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Department of Oncology and Immunology, Newcastle, New South Wales, Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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or Fas ligand, was shown to induce marked
changes in MMP in melanoma, which showed a high correlation with
TRAIL-induced apoptosis. This was associated with activation of
proapoptotic protein Bid and release of cytochrome c
into the cytosol. Overexpression of B cell lymphoma gene 2
(Bcl-2) inhibited TRAIL-induced release of cytochrome
c, changes in MMP, and apoptosis. The pan caspase
inhibitor z-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) and the inhibitor
of caspase-8 (z-Ile-Glu-Thr-Asp-fluoromethylketone; zIETD-fmk) blocked
changes in MMP and apoptosis, suggesting that the changes in MMP were
dependent on activation of caspase-8. Activation of caspase-9 also
appeared necessary for TRAIL-induced apoptosis of melanoma. In
addition, TRAIL, but not TNF- or Fas ligand, was shown to induce
clustering of mitochondria around the nucleus. This process was not
essential for apoptosis but appeared to increase the rate of apoptosis.
Taken together, these results suggest that TRAIL induces apoptosis of
melanoma cells by recruitment of mitochondrial pathways to apoptosis
that are dependent on activation of caspase-8. Therefore, factors that
regulate the mitochondrial pathway may be important determinants of
TRAIL-induced apoptosis of melanoma. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and Fas ligand (FasL), are type
II membrane proteins that can induce apoptotic cell death in a variety
of cell types (1, 2, 3). TRAIL appears to be particularly
important in that it can induce apoptosis in a wide range of cultured
malignant cells but not normal tissues (4, 5, 6, 7, 8). The
potential importance of TRAIL as an anticancer agent has been supported
by studies in animal models showing selective toxicity to transplanted
human tumors but not normal tissues (9, 10). Induction of
apoptosis by TRAIL is believed to be mediated by interaction with two
death receptors on cells referred to as TRAIL-R1 and -R2 (see review
for alternate nomenclature; Ref. 6). Normal cells were
postulated to be protected from TRAIL-induced apoptosis by their
expression of TRAIL receptors R3 and R4, which lack cytoplasmic
death domains and act to sequester TRAIL (decoy receptors) or to
mediate antiapoptotic signals (3, 11).
The mechanism of induction of apoptosis by TRAIL is believed to be
similar to that of TNF- and FasL and to be initiated by
ligand-induced aggregation of TRAIL-R1 and R2 and their death domains
on the cytoplasmic side of the receptors (3, 6). The death
domains in turn orchestrate the assembly of adaptor components such as
Fas-associated protein with death domains (FADD), which activate
caspases by interaction of caspase recruitment domains (CARDs) on the
adaptor proteins with prodomains of the caspases (12). The
adaptor proteins involved in TRAIL-induced apoptosis have been
controversial with some reports, suggesting that FADD and
TNFR-associated death domain protein were not involved
(13), whereas others have demonstrated direct binding of
FADD and TNFR-associated death domain protein to the TRAIL-R (14, 15). Fibroblasts from FADD-knockout mice were shown to undergo
TRAIL-induced apoptosis, which suggests that FADD is not essential for
TRAIL-induced apoptosis (16). The caspases involved also
appear similar to those activated by FasL, with activation of caspase-8
and -10 being early events leading eventually to activation of effector
caspases such as caspase-3 (3, 6). Ectopic expression of
the cowpox virus gene cytokine response modifier A (crmA) was also
shown to inhibit TRAIL-induced apoptosis consistent with involvement of
caspase-1 and/or -8 (13).
The second major pathway to apoptosis in cells results from changes in mitochondrial membrane potential (MMP) due to a number of adverse cellular stimuli such as deprivation of obligatory survival factors or damage to DNA by irradiation, chemotherapy, or other toxins (17, 18). Changes in MMP are believed to be associated with the release of cytochrome c from the mitochondrial intermembrane space together with procaspase-9 and -2 and apoptosis-inducing factor (19, 20). Cytochrome c is believed to bind to apoptotic protease activating factor 1, which is a cytoplasmic scaffolding protein. The latter complex then activates procaspase-9 and -2, leading to activation of the effector caspases-3, -6, and -7, which induce apoptosis (20, 21). However, a clear distinction between these two pathways may not be possible, as activation of caspases, e.g., by interaction of FasL/CD95 may induce changes in MMP by activation of cytoplasmic factors by caspase-8, such as caspase-activated factor CAF (22) or Bid, a proapoptotic B cell lymphoma gene 2 (Bcl-2) family member (21, 22, 23, 24). However, the two pathways can be distinguished experimentally by overexpression of Bcl-2 (25, 26), which inhibits changes in MMP and cytochrome c release from mitochondria (24, 27).
