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Experimental Therapeutics |
Grace Cancer Drug Center, Roswell Park Cancer Institute, Buffalo, New York 14263
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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m) beginning at 24 h
was followed by the release of cytochrome c into the
cytosol at 30 h. Loss of mitochondrial integrity was accompanied
by caspase-3 activation and poly(ADP-ribose) polymerase digestion from
30 to 36 h. The caspase inhibitor
Z-Asp-2,6-dichlorobenzoyloxymethylketone rendered cells
resistant to analogue-induced caspase-3 activation and reduced the
apoptotic response in a dose-dependent manner. Because polyamine
reduction achieved by inhibitors of polyamine biosynthesis inhibited
growth but did not cause apoptosis, we looked for alternative
polyamine-related events, focusing on induction of SSAT. Three DENSPM
analogues that differentially induced SSAT activity but similarly
depleted polyamine pools revealed a close correlation between enzyme
induction and cytochrome c release, caspase
activation, and apoptosis. Dose-dependent inhibition of
polyamine oxidase, an enzyme that oxidizes acetylated polyamines
generated by SSAT and releases toxic by-products such as
H2O2 and aldehydes, prevented cytochrome
c release, caspase activation, and apoptosis. Taken
together, the findings indicate that DENSPM-induced apoptosis is at
least partially initiated via massive induction of SSAT and related
oxidative events and subsequently mediated by the mitochondrial
apoptotic signaling pathway as indicated by cytochrome c
release and caspase activation. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Recent elucidation of the pathways involved in apoptotic signaling
(7)
has provided early end points for mapping analogue
action and relating them to effects involving polyamine homeostasis.
For example, caspases represent a family of cysteine proteases that are
common downstream effectors of apoptosis. Their contributions are
largely through cleavage of a variety of substrates such as PARP,
inhibitor of caspase-activated deoxyribonuclease, Bcl-2, nuclear
lamina, gelsolin, focal adhesion kinase, and p21-activated kinase 2
(8)
. Cytochrome c represents an upstream
activator of the caspases that is released from the mitochondria into
the cytosol, where it binds to Apaf-1 and initiates the caspase cascade
(9, 10, 11)
. Cytochrome c release can be mediated
by different mechanisms including loss of mitochondrial transmembrane
potential (m), alteration of specific ion
or protein channels, mitochondrial swelling, rupture of the outer
membrane, and/or involvement of the Bcl-2 family proteins
(12)
.
In this study, we investigate whether the mitochondrial apoptotic signaling events described above are involved in the cell death response seen in human melanoma SK-MEL-28 cells treated with the polyamine analogue DENSPM. Our findings indicate that activation of caspase-3 is critical for analogue-induced apoptosis, that cytochrome c is one of the main upstream activators of this pathway, and that oxidative events related to analogue induction of SSAT have probable initiating significance.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Cell Culture.
SK-MEL-28 human melanoma cells were maintained in a 5%
CO2 humidified incubator as monolayer cultures in
RPMI 1640 supplemented with 10% Nu-Serum (Collaborative Research
Products, Bedford, MA), 1 mM aminoguanidine, 50 units/ml
penicillin, and 50 µg/ml streptomycin.
Polyamine Enzymes.
ODC, SAMDC, and SSAT activities were assayed as described by Porter
et al. (3)
. SSAT enzyme activity was expressed
as pmol/min/mg protein.
Polyamine Pools.
Intracellular polyamines and polyamine analogues were analyzed by
high-performance liquid chromatography as described previously by this
laboratory (13)
with the following modifications.
Dansylated samples (50 µl) were injected onto a 250 x 3.2-mm ID Econosil C18 column (5 µm particle size; Alltech,
Deerfield, IL) with a column temperature of 50°C and eluted by a
two-solvent gradient using a Waters 616 LC system (Waters, Milford, MA)
and a Waters WISP 710B autosampler. Solvent A contained 55% of 10
mM ammonium phosphate/45% acetonitrile at pH 4.4. Solvent
B contained 100% acetonitrile. At 0.9 ml/min, the gradient began at
100% solvent A and progressed linearly to 82% solvent B over 30 min
with a 15-min hold. Compounds were detected using a McPherson FL-750 BX
fluorescence detector (Acton, MA) with an excitation wavelength of 360
nm and an emission cutoff filter of 500 nm. The data were collected and
analyzed using Waters Millennium 32 chromatography software version
3.05. Polyamine pools were expressed as
pmol/106cells.
Western Blot Analysis.
