Purpose: Many melanoma cell lines and primary cultures are resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. In this study, we investigated the molecular mechanisms that control melanoma cell resistance and searched for chemotherapeutic drugs that could overcome the TRAIL resistance in melanoma cells.

Experimental Design: We examined 21 melanoma cell lines and 3 primary melanoma cultures for their sensitivity to TRAIL-induced apoptosis, and then tested cisplatin, chemptothecin, and etoposide for their synergistic effects on TRAIL sensitivity in resistant melanoma cells.

Results: Of 21 melanoma cell lines, 11 showed various degrees of sensitivity to TRAIL-induced apoptosis through caspase-8-initiated cleavage of caspase-3 and DNA fragmentation factor 45. The remaining cell lines and primary cultures were resistant to TRAIL, but cisplatin, chemptothecin, and etoposide sensitized the resistant cell lines and primary cultures to TRAIL-induced apoptosis, which also occurred through the caspase-8-initiated caspase cascade. Of the two TRAIL death receptors (DR4 and DR5), melanoma cells primarily expressed DR5 on cell surface. Cisplatin treatment had no effects on cell surface DR5 expression or intracellular expression of Fas-associated death domain and caspase-8. Instead, cisplatin treatment down-regulated intracellular expression of the short form of cellular Fas-associated death domain-like interleukin-1β-converting enzyme-like inhibitory protein (c-FLIP) and inhibited phosphorylation of the long form of c-FLIP.

Conclusions: The results presented here indicate that cisplatin inhibits c-FLIP protein expression and phosphorylation to restore TRAIL-induced caspase-8-initiated apoptosis in melanoma cells, thus providing a new combined therapeutic strategy for melanomas.

TRAIL3 is a recently identified member of the TNF family and has two unique features that distinguish it from other members of the TNF family such as TNFα and Fas ligand (FasL/CD95L; Ref. 1). First, TRAIL is expressed in a wide range of normal tissues (2, 3) and is involved in tumor surveillance (4), whereas TNFα and FasL are only transiently expressed in activated cells (1). Second, recombinant soluble forms of human TRAIL trigger apoptosis in various human tumor cells, but not in most normal cells (5, 6, 7, 8, 9, 10), making this ligand a potential candidate for cancer therapy. To explore the therapeutic potential of TRAIL on malignant melanomas, we examined a large number of melanoma cell lines and primary melanoma cultures for their sensitivity to recombinant soluble human TRAIL. The results presented here show that many melanoma cell lines and primary cultures are resistant to TRAIL-induced apoptosis, but this resistance can be overcome by the conventional chemotherapeutic drug cisplatin. Furthermore, we have addressed the molecular mechanisms involved in cisplatin regulation of TRAIL-induced apoptosis in melanoma cells.

TRAIL induces apoptosis through its cognate DRs DR4 (TRAIL-R1; Refs. 11, 12, 13) and DR5 (TRAIL-R2; Refs. 14, 15, 16). DR4 and DR5 are type I transmembrane proteins with cytoplasmic DDs that interact with COOH-terminal DD of an intracellular adaptor FADD (13, 16, 17). FADD has a COOH-terminal DD and an NH2-terminal DED (18, 19) and, through its DED, FADD recruits DED-containing apoptosis-initiating protease caspase-8 to form a DISC (20, 21, 22, 23). Immediately upon recruitment caspase-8 is proteolytically processed to release active subunits into cytoplasm (24). Active caspase-8 subunits subsequently cleave downstream effector caspases such as caspase-3 (25), and the active caspase-3 in turn cleaves its substrate, DFF45 (26), culminating in the execution of programmed cell death. c-FLIP was reported recently to be recruited to the DISC, interrupting DED-DED interaction between FADD and caspase-8 and inhibiting TRAIL-induced apoptosis (23, 27).

Several c-FLIP mRNA species exist, but only two endogenous forms of the proteins have been detected (27, 28, 29). The longer form, c-FLIPL (Mr ∼55 kDa), is structurally similar to caspase-8 and contains two DEDs and a caspase-like domain that lacks catalytic activity because the active-center tyrosine is replaced with cysteine; the short form, c-FLIPS (Mr ∼28 kDa), contains only two DEDs (27). Both forms of c-FLIP are recruited to FasL- and TRAIL-induced DISC, inhibiting FasL- and TRAIL-induced apoptosis (23, 30, 31). We showed recently that c-FLIPL is phosphorylated in resistant glioma cells, and the phosphorylated c-FLIPL is recruited to TRAIL-induced DISC to inhibit apoptosis in the glioma cells (32). In this study, we demonstrated for the first time that cisplatin down-regulates c-FLIPS and inhibits c-FLIPL phosphorylation to facilitate TRAIL-induced and caspase-8-initiated apoptosis in melanoma cells.

Materials and Antibodies.

