Abstract
Past studies have shown that TNF-related apoptosis-inducing ligand (TRAIL) induced apoptosis in a high proportion of cultured melanoma by caspase-dependent mechanisms. In the present studies we have examined whether TRAIL-induced apoptosis of melanoma was mediated by direct activation of effector caspases or whether apoptosis was dependent on changes in mitochondrial membrane potential (MMP) and mitochondrial-dependent pathways of apoptosis. Changes in MMP were measured by fluorescent emission from rhodamine 123 in mitochondria. TRAIL, but not TNF-α 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-α 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.
TNF-related apoptosis-inducing ligand (TRAIL)3 is a member of the TNF family, which, like TNF-α 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-α 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.
Materials and Methods
Cell lines
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-α 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 × 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-α, 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 × 75 mm Falcon polystyrene tube and centrifuged at 200 × 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-α, 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 × 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 × g for 10 min in PBS twice. Cell pellets were resuspended at 1 × 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 × 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.
Results
TRAIL, but not other TNF family members, induces changes in MMP
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-α, 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-α 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.
Relative MMP measured by fluorescent emission from rhodamine 123 taken up by mitochondria. A, Middle open histogram indicates the MMP for MM200; solid histograms indicate the change in MMP after exposure to TRAIL for 20 h. B, MM200 cells transfected with Bcl-2 showing no change in MMP after exposure to TRAIL. The MMP of untreated and TRAIL-treated cells are superimposed.
Percentage apoptosis and changes in the MMPa induced in melanoma cells by members of the TNF family
Melanoma cells transfected with Bcl-2 acquire resistance to TRAIL-induced apoptosis
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⇓.
Flow cytometer histograms of Bcl-2 expression in melanoma cells before and after transfection with cDNA for Bcl-2. A, Me4405. B, MM200. The histogram to the left of the figure is the isotype control, the middle histogram (dashed line) is Bcl-2 expression in the parental cells, and the solid histogram is Bcl-2 expression in the transfected lines.
Inhibition of TRAIL-induced changes in MMP and apoptosis by overexpression of Bcl-2
We found that transfection of Bcl-2 was associated with a marked reduction of changes in MMP induced by TRAIL, illustrated by the flow cytometer histogram in Fig. 1⇑B. Comparison with Fig. 1⇑A 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. 3⇓A 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. 3⇓B 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.
A, Western blot analysis of Bid and cytochrome c in the cytosol of Me4405 melanoma cells before and after exposure to TRAIL (100 ng/ml). Comparison is made between parental Me4405 and Me4405 cells transfected with Bcl-2. Exposure to TRAIL resulted in degradation of Bid and the appearance of cytochrome c in the cytosol. The latter was inhibited by overexpression of Bcl-2. The levels of actin were unchanged over the time course of the experiments. Concentrations of Abs used are described in Materials and Methods. Molecular weights of cytochrome c, Bid, and Actin were 15, 22, and 42 kDa. B, Exposure of Me4405 cells to TRAIL (100 ng/ml) for 3 h induced the appearance of activated caspase-9 in the cytosol. The polyclonal rabbit Ab against activated caspase-9 was used at a dilution of 1:2000. Molecular mass of activated caspase-9 was 37 kDa.
Changes in MMP induced by TRAIL are dependent on activation of caspase-8
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.
Inhibition of TRAIL-induced apoptosis and changes in MMP by a pan caspase inhibitor and inhibitors of caspase-8
TRAIL induces activation of caspase-9, which is blocked by overexpression of Bcl-2 and inhibitors of caspase-8
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. 3⇑B). 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.
TRAIL-induced activation of caspase-9 is blocked by an inhibitor of caspase-8 and by overexpression of Bcl-2a
Inhibition of apoptosis induced by TRAIL with inhibitors of caspase-9a
TRAIL induces clustering of mitochondria around the nucleus
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-α 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.
Confocal microscopy showing that TRAIL induces perinuclear clustering of mitochondria (stained with Mitotracker red) in melanoma cells (Me4405) sensitive to TRAIL-induced apoptosis but not in Me10538 cells that lack TRAIL-R or in the MRC-5 fibroblasts, which are resistant to TRAIL. FasL and TNF-α were unable to induce similar changes. Photographs under Me4405, Me10538, and MRC-5 indicate studies on cells from these lines. The treatment of the cells before confocal photography is indicated on the left.
The TRAIL-induced movement of mitochondria appeared to be caspase dependent in that pretreatment of the melanoma cells with the pan caspase inhibitor zVAD-fmk inhibited movement of the mitochondria (Fig. 5⇓). 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.
Confocal microscopy showing that perinuclear clustering of mitochondria was not inhibited by overexpression of Bcl-2 but was by the pan caspase inhibitor zVAD-fmk and the tubulin poison nocodazole at 50 nM. A, Me4405 transfected with Bcl-2 30 min after exposure to TRAIL (100 ng/ml). B, Me4405 cells pretreated with zVAD-fmk (20 μM) for 30 min and then exposed to TRAIL as in A. C, Me4405 cells pretreated with nocodazole (50 nM) 1 h before addition of TRAIL.
Nocodazole induces delay in TRAIL-induced apoptosis of melanoma cells
The TRAIL-induced clustering of mitochondria did not appear to be due to direct interaction of TRAIL with the mitochondria as confocal microscopy showed no colocalization of mitochondria and TRAIL. The latter was identified by M15 MAb against the leucine zipper on TRAIL (data not shown).
Discussion
The studies above have used assays of MMP based on uptake of the lipophilic cation rhodamine 123 by mitochondria. The dye binds to the inner and outer membrane of mitochondria and undergoes a red shift (decrease) in fluorescence during membrane depolarization (35). Use of this assay revealed that exposure of melanoma cells to TRAIL induced marked changes in MMP that showed a high correlation with the levels of apoptosis induced by TRAIL. These results were unexpected in that TRAIL-induced apoptosis was considered to be dependent on the caspase cascade resulting from activation of caspase-8 (3) but independent of the mitochondrial pathway to apoptosis (39).
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-α 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-α 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-α 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-α 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.
Acknowledgments
We thank the staff of the Electron Microscope Unit, University of Sydney, for assistance with confocal microscopy.
Footnotes
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↵1 This work was supported by the Melanoma and Skin Cancer Research Institute, Sydney, Australia and the New South Wales State Cancer Council.
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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. E-mail address: Peter.Hersey{at}newcastle.edu.au
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↵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 April 24, 2000.
- Accepted August 25, 2000.
- Copyright © 2000 by The American Association of Immunologists
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