SK-MEL-28 cells were treated with 10 µM DENSPM for the
indicated time. Cells were harvested, and proteins (40 µg/lane) were
run on 7.5–15% SDS-PAGE gels followed by transfer to polyvinylidene
difluoride membrane and immunoblotted with antibodies as indicated.
Detection was performed using enhanced chemiluminescence from Amersham
Pharmacia Biotech. (Arlington Heights, IL). Polyclonal rabbit
anti-caspase-3, polyclonal rabbit antihuman caspase-8, anti-Bax,
polyclonal rabbit anti-Bid, anti-Bcl-2, and anti-cytochrome
c antibodies were purchased from PharMingen (San Diego, CA).
The anti-PARP antibody was obtained from Boehringer Mannheim Corp.
(Indianapolis, IN), and the monoclonal caspase-9 antibody and
polyclonal Bcl-XL antibody were bought from
Oncogene Research Products (Boston, MA).
Cell Growth and Apoptosis.
Control and analogue-treated cells were collected from flasks and
counted by using a Model ZM Coulter (Coulter Electronics, Hialeah, FL).
Apoptotic cells were detected by annexin V/FITC staining using a kit
obtained from R&D Systems (Minneapolis, MN). Stained cells were
analyzed using a Becton Dickinson FACScan (Flow Cytometry Facility,
Roswell Park Cancer Institute), and data were analyzed using the
Winlist program (Verity House, Topsham, ME).
Mitochondrial Transmembrane Potential.
The electron gradient across the mitochondrial membrane space
during normal respiration is called the mitochondrial transmembrane
potential (m). Loss of
m was measured by flow cytometry after
staining with a MitoCapture kit (Biovision Research Products, Palo
Alto, CA). This technique uses a cationic dye that fluoresces red as it
aggregates inside healthy mitochondria. In apoptotic cells, if the
m collapses, the dye stays as a monomer in
the cytoplasm and emits green light. Cells were stained according to
manufacturer’s protocol and analyzed by FACScan.
Cytochrome c Release.
Cells incubated in the presence and absence of 10 µM
analogue for different periods of time were harvested for separation of
mitochondria and cytosol according to the method reported by Yang
et al. (14)
.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The annexin V assay measures phospholipid turnover from
the inner to the outer lipid layer of the plasma membrane, an event
typically associated with apoptosis (16)
. As shown in Fig. 2
, the percentage of apoptotic cells was negligible at 24 h but
became significant by 36 h, with cells appearing in the early and
late apoptotic quadrants. Thereafter, apoptosis, as indicated by
positive annexin V staining, increased to 52% by 48 h and to 85%
by 72 h. As described below, major reductions in polyamine pools
were apparent by 16 h.
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. Rather, these compounds produced a sustained
inhibition of cell growth that was first apparent at 48 h (Fig. 1B)
. Thus, the polyamine pool reduction produced by enzyme
inhibitors (Table 1)
was cytostatic rather than cytotoxic.
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4 times higher than
that of Spd (Table 1)
. By comparison, the Spm:Spd ratio of MALME-3M
human melanoma cells is closer to 1 (5)
. As shown in Table 1
, polyamine pools were rapidly and profoundly affected by treatment
with 10 µM DENSPM. By 8 h, intracellular Spd and Spm
pools were reduced by >50%. By 16 h, these pools were almost
completely depleted, whereas the Put pool was increased 5-fold,
presumably because of back conversion of the higher polyamines by SSAT
and PAO. As further evidence for SSAT activity, there was a substantial
increase in the SSAT products,
N1-acetyl-Spd and
N1-acetyl-Spm. In addition, we noted
the appearance of significant levels of DiAcSpm for the first time in
DENSPM-treated cells (Fig. 3)
. In an earlier study of stably transfected cells that conditionally
overexpress SSAT (17)
, we identified the same metabolite
by spiking samples with authentic DiAcSpm kindly provided by Dr.
Nikolaus Seiler (IRCAD, Strasbourg, France).
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15 pmol/min/mg
to 4200 pmol/min/mg by 8 h of treatment (Table 1)
and continued
to rise rapidly until 16 h, where it plateaued at 1500 times
basal levels. The greatest decrease of polyamine pools and 20% of
the total SSAT induction occurred at 8 h, when intracellular
analogue levels were comparable with the Spm pool in untreated cells.
