, HL60 cells
were pretreated with 3-aminobenzamide (0.5 mM) for 1 h
prior to the addition of C2 (50 µg/ml for 4 h) or
H2O2 (138 µM for 30 min) and
pHi was determined by BCECF loading as described
under "Experimental Procedures." b, HL60 cells were
incubated with C2 (50 µg/ml for 4 h) or
H2O2 (138 µM for 30 min) prior to
the addition of 10 µM amiloride (30 min).
pHi was determined as described under
"Experimental Procedures." There was no significant difference in
pHi upon addition of amiloride after C2
compared with C2 alone (*) and upon addition of amiloride following
H2O2 over H2O2 alone
(**).
|
|
H2O2 Production and Acidification by C2 Is
Upstream of Caspase Activation--
Having shown that C2 triggered
H2O2 production and intracellular
acidification, we next assessed the temporal relationship between
caspase activation and H2O2
production/acidification. In a previous report, we have shown that
apoptosis induced by C2 in HL60 cells was caspase
8-dependent (6); therefore, we asked if incubation of cells
with inhibitors of caspases 8, or executioner caspase 3 could block
H2O2 production by C2. Flow cytometric analysis
of cells treated with 50 µg/ml C2 in the presence of cell permeable
caspase inhibitors (IETD-CHO for caspase 8 or DEVD-CHO for caspase 3)
did not inhibit intracellular H2O2 production (Fig. 7a). In addition,
preincubation of cells with inhibitors of caspases 8, 3, and 9 (LEHD-CHO) did not significantly inhibit intracellular acidification
induced by C2 (Table I). On the contrary, incubation of cells with catalase (1000 units/ml) or EUK-8 (30 µM) resulted in almost complete inhibition of both
caspase 8 and 3 activities, and mitochondrial-dependent
caspase 9 activity triggered in HL60 cells by 50 µg/ml C2 (Fig.
7b). These results indicate that intracellular
H2O2 production and acidification induced upon exposure of HL60 cells to C2 occurred upstream of caspase
activation.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 7.
Drug-induced
H2O2 production and acidification occurs
upstream of caspase activation. a, HL60 cells (1 × 106/ml) were treated with C2 (50 µg/ml) for 4 h,
or preincubated for 1 h with 100 µM caspase
inhibitors DEVD-CHO or YVAD-CHO followed by incubation with C2 (50 µg/ml) for 4 h. Intracellular H2O2 was
detected by dichlorofluorescein fluorescence by flow cytometry.
b, HL60 cells were incubated with C2 (50 µg/ml) for 8 h in the presence or absence of catalase (1000 units/ml) or EUK-8 (30 µM) and caspase 3, 8, and 9 activities were measured in
the lysates. Data shown are the mean ± S.D. of three independent
experiments and show -fold (×) increase in caspase activity over the
untreated cells (1×).
|
|
View this table:
[in this window]
[in a new window]
|
Table I
Acidification induced by C2 is independent of caspase activation
HL60 cells (1 × 106/ml) were incubated with 50 µg/ml C2
for 4 h in the presence or absence of 100 µM
specific inhibitors of caspases 8 (IETD), 9 (LEHD), or 3 (DEVD). Cells
were then loaded with BCECF and pHi was determined as described
under "Experimental Procedures." The change in pHi
( pHi) was calculated from the mean pH values obtained from
each set of experiments. Data shown are mean ± S.D. of three
independent experiments.
|
|
C2 Triggers H2O2 Production via
Mitochondrial O2
Generation--
Intracellular H2O2 is a
dismutation product of O2
; hence, we
first asked if the production of H2O2 by C2 was
secondary to O2
generation. A
lucigenin-based chemiluminescence assay was used to detect
intracellular O2
following incubation
of HL60 cells to C2 (50 µg/ml) for 1-4 h. A kinetic analysis of
O2
generation upon exposure to C2
revealed a significant increase in the intracellular concentration of
O2
with the maximum concentration
attained at 2 h following C2 treatment, which dropped to base-line
levels by 4 h (Fig.