We have previously shown that TRAIL induces apoptosis in approximately two-thirds of melanoma lines (4, 5). It is known from previous studies (6) that TRAIL activates caspases in melanoma cells and that inhibitors of caspases prevent TRAIL-induced apoptosis. However, the extent to which changes in MMP may be involved in TRAIL-induced apoptosis of melanoma is not known. To answer this question, we have used assays of changes in MMP in melanoma cells and transfection of cDNA for Bcl-2 to assess the role of these changes in TRAIL-induced apoptosis of melanoma. The results indicate that the mitochondrial pathway appears to be a major mediator of TRAIL-induced apoptosis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Melanoma cell lines with the prefix Mel were isolated from fresh surgical biopsies from patients attending the Sydney and Newcastle Melanoma Units and established in the laboratory. FH and CV were from lymph nodes. RM and JG were from bowel. The cell lines had been in culture for 2–6 mo at the time of these studies. MM200, Me10538, Me4405, and IgR3 cell lines were obtained from primary melanoma and are described elsewhere (4, 5). SK-Mel-28 (28) was a gift from Dr. Ralph (Department of Biochemistry and Molecular Biology, Monash University, Victoria, Australia). All melanoma cell lines were positive for tyrosinase and MART-1 mRNA by RT-PCR tests described elsewhere (29). MRC-5 lung fibroblasts were obtained from BioWhittaker (Walkersville, MD). The cell lines were cultured in DMEM containing 5% FCS (Commonwealth Serum Laboratories, Melbourne, Victoria, Australia). Melanocytes were purchased from Clonetics (San Diego, CA) and cultured in medium supplied by Clonetics (EK Medical, Waterloo, New South Wales, Australia).
Monoclonal Abs, recombinant proteins, and other reagents
Recombinant human TRAIL (lot 6321-19) prepared as described
elsewhere (1) and human CD40 ligand (CD40L; lot 5753-56)
were supplied by Immunex (Seattle, WA). Each preparation was supplied
as a leucine zipper fusion protein, which required no further
cross-linking for maximal activity. The MAb (M15) against the leucine
zipper on TRAIL was purchased from Immunex and is described elsewhere
(30). Recombinant human FasL (huFasL), produced from
isolated cDNA (GenBank accession no. U08137) in vector pDC409 and
transfected into COS cells, was supplied as sterile supernatants by
Immunex. It produced 50% lysis of Jurkat T cells at dilutions of 1:150
(4). rTNF- cytokines and control MAb antitrinitrophenyl
(anti-TNP;IgG1) were purchased from PharMingen (Bioclone,
Marrickville, New South Wales, Australia). The MAb against
Bcl-2 (Ab-3) was obtained from Calbiochem (catalog no. OP91;
Alexandria, Sydney, New South Wales, Australia) and used at a dilution
of 10 µg/ml. The Ab against activated caspase-9 (catalog no. 95015)
was purchased from New England Biolabs (Beverly, MA) and used in flow
cytometry at a dilution of 1:50. Mouse anti-cytochrome c
mAbs and rabbit anti-Bid polyclonal Abs were purchased from
PharMingen (Becton Dickinson, Lane Cove, Australia) and used at 1–5
µg/ml and at a dilution of 1:1000, respectively. Mouse anti-actin
IgM and peroxidase goat anti-mouse IgM were purchased from Oncogene
Research (Calbiochem-Novabiochem, Croydon, Australia) and used at
dilutions of 1:5000 and 1:3000, respectively. Goat anti-rabbit and
anti-mouse IgG were purchased from Bio-Rad (Hercules, CA) and used
at dilutions of 1:3000 and 1:2000, respectively. The caspase peptide
inhibitors, z-Ile-Glu-Thr-Asp-fluoromethylketone (zIETD-fmk),
z-Leu-Glu-His-Asp-fluoromethylketone (zLEHD-fmk) against caspase-8 and
-9, respectively, and the pan caspase inhibitor
z-Val-Ala-Asp-fluoromethylketone (zVAD-fmk) were purchased from
Calbiochem (catalog nos. 235420, 218759, 218761, and 627610).
Nocodazole was obtained from Sigma (catalog no. 152405).