More specifically, DENSPM levels at 8 h were 8000
pmol/106 cells, which was comparable to the basal
Spm pool of 7000 pmol/106 cells. DENSPM
continued to accumulate steadily over the 28-h period and eventually
attained an intracellular level that was 4-fold that of the original
Spm pool. Taken together, the data indicate that although DENSPM
accumulates steadily, most of the analogue effects on polyamine
metabolism were established during the first 16 h.
Caspase-3 Activation.
Because our preliminary studies indicated that caspase effects occurred
after 24 h, the present experiments focused on the period from 24
to 36 h. As shown in Fig. 4
, DENSPM-induced apoptosis was accompanied by activation of caspase-3 as
indicated by the presence of Mr 20,000
and Mr 17,000 fragments at
30 h of treatment. The appearance of these protein bands at
30 h slightly preceded the onset of apoptosis at 36 h as
indicated by annexin V staining (Fig. 2)
. As further confirmation of
caspase-3 activation, we also assayed PARP cleavage (Fig. 4)
. Beginning
at 30 h, there was a decline in the
Mr 116,000 intact PARP protein
accompanied by the appearance of a Mr
85,000 cleaved product that was fully evident at 36 h. Thus, PARP
cleavage coincides with caspase-3 activation at 30 h and with
onset of apoptosis at 36 h. We next examined whether either of two
upstream proteases, caspase-8 and -9, was activated by DENSPM
treatment. Caspase-8 is activated through death receptor-mediated
apoptosis (18
, 19)
and is known to activate caspase-3
(20)
. The polyclonal antihuman caspase-8 antibody was used
to measure both the proform (Mr
50,000) and the cleaved forms (40/36 doublet) of caspase-8. As shown In
Fig. 4
, caspase-8 was found in low abundance in these cells, and it did
not appear to be activated. Examination of caspase-9 (Fig. 4)
showed
that the proform decreased steadily to 62% of control at 24 h,
64% at 30 h, and 7% at 36 h, suggesting that activation of
caspase-3 is most likely mediated by caspase-9 rather than caspase-8.
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, cells were treated for 36 h with 10 µM
DENSPM to ensure obvious activation of caspase-3 and cleavage of PARP.
As shown in Fig. 5
, we found that Z-D-DBMK interferes with DENSPM-induced caspase-3
activation and PARP cleavage in a concentration-dependent manner (Fig. 5A)
. The effect was most apparent by the reduction of
cleaved fragments accompanied by an increase in the intact proteins.
Using these criteria, 100 µM Z-D-DBMK provided
near total prevention of caspase-3 activation. It is significant,
therefore, that this caspase inhibitor also protected cells from
DENSPM-induced apoptosis as measured by annexin V staining at 48 h
(Fig. 5B)
. Taken together with the temporal sequence data,
the present findings establish a causal role for caspase-3 activation
in DENSPM-induced apoptosis.
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, cytochrome c was largely located in the
mitochondria of untreated cells. The protein shifted from the
mitochondria to the cytosol beginning at 24 h and rapidly
accumulated to significant levels by 30 h of treatment.
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Both Bcl-2 cleavage and cytochrome c release were blocked by
addition of the general caspase inhibitor Z-D-DBMK (Fig. 6B)
, suggesting that this cleavage may be mediated by
caspases (25)
.
Loss of m Precedes Cytochrome c
Release.
Disruption of the m is an early event
associated with apoptosis and has been suggested to be one of several
factors responsible for cytochrome c release (26
, 27)
. Using MitoCapture staining and flow cytometry, we analyzed
the m in DENSPM-treated SK-MEL-28 cells. In
healthy cells, the dye accumulates and aggregates in mitochondria,
where it fluoresces bright red. In apoptotic cells, however, it cannot
enter mitochondria if the m is altered and
fluoresces bright green as a cytoplasmic monomer. Analogue treatment
caused the loss of m in a time-dependent
manner (Fig. 7)
, as shown by the shift in the cell population from low to high
green fluorescence. First apparent at 24 h, this effect preceded
the release of cytochrome c at 30 h. These observations
suggest that loss of m may be associated
with release of cytochrome c into the cytosol. To further
examine the correlation of m with
cytochrome c release and its contribution to caspase-3
activation, we used CsA, a known inhibitor of the mitochondrial
permeability transition pore, previously shown to block apoptosis in
several other systems (28
, 29)
. Preincubation of SK-MEL-28
cells with 5 µM CsA significantly reduced
DENSPM-induced cytochrome c release, caspase activation, and
apoptosis (Fig. 8)
, indicating that the majority of caspase-3 activation is occurring
through cytochrome c release.