8a).

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 8.
Intracellular surge of
O2 in HL60 cells treated with
C2. a, HL60 cells (2 × 106) were
incubated with 50 µg/ml C2 for 1, 2, or 4 h. Intracellular
O2 was measured by a lucigenin-based
chemiluminescence assay as described under "Experimental
Procedures." Data shown are mean ± S.D. of three independent
experiments done in duplicates and are expressed as RLU/60 s.
b, HL60 cells were preincubated with the NADPH oxidase
inhibitor DPI (10 µM) for 1 h followed by 2 h
of treatment with 50 µg/ml C2 or phorbol 12-myristate 13-acetate (1 µM) and intracellular O2
was measured as described. Data are shown as RLU/60 s and expressed as
percentage of control. c, cells (1 × 106/ml) were pretreated with DPI (10 µM) for
1 h, followed by incubation for 8 h with 50 µg/ml C2, and
lysates were assayed for caspase 3 activity.
|
|
Intracellular sources of O2
include
the NADPH oxidase and the mitochondria (52, 53). Whereas inhibition of
NADPH oxidase by incubation of cells with 10 µM
diphenyleneiodonium (DPI) blocked phorbol 12-myristate
13-acetate-induced intracellular production of
O2
, there was no significant
inhibition of intracellular O2
production triggered by C2 (Fig. 8b), which correlated with
the inability of DPI to inhibit caspase 3 activation (Fig.
8c), and percentage of cell death assessed by PI staining
(data not shown) induced by C2. We then resorted to the mitochondria,
as the potential source of intracellular
O2
in response to C2. Purified mouse
liver mitochondria were incubated with 50 µg/ml C2 or 150 µg/ml C5
for 0-20 min and O2
was measured as
described before. Results showed that, within 5 min of incubation with
C2, the mitochondrial O2
was
significantly increased, peaked by 10 min, and returned to base-line
levels by 20 min (Fig. 9a). In
contrast, treatment with C5 had no effect on mitochondrial
O2
concentration (Fig.
9a).

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 9.
Drug-induced mitochondrial generation of
O2 triggers cytochrome
c release, without the induction of MPT. a,
purified mouse liver mitochondria (0.1 mg) were incubated with 50 µg
of C2 or 150 µg of C5 for 5, 10, or 20 min at 30 °C and
O2 was determined by the lucigenin
chemiluminescence assay. Data shown are mean ± S.D. of three
experiments. b, 0.5 mg purified mitochondria were incubated
with 100 µg of C2 in the absence or presence of
H2O2 inhibitors catalase (1000 units) or EUK-8
(30 µM) at 30 °C for 1 h and centrifuged at
2500 × g. Supernatants were then subjected to SDS-PAGE
and Western blot analysis using anti-cytochrome c antibody.
c, induction of the MPT pore was assessed by monitoring
mitochondrial matrix swelling spectrophotometrically at 540 nm after
the addition of 100 µg of C2 or 150 µg of C5. Where indicated CsA
(10 µM) or catalase (1000 units) were added before the
addition of the drugs.
|
|
H2O2-mediated Release of Cytochrome c Is
Independent of the MPT Pore--
We recently showed that cytochrome
c release triggered by C5 was dependent upon induction of
MPT and opening of the CsA-inhibitable inner membrane pore, whereas the
mechanism of C2-induced cytochrome c release was independent
of the mitochondrial pore (41). Having shown that mitochondria were the
source of O2
generated upon exposure
of HL60 cells to C2, we next evaluated the effect of scavenging
intracellular H2O2 on the release of cytochrome
c, and the induction of permeability transition. Our results
showed that preincubation of mitochondria with catalase or EUK-8
completely inhibited the release of cytochrome c triggered by exposure to 50 µg of C2 (Fig. 9b). In contrast,
although CsA inhibited C5-induced mitochondrial swelling and MPT pore
opening, catalase had no effect on the induction of MPT and matrix
swelling triggered by C5 (Fig. 9c). Similarly, the
CsA-inhibitable release of cytochrome c from C5-treated
mitochondria was unaffected by prior incubation with the radical
scavengers (data not shown).