Plasmid vectors and transfection
Stable transfectants of Bcl-2 were established by electroporation of the PEF-puro vector carrying human Bcl-2 provided by Dr. David Vaux (Walter and Eliza Hall Institute, Melbourne, Victoria, Australia) and described elsewhere (31). Electroporation was conducted with a gene pulser (Bio-Rad) with settings as follows: MM200 (0.36 kV and 960 µF), Mel-RM (0.32 kV and 500 µF), Mel-FH (0.36 kV and 960 µF), SK-Mel-28 (0.32 kV and 960 µF), Mel-CV (0.36 kV and 500 µF), and Me4405 (0.30 kV and 500 µF). Twenty four hours after electroporation, puromycin was added to a final concentration of 2 µg/ml for 14 days or until colonies appeared on the plate.
Fluorescence confocal microscopy
Melanoma cells were seeded onto sterile glass coverslips in 24-well plates (Falcon 3047; Becton Dickinson) 16–24 h before fixation. Mitotraker Red CMXRos (50 nM) (catalog no. M-7512; Molecular Probes, Eugene, OR) was added to the culture medium at 37°C for 30 min to stain mitochondria. When necessary, the desired cytokine was added at the same time. After washing with warm PBS, cells were fixed with 2% paraformaldehyde. Coverslips were then rinsed in PBS and, for double labeling, permeabilized by incubating in ice-cold acetone for 10 min. After blocking with 10% human AB serum for 10 min, cells were incubated with primary Abs diluted in PBS containing 1% human AB serum at 4°C for 45 min. Cells were then washed with PBS followed by incubating with secondary Ab at 4°C for 45 min. Coverslips were mounted in Gel-mount (Biomedia, Foster City, CA). All slides were examined using a Zeiss Axiophot microscope (Oberkochem, Germany) coupled to a Bio-Rad (MRC 600; Hemel Hempstead, U.K.) confocal attachment and COMOS software (Bio-Rad). Mitotraker Red CMXRos was dissolved in DMSO and made up in a stock solution of 1 mM. Nocodazole was dissolved in DMSO and made up into a stock solution of 1 mg/ml and used at 50 µM.
Flow cytometry
Analysis was conducted using a Becton Dickinson (Mountain View, CA) FACSscan flow cytometer. Adherent cells were removed by trypsinization in 0.25% trypsin at 37°C for 5 min, then washed twice in cold DMEM and once in PBS at 4°C. Studies on permeabilized cells were similar to the methods of Jung et al. (32). The cells were fixed in 4% paraformaldehyde, permeabilized in 0.1% saponin in permeabilization buffer, and the primary Ab was added for 30 min at 4°C. The cells were washed and then stained by FITC-labeled F(ab)2 fraction of affinity-isolated sheep anti-mouse Ig (Silenus; Amrad Biotech, Boronia, Victoria, Australia) at 1:100 dilution for 30 min at 4°C. After washing, the cells were analyzed by flow cytometry. The percentage of cells expressing the receptors was calculated as the difference in positive area between the positive and negative control histograms. The positive area was that to the right of the intersection of the two curves (33).
Measurement of apoptosis
Apoptotic cells were determined by the propidium iodide method
(34). In brief, melanoma cells were adhered overnight in a
24-well plate (Falcon 3047; Becton Dickinson, Lane Cove, Australia) at
a concentration of 1 x 105/well in 10%
FCS. Cells in suspension were added on the day of the assay. Medium was
removed and cells were treated for 1 h with different
concentrations of nocodazole or caspase inhibitors in 500 µl of fresh
media + 10% FCS before further treatment for 20 h at 37° with
the inhibitors in the presence of the reagents TRAIL, FasL, TNF-, or
CD40L. The medium was removed, and adherent and suspended cells were
washed once with PBS. The medium and PBS were placed in a 12 x 75
mm Falcon polystyrene tube and centrifuged at 200 x g.
A hypotonic buffer of 1 ml (propidium iodide, 50 µg/ml, in 0.1%
sodium citrate plus 0.1% Triton X-100; Sigma, St. Louis, MO) was added
directly to the cell pellet of cells grown in suspension or to adhered
cells in the 24-well plate, gently pipetted off, then added to the
appropriate cell pellet in the Falcon tube. The tubes were placed in
4°C in the dark overnight before flow cytometric analysis. The
propidium iodide fluorescence of individual nuclei was measured in the
red fluorescence using a FACSscan flow cytometer (Becton Dickinson,
Mountain View, CA) and the data were registered in a logarithmic scale.
At least 104 cells of each sample were analyzed.
Apoptotic nuclei appeared as a broad hypodiploid DNA peak, which was
easily distinguished from the narrow hyperdiploid peak of nuclei in the
melanoma cells.
Mitochondrial membrane potential
Tumor cells were cultured in 24-well plates and allowed to reach
exponential growth for 24 h before treatment. The cells were
harvested 20 h after treatment with the reagents TRAIL, FasL,
TNF-, or CD40L. Caspase inhibitors were added 1 h before the
addition of reagents. Changes in MMP were measured by uptake of the
lipophilic cation rhodamine 123 into mitochondria (35).