Recombinant nontagged, native sequence soluble form of human TRAIL (amino acids 114–281) was a kind gift from PeproTech, Inc. (Rocky Hill, NJ). The tetrapeptide caspase inhibitor z-VAD-fmk (R&D Systems, Minneapolis, MN) was prepared as a 20 mm stock in DMSO and stored at −20°C in aliquots until use. Cisplatin, CPT, and VP16 (Sigma-Aldrich Canada Ltd., Oakville, Ontario, Canada) were prepared as 100 mg/ml stock in DMSO with the final concentration of DMSO not exceeding 0.1% (v/v). Protein quantification reagents were obtained from Bio-Rad Laboratories, Inc. (Hercules, CA), and enhanced chemiluminescence reagents for Western blot analysis were purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Mouse monoclonal antibodies included antihuman FADD (Transduction Laboratories, Lexington, KY), antihuman caspase-8 (MBL, Nagoya, Japan), antihuman DFF45 (StressGen Biotechnologies In., Victoria, British Columbia, Canada), and antihuman c-FLIP NF6 (kind gift from Dr. Peter H. Krammer, German Cancer Research Center, Heidelberg, Germany). Rabbit polyclonal antibodies to human caspase-3, c-FLIP, and ERK1/2 were purchased from StressGen. Phycoerythrin-conjugated antihuman DR4 and DR5 mouse IgG1 were purchased from eBioscience (San Diego, CA), and phycoerythrin-conjugated IgG1 was from BD PharMingen (San Diego, CA). HRP-conjugated goat antimouse IgG2b antibody was purchased from Southern Biotechnology (Birmingham, AL) and HRP-conjugated goat antirabbit antibody was from Jackson IR Labs (West Grove, PA). All of the other chemicals used were of analytical grade and purchased from Sigma-Aldrich.

Melanoma Cell Lines and Primary Melanoma Cultures.

The human primary melanoma cell lines WM793, WM278, WM1366, SBC12, WM9832A, WM902B, WM1552C, WM35, WM115, WM1341, WM3248, WM39, and WM3211, and metastatic melanoma cell lines WM164, WM1232, WM1617, WM9, WM852, 1205Lu, 451Lu, and WM239A were cultured in RPMI 1640 (Invitrogen) supplemented with 10% FBS, 5 μg/ml insulin, and 1% antibiotics (33). Early passages of primary melanoma cultures were established from fresh operative tumor samples, as described previously (34). They were cultured in RPMI 1640 supplemented with 10% FBS, 1% non-essential amino acids, 2 mm l-glutamine, 1 mm sodium pyruvate, and 1% antibiotics.

Flow Cytometric Analysis.

Cell surface expression of DR4 and DR5 was measured by flow cytometry. In brief, 0.1 μg/ml of phycoerythrin-conjugated antihuman DR4 and DR5 (mouse IgG1) or mouse IgG1 (a negative control) were added to the 106 cells in 200 μl of immunofluorescence buffer (PBS containing 2% FBS and 0.02% sodium azide; Sigma-Aldrich). After 1 h of incubation in the dark at 4°C, the cells were washed with immunofluorescence buffer and then dispersed 500 μl PBS. For all of the tested cell samples, 10,000 cells were analyzed using a Becton and Dickinson FACScan (Mountain View, CA), and the data were processed by using Cell Quest software (Becton Dickinson).

Cell Death Assays and Detection of Cleavage of Caspases.

Cell death was evaluated by crystal violet assay as described previously (9). In brief, cells were seeded at 3 × 104 cells/well in 96-well plates overnight and then treated with 100 μl fresh medium containing various doses of cisplatin, as indicated in “Results,” at 37°C for 16 h. The cells were then treated with 100 ng/ml TRAIL at 37°C for an additional 16 h, and the plates were analyzed using a microplate reader (Bio-Rad). The effects of the treatments were quantified as percentage of cell death using untreated cells as a control. For cellular apoptosis, cells were treated and examined under phase contrast microscopy (9). For cleavage of caspases, subconfluent cells were treated with 100 ng/ml TRAIL, alone or in the presence of cisplatin or other chemotherapeutic drugs for the times as indicated in “Results,” and the cells were subjected to Western blotting.

Western Blot Analysis.

Cells in cultures were harvested by trypsinization and lysed in 50 mm Tris-HCl (pH 7.4) containing 150 mm NaCl, 2 mm EDTA, 10% glycerol, 1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, and 0.2% protease inhibitor mixture. The lysed cells were centrifuged at 18,000 × g to remove cellular debris. Protein concentrations of the extracts were determined by the Bradford protein assay following the manufacturer’s protocol (Bio-Rad). Equal amounts of protein were separated by SDS-PAGE, transferred to nitrocellulose membranes (Bio-Rad), and blocked with 5% nonfat dry milk in TBST [20 mm Tris-HCl (pH 7.4) with Tween 20 (0.05%, v/v)] for 1 h at room temperature. The membranes were then incubated overnight with either anticaspase-8 (diluted 1:1000), anti-caspase-3 (diluted 1:5000), anti-FADD (diluted 1:1000), anti-ERK1/2 (diluted 1:1000), or c-FLIP NF6 (diluted 1:10). After several washes, the membranes were incubated with either HRP-conjugated goat antimouse or HRP-conjugated goat antirabbit antibodies (diluted 1:5,000) for 1 h and then developed by an enhanced chemiluminescence.