 |
DISCUSSION |
Intracellular acidification has been described in cells undergoing
apoptosis through the cell surface receptor CD-95 or in response to
anti-cancer drug treatment (26, 27, 29, 32). Thus, intracellular
acidification appears to represent a common event in the pathway of
apoptotic cell death. Indeed, our data show that acidification of the
intracellular milieu is critical to induce efficient caspase activation
and cell death following drug-induced apoptosis. Moreover, we provide
evidence that acidification of the intracellular milieu is due to
mitochondrial production of radical oxygen species, in particular
H2O2, upstream of caspase activation.
We used two novel compounds derived from photo-oxidation of MC540,
namely C2 and C5, because in a recent report we had shown that, despite
their similar ability to induce release of mitochondrial cytochrome
c in tumor cells, C5 was unable to trigger efficient apoptotic cell death, which was due to inefficient
processing/activation of cytosolic caspase (41). Hence, we hypothesized
that the mere release of cytochrome c and induction of
permeability transition was insufficient for effective apoptosis in
HL60 cells, and that an additional factor with cytochrome c
could explain for the efficient apoptotic signaling by C2. We show here
that the intracellular milieu of HL60 cells acidified upon exposure to
C2. On the contrary, cells incubated with C5 failed to show any
significant change in the pHi with minimal
caspase activation and apoptosis; however, the apoptotic activity of C5
was significantly enhanced upon acidification of the intracellular
milieu. These data strongly support that cytochrome c
release without acidification is not efficient for effective apoptotic
cell death, and that acidification creates an intracellular environment
permissive for caspase activation.
Although intracellular acidification during apoptosis has been
attributed to a selective loss of pH regulation, the factor(s) that
trigger acidification during induction of apoptosis are not well
understood. A recent report (37) and data shown in Fig. 3 demonstrate
that apoptotic signaling by H2O2 is mediated by intracellular acidification. A similar effect of
H2O2 on pHi has also
been demonstrated in several other cell types, including renal
epithelial cells (54), rat cardiac myoblasts, and rat cardiac myocytes
(55). Interestingly, apoptosis induced by commonly used anti-cancer
drugs has been shown to involve H2O2 production (39). Stimulated by these observations, we asked if
H2O2 production could explain for intracellular
acidification following HL60 treatment with C2. Indeed, we observed a
surge of H2O2 production within 4 h of
incubation of HL60 cells with C2, which was absent in C5-treated cells.
Moreover, scavenging intracellular H2O2
abrogated the shift in response to C2 and the drop in
pHi, which correlated with the absence of
caspase activation and cell death. In addition, the critical role of
H2O2 dependent intracellular acidification in
the apoptotic process is further highlighted by our findings that the
addition of low concentrations of H2O2 (69 µM) to cells for 4 h after 18 h of incubation
with C5 resulted in intracellular acidification, enhanced caspase
activation, and DNA fragmentation. These results strongly suggest that
production of H2O2 is critical in the induction
of intracellular acidification and activation of caspase following
cytochrome c release.
Investigations on the temporal relationship of
H2O2 to the caspase cascade during the
apoptotic process have yielded conflicting results. One set of data
suggests that oxygen radicals such as H2O2,
function upstream of caspase 3 activation (56), and another supports
caspase 3-dependent production of
H2O2 in drug-induced apoptosis (39).