Untreated control cells were used to determine the normal uptake of
this cation, and the percentage of treated cells with a low, normal,
and high MMP was then calculated. Briefly, the medium was removed and
the adherent cells trypsinized. Both the medium and adherent cells were
placed in a 75-mm Falcon polystyrene tube, and the cells were pelleted
by centrifugation at 800 x g for 5 min at room
temperature and washed once in PBS. The cells were resuspended in 1 ml
of rhodamine 123 (10 µg/ml) for 30 min at room temperature and washed
with PBS twice and resuspended in PBS. The samples
(104 events) were analyzed for fluorescence (FL1
detector, filter 430/30 nm band pass) using a FACScan (Becton
Dickinson, Sunnyvale, CA). Histograms were analyzed using Cell Quest
software, and compared with histograms of control untreated cells.
Preparation of cytosolic extracts
Cells were induced to undergo apoptosis by exposure to TRAIL (100
ng/ml) and harvested at 6 h by centrifugation at 300 x
g for 10 min in PBS twice. Cell pellets were resuspended at
1 x 108 cells/ml in hypotonic buffer (20 mM
HEPES, pH 7.4; 10 mM KC1; 2 mM MgCl2; 1 mM EDTA)
with the addition of PMSF (0.1 mM) and incubated on ice for 15 min.
Cells were homogenized by passing the cells through a syringe (G21)
20 times. The membranes were isolated by 2-fold centrifugation at
10,000 x g at 4°C for 10 min, and the supernatant of
the second centrifugation was used as cytosolic extract
(36). Protein assays were performed on the cytosolic
fractions by the Bradford assay (Bio-Rad).
SDS-PAGE and Western blot analysis
Methods used were as described previously with minor modifications (5). Equal amounts of protein (100 µg) were loaded on each lane of the gel. Proteins were then separated under reducing conditions in a 16% SDS-PAGE gel and transferred to nitrocellulose membranes. Membranes were blocked and probed with an appropriate amount of primary Ab for 2 h. Blots were then washed in several changes of TBST, followed by probing for a further 1.5 h with the appropriate HRP-conjugated secondary Abs. Bound Ab was detected by chemiluminescence using Renaissance Western Blot Chemiluminescence Reagent (NEN, Boston, MA) and exposed onto Hyper MP autoradiography film (Amersham, Arlington Heights, IL). Blots were erased for reprobing by incubation in 0.2 M NaOH for 5 min at room temperature to strip Abs, followed by a brief washing in distilled water. Blots were then reblocked and probed with fresh Ab as described. The methods used for analysis of activated caspase-9 were as described by the supplier of the Ab against activated caspase-9 (New England Biolabs). The melanoma cells were incubated for 3 h with TRAIL and then washed 2 times with PBS. The cell extract buffer (50 mM PIPES/NaOH, pH 6.5; 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; and protease inhibitor cocktail (1697498; Boehringer-Mannheim)) was added, the cells were freeze-thawed 3 times and spun down, and the supernatant was tested. Cleaved caspase-9 was assayed on a 10% SDS-PAGE gel with 30 µg of protein per lane of the gel.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether TRAIL induced changes in MMP, we treated
melanoma cells with TRAIL for 20 h and then exposed the cells to
the lipophilic rhodamine 123 dye that is taken up by mitochondria and
undergoes a red shift in emission spectrum during changes in MMP. A
typical fluorescent profile is shown in Fig. 1
A for cells from the MM200
line before and after exposure to TRAIL. Comparisons were made with
other members of the TNF family, TNF-, FasL, and CD40L. The results
shown in Table I from studies on six
melanoma lines indicate that TRAIL, but not the other TNF family
members, induced marked changes in MMP in melanoma that correlated with
the degree of apoptosis induced by TRAIL (levels of apoptosis are shown
in brackets). Regression analysis of apoptosis vs percentage of cells
showing reduction in MMP gave an r2
value of 0.95 (p = 0.0008). However, FasL and
TNF- did induce changes in MMP in Jurkat T cells. CD40L at the
concentrations used did not induce apoptosis or changes in MMP. The
results shown are representative of three similar experiments.