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, we found that analogues (DE-444 and DE-443) that induce relatively low
levels of SSAT (26- and 149-fold, respectively) failed to cause
cytochrome c release or caspase-3 activation after 36 h
of treatment. Similarly, they did not cause apoptosis by annexin V
staining by 48 h. DE-343, which induced relatively high levels of
SSAT (270-fold), did, similar to DENSPM, lead to cytochrome
c release, caspase activation, and resulted in apoptosis in
34% of the cells. By comparison, DENSPM induced SSAT by 912-fold and
caused apoptosis in 68% of the cells. We therefore deduce that
differential induction of SSAT correlates with differential activation
of caspase-3 and induction of apoptosis. Although not definitive, the
findings strongly implicate massive SSAT induction as a fundamental
effector of the apoptotic response by DENSPM.
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, we observed that both NAC and MDL-72527 prevented cytochrome
c release and caspase-3 cleavage as well as apoptosis in a
dose-dependent manner. Toward this end, MDL-72527 was more efficient
than NAC. More specifically, 40 mM NAC reduced
annexin V-stained cells in DENSPM-treated cultures by 63%, and 250
µM MDL-72527 reduced apoptosis by 79%. The
near complete prevention with MDL-72527 would suggest that the other
metabolite generated by PAO, 3-acetamidopropanal, is also contributing
to apoptosis. Cells treated with 5 and 20 mM NAC
alone were similar to untreated cells; however, 40
mM NAC showed some toxicity by annexin V
staining. NAC or MDL-72527 did not prevent cellular accumulation of
DENSPM or its ability to reduce polyamine pool (data not shown).
Combining both H2O2
modulators with DENSPM did not enhance their protective effects and
gave results similar to that seen with DENSPM plus MDL-72527 alone
(data not shown). Taken together with the differential analogue effects
on SSAT, these findings suggest that a significant portion of
DENSPM-induced apoptosis derives from analogue induction of SSAT and
the downstream consequences of
H2O2 and
3-acetamidopropanal liberation via the PAO reaction.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Time course analysis implicates disruption of polyamine homeostasis as the earliest DENSPM-mediated event. Major decreases in intracellular Spm and Spd pools began at 8 h and approached completion at 16 h. As shown previously (2, 3, 4) , this pool depletion is preceded by down-regulation of the biosynthetic enzymes ODC and SAMDC and potent up-regulation of SSAT (up to a 1000-fold increase in activity). The induction of SSAT is further evidenced by a distinct rise in acetylated polyamines and by the increase in Put due to back conversion of Spm and Spd. In addition to monoacetylated polyamines, we also detected DiAcSpm, a novel metabolite recently identified in MCF-7 cells that conditionally overexpress SSAT (17) . Although this metabolite was originally observed in bacterial and acellular systems (35 , 36) , this is the first report to describe its formation in analogue-treated mammalian cells. The cellular implications of DiAcSpm need to be further studied.
Having characterized the early polyamine-related responses, we
sought to define the downstream signaling pathways that lead to
apoptosis. Because impressive progress has been made in delineating
apoptotic signaling, our strategy was to identify reliable end points
and work backwards to investigate possible polyamine-related upstream
events. We began with the caspases, a family of cysteine proteases
regarded as the executioners of apoptosis (8)
. Our data
demonstrate that caspase-3 is activated in DENSPM-treated cells and
that it is a necessary component of apoptosis, as indicated by the
finding that the general caspase inhibitor Z-D-DBMK interfered with
apoptosis (Fig. 5)
. Further definition of upstream caspase effectors
revealed that caspase-9, which is typically initiated by mitochondrial
events (9
, 10)
, was activated via cytochrome c
release, whereas caspase 8, which is usually stimulated by cytosolic
apoptotic events (37)
, was not activated.
An important role of mitochondria in apoptosis involves the liberation
of factors such as cytochrome c into the cytosol
(38)
, where it binds to Apaf-1 and activates the caspase
cascade (9, 10, 11)
. Indeed, our studies found that the
location of cytochrome c shifted from the mitochondria to
the cytosol during DENSPM treatment and that this effect preceded the
activation of caspase-3 (Fig. 6A)
. This is accompanied by a
time-dependent loss of the mitochondrial transmembrane potential (Fig. 7)
. CsA, the inhibitor of membrane pores, not only prevented cytochrome
c release by DENSPM but also reduced caspase activation and
apoptosis (Fig. 8)
. Others have demonstrated that caspases can act in a
feed-forward pathway to cleave and inactivate the antiapoptotic Bcl-2
protein to promote further cytochrome c release
(25)
. Our finding that Bcl-2 cleavage temporally followed
caspase activation and was inhibited by the caspase inhibitor (Fig. 6B)
supports this feed-forward possibility. Interestingly,
other Bcl-2 family proteins such as Bcl-XL, Bax,
and Bid were unaffected by DENSPM treatment (data not shown).