Furthermore, a recent report has demonstrated that intracellular
acidification occurs downstream of caspase 8 activation (57). In our
model, inhibiting caspase activity did not block
H2O2 production triggered by exposure to C2;
however, scavenging intracellular H2O2
inhibited C2-induced caspase 8, 9, and 3 activities. In addition, the
intracellular acidification induced by C2 was completely inhibited by
blocking H2O2. These data suggest a sequence of
events whereby generation of intracellular H2O2
induced by C2 dysregulates pHi and the resulting acidification facilitates the downstream activation of the caspase cascade.
Intracellular acidification induced by H2O2 has
been linked to the inhibition of the membrane
Na+/H+ exchanger in a variety of cell types
(50, 54, 58, 59), which appears to be mediated by the activation of the
nuclear repair enzyme PARP (50, 60).
H2O2-mediated PARP activation could rapidly
decrease cellular NAD+ and, subsequently, ATP levels (60).
Depletion of ATP could then inhibit the ATP-dependent
Na+/H+ exchanger (61, 62) and induce
acidification. Indeed, prior inhibition of PARP by 3-aminobenzamide or
phenanthridinone completely blocked acidification induced by C2 or
H2O2. In addition, we provide evidence that the
pH dysregulation induced by C2 and H2O2, at least in part, involved inhibition of the
Na+/H+ exchanger. Interestingly, it has been
previously reported that photoproducts of MC540 induce rapid reduction
in cellular ATP levels in tumor cells (63). These results suggest that
intracellular acidification induced by C2 is dependent upon
H2O2-mediated PARP activation, and may involve
inhibition of the Na+/H+ exchanger.
Intracellular H2O2 is produced via the
enzymatic dismutation of O2
by
Cu/ZnSOD or the mitochondrial MnSOD. Indeed, our results showed that
intracellular H2O2 production in HL60 cells was
preceded by a surge in intracellular
O2
, which returned to base-line levels
by 4 h following drug exposure. This is consistent with our
results on H2O2 production in HL60 cells, which
could be detected as early as 2 h (data not shown) and peaked at
4 h after the addition of C2. The two major intracellular sources
of O2
are the membrane-bound NADPH
oxidase system, and the mitochondrial electron transport chain. Whereas
inhibitor of NADPH oxidase DPI (64) had no effect on
O2
production, caspase activation, and
apoptosis triggered in HL60 cells by C2, incubation of isolated
mitochondria with C2 showed a significant increase in
O2
. Contrarily, C5 had no effect on
mitochondrial O2
production. The
O2
thus generated could then be
converted by the mitochondrial MnSOD to H2O2,
which can readily diffuse through mitochondrial and other cellular
membranes and become distributed throughout the cell. These data are
supported by published evidence of H2O2 release from the mitochondria (53).
One of the critical steps in apoptosis is the cytosolic translocation
of cytochrome c (12), which is an amplification factor for
the caspase cascade (24). Here we provide evidence for the involvement
of mitochondrial-derived oxygen radicals in C2-induced cytosolic
translocation of cytochrome c. Whereas scavenging
H2O2 completely inhibited cytochrome
c release induced by C2, there was no effect on
mitochondrial swelling and MPT pore-dependent cytochrome
c release triggered by C5. Thus, mitochondrial generation of
H2O2 might induce the dissociation of
cytochrome c from the inner membrane, independent of the MPT
pore and matrix swelling. In this respect, a previous report has
demonstrated that exogenous H2O2 triggered
pro-caspase activation via cytosolic translocation of cytochrome
c before any loss in
m in Jurkat T cells
(65).
Taken together, these data suggest that acidification triggered
by mitochondrial-derived H2O2, coupled with the
release of cytochrome c, could be a strong amplification
signal for the efficient activation of the caspase cascade during
drug-induced apoptosis. However, with respect to caspase 9 activation,
our results do not rule out the possibility that, although dependent
upon H2O2 production and cytochrome
c release, activation could occur independent of
acidification. This is corroborated by our published findings that
slight caspase 9 activation, in the absence of caspase 3 and 8 activation, is induced with MPT pore-dependent release of cytochrome c by the other photoproduct C5 (41).