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To determine whether the changes in MMP were responsible for
TRAIL-induced apoptosis we transfected Bcl-2 cDNA into the
melanoma cells because it was shown previously that overexpression of
Bcl-2 blocked the mitochondrial but not the transmembrane
signaling pathway (25, 26). Bcl-2 expression
was measured by interaction with MAbs specific to Bcl-2 in
permeabilized melanoma cells and measured by flow cytometry. The
histograms in Fig. 2 show that there was
a marked increase in median fluorescent intensity (MFI) in the
Bcl-2-transfected melanoma cells consistent with
overexpression of Bcl-2. Bcl-2 expression in the
vector-alone control was similar to that in the parental cells. There
was no increase in MFI with the isotype control MAb. The results for
Bcl-2 expression in the panel of melanoma cells under study
are shown in Table II.
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B. Comparison with Fig. 1A shows that the TRAIL-induced decrease in MMP in the
untransfected MM200 cells was not seen in MM200 cells transfected with
Bcl-2. Cells transfected with the vector alone had the same
changes in MMP as in the parental, untransfected cells. Table II
summarizes the results of similar studies on changes in MMP in the
other cell lines. The importance of changes in MMP to the induction of
apoptosis by TRAIL is shown by the marked reduction in apoptosis in the
Bcl-2-transfected cells shown in Table II. Similar results
were obtained in a repeat of the experiment. TRAIL induces cleavage of Bid and release of cytochrome c into the cytosol
Previous studies on human lymphoma cells have shown that TRAIL
induced cleavage of the proapoptotic protein Bid (23),
which was shown by others to induce cytochrome c release
from mitochondria (24). In view of this, we prepared
cytosolic extracts from Me4405 melanoma cells before and after exposure
to TRAIL for 6 h and conducted Western blots to detect Bid and
cytochrome c. Fig. 3A shows that exposure to
TRAIL resulted in a loss of the 23-kDa form of Bid and the appearance
of cytochrome c in the cytosol. (The Ab used did not
recognize efficiently the cleaved 15-kDa fragment of Bid.) Fig. 3 also
shows that overexpression of Bcl-2 in the Me4405 melanoma
cells partially suppressed the release of cytochrome c but
did not inhibit the degradation of Bid. Similar results were obtained
in studies on cells from the MM200 line that had or had not been
transfected with Bcl-2 (results not shown). Fig. 3B shows that TRAIL induced the appearance of activated
caspase-9 in the cells, and that transfection of the melanoma cells
with Bcl-2 inhibited activation of caspase-9. Similar
results were obtained in studies on the Mel-CV line.
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Previous studies have suggested that changes in MMP induced by
FasL were due to activation of caspase-8 in the death-inducing signal
complex (DISC), which then acted on cytoplasmic proteins such as Bid,
which induced changes in MMP. To investigate the role of caspases and,
in particular, activation of caspase-8 in TRAIL-induced changes in MMP,
we used the pan caspase inhibitor zVAD-fmk and a specific inhibitor of
caspase-8. The results shown in Table III
are representative of three similar studies and indicate that the pan
caspase inhibitor and the inhibitor of caspase-8 blocked, almost
completely, TRAIL-induced changes in MMP in the panel of melanoma cell
lines.
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Caspase-9 is believed to be released from mitochondria during
changes in MMP and to be activated by the complex of apoptotic protease
activating factor 1 and cytochrome c (20). To
establish whether this may also occur following exposure to TRAIL we
studied activation of caspase-9 with an Ab that recognizes the
activated form of caspase-9 by flow cytometry on permeabilized melanoma
cells. A representative result of two experiments is shown in Table IV and indicates that exposure of
melanoma cells to TRAIL led to the appearance of activated caspase-9
(see also Fig. 3B). This was inhibited in cells transfected
with Bcl-2. Activation of caspase-9 appeared dependent on
activation of caspase-8 in that inhibition of caspase-8 also completely
inhibited TRAIL-induced activation of caspase-9. Furthermore,
activation of caspase-9 appeared important in the induction of
apoptosis in that TRAIL-induced apoptosis was inhibited almost
completely by an inhibitor of caspase-9 (zLEHD-fmk) in two of the
melanoma lines and substantially in another two lines, as shown in
Table V. Similar results were found in a
repeat of the studies.
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We have shown previously, by confocal microscopy, that TRAIL
induces endocytosis of TRAIL receptors R1 and R2 and export of R1 and
R2 from the trans Golgi network, whereas the decoy receptors R3 and R4
were located in the nucleus and were relocated to the cytosol and cell
membrane after exposure to TRAIL (37). In these studies,
confocal microscopy was used to examine the location of mitochondria
identified with the Mitotraker dye, which binds permanently to
mitochondria. Fig. 4 shows that
mitochondria in the Me4405 cells became clustered around the nucleus
after exposure to TRAIL for 30 min. These changes were not seen in
response to TNF- or FasL and were not induced in the Me10538 line,
which does not express receptors for TRAIL or the MRC-5 fibroblasts,
which are resistant to TRAIL-induced apoptosis. Similar clustering of
mitochondria was seen in response to TRAIL in cells from the MM200,
Mel-FH, and Mel-RM lines.