It was of interest to determine whether the observed apoptotic effects
were related to analogue-induced polyamine perturbations. As noted
above, we found that DENSPM caused potent SSAT induction by 16 h
and complete polyamine pool depletion by 20 h, both of which
happened before the apoptosis occurring between 24 and 48 h (Fig. 2)
. The data suggest that SSAT induction, polyamine pool depletion, or
both could be initiating events in apoptosis. To discern the relative
contribution of these two components (SSAT induction and polyamine pool
depletion), we used polyamine analogues that differentially induce SSAT
but similarly deplete polyamine pools and accumulate to comparable
intracellular levels (5)
. As shown in Fig. 9
, the
analogues differentially affected apoptosis in a manner consistent with
their ability to induce SSAT, but not with their ability to deplete
polyamine pools. Thus, the data are consistent with the possibility
that SSAT induction may contribute to apoptosis, a conclusion
reinforced by studies with polyamine enzyme inhibitors. More
particularly, treatment with both biosynthetic inhibitors, DFMO and
MDL-73811, markedly lowered polyamine pools in the absence of SSAT
induction and produced cell cycle arrest but not apoptosis. The
relative contributions of complete pool depletion as well as direct
analogue effects (Fig. 11)
are still being investigated.
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79%), it is possible that other
contributing events may be involved. One possibility is
3-aminopropanal, a by-product of SSAT-independent Spm oxidation that
has been shown to be involved in glial cell death induced by cerebral
ischemia (41)
. In further support of the above-mentioned
findings, we also observed that the glutathione precursor and
antioxidant NAC (33)
prevented DENSPM-induced apoptosis
(63%), presumably by reducing oxidative events related to
H2O2. Taken together, the
data strongly implicate oxidative products related to SSAT induction to
be among the events leading to apoptosis via mitochondrial damage and
the caspase cascade (Fig. 11)
. Although Ha et al. (42)
reported cytochrome
c release and caspase activation in cells treated with an
asymmetrically modified polyamine analogue, these effectors were not
linked to apoptosis per se. This particular class of
analogue has been shown to uniquely affect tubulin polymerization
(43)
, which could account for the apoptotic response. Having begun to define the apoptotic pathways involved in analogue-induced apoptosis, we were interested in using these effectors as end point markers for determining other upstream signaling events. In an ongoing study,4 we noted that Egr-1 mRNA was increased in DENSPM-treated SK-MEL-28 cells analyzed by microarray cDNA analysis and confirmed by Northern blots. This, in turn, implicated the mitogen-activated protein kinase pathway, which is known to activate Egr-1 transcription (44) . We are currently investigating the role of mitogen-activated protein kinase signaling in SK-MEL-28 apoptosis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by Grant NCI RO1 CA-22153 (to
C. W. P.) and Institute Core Grant CA-16056 that partially funds the
Flow Cytometry, Cell Analysis, and NMR Facilities. 
2 To whom requests for reprints should be
addressed, at Grace Cancer Drug Center, Roswell Park Cancer Institute,
Elm and Carlton Streets, Buffalo, NY 14263. Phone: (716) 845-3002;
Fax: (716) 845-8857; E-mail: carl.porter{at}roswellpark.org
3 The abbreviations used are: DENSPM,
N1,N11-diethylnorspermine;
CsA, cyclosporin A; DiAcSpm,
N1,N12-diacetylspermine;
NAC, N-acetylcysteine; ODC, ornithine decarboxylase;
PARP, poly(ADP-ribose) polymerase; PAO, polyamine oxidase; Put,
putrescine; SAMDC, S-adenosylmethionine decarboxylase;
SSAT, spermidine/spermine
N1-acetyltransferase; Spd, spermidine; Spm,
spermine; Z-D-DBMK,
Z-Asp-2,6-dichlorobenzoyloxymethylketone; DFMO,
-difluoromethylornithine.
4 Y. Chen, K. Alm, S. Vujcic, D. Kramer, K. Kee, P. Diegelman, and C. W. Porter,
unpublished data.
Received 3/16/01. Accepted 6/29/01.
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REFERENCES |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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