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). Similarly, pretreatment of the cells
with nocodazole, which disrupts microtubular structure
(38), was shown to inhibit relocation of the mitochondria,
but overexpression of Bcl-2 had no effect (Fig. 5). Similar
results were obtained in studies on Mel-RM and Mel-FH. We examined
whether nocodazole would also inhibit apoptosis by pretreatment of the
cells for 1 h with nocodazole 50 nM before the addition of TRAIL.
As shown in Table VI, nocodazole
partially inhibited TRAIL-induced apoptosis when measured after 5
h of culture but had little effect after 18 h of culture. Similar
results were obtained in a repeat of the experiments.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The main evidence for involvement of mitochondria in TRAIL-induced
apoptosis of melanoma was the marked reduction in TRAIL-induced changes
in MMP and apoptosis in melanoma cells transfected with
Bcl-2. The latter is believed to bind to the permeability
transition pore in mitochondria and to prevent release of cytochrome
c (24, 26) and caspase-2 and -9
(20). Overexpression of Bcl-2 was found in
previous studies to inhibit apoptosis mediated by the mitochondrial
pathway but did not have any effects on apoptosis induced by TNF- or
FasL that did not involve the mitochondrial signaling pathway
(22, 25, 39, 40). In studies on isolated mitochondria,
changes in MMP were shown to be dependent on exposure to a factor in
the cytosol activated by caspase-8 but caspase-1, -3, -6, -7, and -11
could not initiate these changes in MMP (22). Previous
studies have shown that both CD95 (21) and TRAIL
(23) are known to cleave Bid, a Bcl-2 family
member protein that induces changes in MMP, and result in release of
cytochrome c (24). These studies are consistent
with the results of these studies in that the pan caspase inhibitor
zVAD-fmk and the inhibitor of caspase-8 were able to markedly reduce
TRAIL-induced changes in both MMP and apoptosis. Exposure to TRAIL was
also shown to result in the disappearance of the proapoptotic protein
Bid from the cytosol of the melanoma cells, consistent with its
activation by TRAIL, and was associated with release of cytochrome
c into the cytosol of the melanoma cells. Overexpression of
Bcl-2 was shown to inhibit release of cytochrome
c into the cytosol of melanoma cells, consistent with
similar results reported in studies on Bid-induced cytochrome
c release in hepatocytes (24). These results
were at variance with studies on CEM leukemia cells where expression of
Bcl-2 did not inhibit TRAIL-induced release of cytochrome
c (41). The reason for these differences
between cell types is not understood.
Additional evidence for involvement of the mitochondrial pathway in TRAIL-induced apoptosis came from the studies showing that TRAIL activated caspase-9 and that this activation was inhibited by overexpression of Bcl-2 and by the specific peptide inhibitor of caspase-8 (42, 43). Activation of caspase-9 appeared important for TRAIL-induced apoptosis in that inhibition of caspase-9 with the peptide inhibitor zLEHD-fmk markedly suppressed TRAIL-induced apoptosis. At the concentration used, the inhibitor is believed to be specific for caspase-9 (with some activity against caspase-4 and -5) (43); hence, it is unlikely that the results were due to inhibition of other initiator caspases such as caspase-8.
These results are consistent firstly with the notion that changes in MMP were induced by TRAIL via activation of caspase-8 and that these changes in MMP resulted in activation of caspase-9 via the formation of apoptotic protease activating factor/cytochrome c complexes. Secondly, they support the view that caspase-9 is released following changes in MMP (20) and that once activated it is an initiator of apoptosis induced by TRAIL. Previous studies in knockout mice have suggested that it is a key factor in mitochondrial pathways to apoptosis (44). Prior studies have shown that the ability of Bcl-2 to block apoptosis induced by transmembrane signaling depended on the concentration of caspase-8 at the receptor complex. When this was high (type 1 cells) there was direct activation of effector caspases and Bcl-2 did not inhibit apoptosis. When the concentration of caspase-8 at the receptor complex was low, apoptosis proceeded only via changes in mitochondria (type II cells) and apoptosis was inhibited by Bcl-2 (22, 25, 39). Therefore, our results suggest that the response of melanoma to TRAIL-induced apoptosis is consistent with that of type II cells in that amplification of caspase activation through the mitochondrial pathway appears necessary for TRAIL to induce apoptosis.
Previous studies have shown that TNF- may localize to mitochondria
in human endothelial cells (45). In view of this, we
examined melanoma cells, exposed to TRAIL, by confocal microscopy and
two-color fluorescence to identify TRAIL and mitochondria within the
cells. This did not show colocalization of TRAIL with mitochondria but
did show clustering of mitochondria around the nucleus. The latter was
not seen in TRAIL receptor-negative melanoma cells or in cells not
sensitive to TRAIL-induced apoptosis. It was also not seen in cells
exposed to TNF- or FasL. These results are similar to those reported
for clustering of mitochondria in L929 cells in response to activation
of the TNF-R55 receptor, which was found to correlate with sensitivity
to apoptosis induced by TNF (46). In the absence of
clustering, there was delay in induction of apoptosis
(46). Perinuclear clustering of mitochondria was also
reported in Daudi cells after treatment with TNF- or ceramide
(47). We found that clustering of mitochondria was not
inhibited by overexpression of Bcl-2 but was inhibited by
pan caspase inhibitors and nocodazole. The latter is known to inhibit
polymerization of tubulin (38). Melanoma cells treated
with nocodazole underwent a delay in TRAIL-induced apoptosis, which
supports the view that the clustering of mitochondria around the
nucleus may facilitate apoptosis perhaps by providing relatively high
concentrations of proapoptotic molecules from mitochondria near target
structures in the nucleus, e.g., similar to the localization of
caspase-9 in nuclei of neurons undergoing apoptosis (47).
The role of caspase activation in clustering of the mitochondria is yet
to be elucidated but is presumably due to events upstream of changes in
mitochondria as inhibition of changes in MMP by overexpression of
Bcl-2 did not inhibit clustering of mitochondria.
In summary, these studies show that TRAIL appears to induce apoptosis in melanoma cells largely by induction of changes in mitochondria rather than by direct activation of effector caspases. TRAIL also induces a clustering of mitochondria around the nucleus, and this appears to facilitate apoptosis perhaps by generation of high concentrations of the caspases around the nucleus. The predominantly mitochondrial pathway of TRAIL-induced apoptosis implies that more attention may need to be given to regulators of this pathway, such as that by the Bcl-2 family, e.g., suppression of Bcl-2 activity may be an important therapeutic strategy to increase the sensitivity of melanoma to TRAIL-induced apoptosis. Similarly, levels of inhibitors of apoptosis 1 and 2 and X-linked inhibitor of apoptosis, which are known to inhibit caspase-9 (48), may be important determinants of TRAIL-induced apoptosis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Peter Hersey, Department of Oncology and Immunology, Room 443, David Maddison Building, King and Watt Streets, Newcastle, New South Wales 2300, Australia.
3 Abbreviations used in this paper: TRAIL, TNF-related apoptosis-inducing ligand; TRAIL-R, receptor for TRAIL; MMP, mitochondrial membrane potential; MFI, median fluorescent intensity; Bcl-2, B cell lymphoma gene 2; zVAD-fmk, z-Val-Ala-Asp-fluoromethylketone; zLEHD-fmk, z-Leu-Glu-His-Asp-fluoromethylketone; FasL, Fas ligand; FADD, Fas-associated protein with death domains; CD40L, CD40 ligand.
Received for publication April 24, 2000. Accepted for publication August 25, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B. Immunity 7:831.[Medline]
-converting enzyme-mediated apoptosis of mammalian cells by baculovirus IAP. Proc. Natl. Acad. Sci. USA 93:13786.This article has been cited by other articles:
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  J. H. Song, K. Kandasamy, and A. S. Kraft ABT-737 Induces Expression of the Death Receptor 5 and Sensitizes Human Cancer Cells to TRAIL-induced Apoptosis J. Biol. Chem., September 5, 2008; 283(36): 25003 - 25013. [Abstract] [Full Text] [PDF]
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 K. A. Avery-Kiejda, X. D. Zhang, L. J. Adams, R. J. Scott, B. Vojtesek, D. P. Lane, and P. Hersey Small Molecular Weight Variants of p53 Are Expressed in Human Melanoma Cells and Are Induced by the DNA-Damaging Agent Cisplatin Clin. Cancer Res., March 15, 2008; 14(6): 1659 - 1668. [Abstract] [Full Text] [PDF]
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 N. Chetoui, K. Sylla, J.-V. Gagnon-Houde, C. Alcaide-Loridan, D. Charron, R. Al-Daccak, and F. Aoudjit Down-Regulation of Mcl-1 by Small Interfering RNA Sensitizes Resistant Melanoma Cells to Fas-Mediated Apoptosis Mol. Cancer Res., January 1, 2008; 6(1): 42 - 52. [Abstract] [Full Text] [PDF]
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 F. A. Sinicrope and R. C. Penington Sulindac sulfide-induced apoptosis is enhanced by a small-molecule Bcl-2 inhibitor and by TRAIL in human colon cancer cells overexpressing Bcl-2 Mol. Cancer Ther., October 1, 2005; 4(10): 1475 - 1483. [Abstract] [Full Text] [PDF]
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 L. Liu, A. Vo, G. Liu, and W. L. McKeehan Distinct Structural Domains within C19ORF5 Support Association with Stabilized Microtubules and Mitochondrial Aggregation and Genome Destruction Cancer Res., May 15, 2005; 65(10): 4191 - 4201. [Abstract] [Full Text] [PDF]
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S. Gillespie, X. D. Zhang, and P. Hersey Variable expression of protein kinase C{varepsilon} in human melanoma cells regulates sensitivity to TRAIL-induced apoptosis Mol. Cancer Ther., April 1, 2005; 4(4): 668 - 676. [Abstract] [Full Text] [PDF]
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F. A. Sinicrope, R. C. Penington, and X. M. Tang Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand-Induced Apoptosis Is Inhibited by Bcl-2 but Restored by the Small Molecule Bcl-2 Inhibitor, HA 14-1, in Human Colon Cancer Cells Clin. Cancer Res., December 15, 2004; 10(24): 8284 - 8292. [Abstract] [Full Text] [PDF]
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 W.-H. Tsai, C.-W. Chang, W.-J. Chuang, Y.-S. Lin, J.-J. Wu, C.-C. Liu, W.-T. Chang, and M. T. Lin Streptococcal Pyrogenic Exotoxin B-Induced Apoptosis in A549 Cells Is Mediated by a Receptor- and Mitochondrion-Dependent Pathway Infect. Immun., December 1, 2004; 72(12): 7055 - 7062. [Abstract] [Full Text] [PDF]
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A. Kosta, C. Roisin-Bouffay, M.-F. Luciani, G. P. Otto, R. H. Kessin, and P. Golstein Autophagy Gene Disruption Reveals a Non-vacuolar Cell Death Pathway in Dictyostelium J. Biol. Chem., November 12, 2004; 279(46): 48404 - 48409. [Abstract] [Full Text] [PDF]
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 E. Mezosi, S. H. Wang, S. Utsugi, L. Bajnok, J. D. Bretz, P. G. Gauger, N. W. Thompson, and J. R. Baker Jr. Interleukin-1{beta} and Tumor Necrosis Factor (TNF)-{alpha} Sensitize Human Thyroid Epithelial Cells to TNF-Related Apoptosis-Inducing Ligand-Induced Apoptosis through Increases in Procaspase-7 and Bid, and the Down-Regulation of p44/42 Mitogen-Activated Protein Kinase Activity J. Clin. Endocrinol. Metab., January 1, 2004; 89(1): 250 - 257. [Abstract] [Full Text] [PDF]
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A. B. Werner, E. de Vries, S. W. G. Tait, I. Bontjer, and J. Borst TRAIL Receptor and CD95 Signal to Mitochondria via FADD, Caspase-8/10, Bid, and Bax but Differentially Regulate Events Downstream from Truncated Bid J. Biol. Chem., October 18, 2002; 277(43): 40760 - 40767. [Abstract] [Full Text] [PDF]
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J. H. L. Velthuis, K. M. A. Rouschop, H. J. G. M. de Bont, G. J. Mulder, and J. F. Nagelkerke Distinct Intracellular Signaling in Tumor Necrosis Factor-related Apoptosis-inducing Ligand- and CD95 Ligand-mediated Apoptosis J. Biol. Chem., June 28, 2002; 277(27): 24631 - 24637. [Abstract] [Full Text] [PDF]
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X. D. Zhang, X. Y. Zhang, C. P. Gray, T. Nguyen, and P. Hersey Tumor Necrosis Factor-related Apoptosis-inducing Ligand-induced Apoptosis of Human Melanoma Is Regulated by Smac/DIABLO Release from Mitochondria Cancer Res., October 1, 2001; 61(19): 7339 - 7348. [Abstract] [Full Text] [PDF]
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 P. Secchiero, A. Gonelli, C. Celeghini, P. Mirandola, L. Guidotti, G. Visani, S. Capitani, and G. Zauli Activation of the nitric oxide synthase pathway represents a key component of tumor necrosis factor-related apoptosis-inducing ligand-mediated cytotoxicity on hematologic malignancies Blood, October 1, 2001; 98(7): 2220 - 2228. [Abstract] [Full Text] [PDF]
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