Two-dimensional-PAGE Immunoblot.

Two-dimensional-PAGE was carried out as described previously (23). In brief, cells were treated with 100 ng/ml of TRAIL, either in the absence or presence of 10 μg/ml cisplatin for 16 h and were lysed in 40 mm Tris-HCl (pH 8.0) containing 65 mm DTT, 1% Triton X-100, 1 mm phenylmethylsulfonyl fluoride, and 0.2% protease inhibitor mixture. After centrifugation, the supernatants were examined by protein assay and were precipitated with chilled acetone. The isoelectric focusing using an IPG strip was carried out by loading 10 mg protein in 250 μl (complemented with 0.5% v:v IPG-buffer 3–10 nonlinear) onto a 13-cm IPG-strip 3–10 nonlinear (Amersham). Focusing was performed to a total of 48.8 kVh using an IPGphor unit (20°C), and then the strips were equilibrated with 50 mm Tris-HCl (pH 8.8) containing 6 m urea, 30% glycerol, 2% SDS, and 10 mg/ml DTT. The second dimension was carried out in a 12.5% SDS-PAGE gel followed by nitrocellulose membrane transferring (Bio-Rad) and immunoblotting with rabbit polyclonal c-FLIP antibody (1:1000). After washing, the membranes were incubated with HRP-conjugated goat antimouse antibody (diluted 1:5000) for 1 h and then developed by an enhanced chemiluminescence.

TRAIL-induced Apoptosis Occurs Through Caspase-8-initiated Caspase Cascade in Sensitive Melanoma Cells.

Although melanoma cells express cell surface TRAIL-Rs, some of the melanoma cell lines are susceptible, whereas other cell lines are resistant to TRAIL (8, 35, 36). To explore the regulatory mechanisms that control TRAIL-induced apoptosis, we examined 21 human melanoma cell lines for their sensitivity to a recombinant nontagged soluble form of human TRAIL (amino acids 114–281). Cell death analysis by crystal violet assay showed that TRAIL induced various degrees of cell death in 11 cell lines; 5 (WM793, WM278, WM1366, SBC12, and WM9832A) were remarkably sensitive to TRAIL (>60% cell death), and 6 (WM9, WM239A, WM1232, WM1617, WM902B, and WM1552C) were moderately sensitive (30–60% cell death). The remaining 10 cell lines (WM164, WM35, WM115, WM852, WM1341, 1205Lu, WM3248, WM39, 451Lu, and WM3211) were resistant to TRAIL (<20% cell death; Fig. 1).

We additionally examined the TRAIL-sensitive cell lines to determine whether TRAIL-induced cell death occurred through caspase-8-initiated caspase cascade. First, we examined the ability of caspase inhibitors to prevent TRAIL-induced cell death in TRAIL-sensitive WM793 cell line. WM793 cells were killed by TRAIL in a dose-dependent manner (Fig. 2,A). However, pretreatment of WM793 with a pan-caspase inhibitor z-VAD-fmk (20 μm) for 2 h completely protected WM793 cells from TRAIL-induced cell death (Fig. 2,A). Next, we examined this cell line for TRAIL-induced cleavage of caspase-8, caspase-3, and DFF45. Caspase-8 cleavage is reported to occur in two consecutive steps with the first-step cleavage producing p12, p43, and p41 subunits from p55 and p53 precursors, and second-step generating a prodomain, and active p18 and p10 subunits (37, 38). The active caspase-8 subunits are thereafter released into the cytoplasm to cleave downstream caspase-3 (p32 precursor) into two large (p20 and p17) and one small (p10) subunits (39), which in turn proteolytically process DFF45, a substrate of caspase-3 (26). Western blots detected p55/p53 caspase-8 precursors, p43/p41 (i.e., the first step cleavage products) and the p18 active subunit (the second step cleavage product) within 0.5 h. The caspase-3 p20 and p17 cleavage products were detected within 2 h after exposure of the cells to 100 ng/ml TRAIL (Fig. 2,B). Two forms of DFF45, the long form and the short form (26, 40), were endogenously expressed in the melanoma cells, and proteolytically cleaved into p17 and p11 cleavage products within 2 h upon treatment with 100 ng/ml TRAIL (Fig. 2 B).

Next, we examined TRAIL-induced cleavage of caspase-8, caspase-3, and DFF45 in 4 additional sensitive cell lines (SBC12, WM1366, WM278, and WM9). These cell lines were susceptible to TRAIL-induced cell death in a dose-dependent manner, as determined by crystal violet cell death assay (Fig. 2,C). Additional Western blot analysis revealed caspase-8 cleavage p43, p41, and p18 products, caspase-3 cleavage p20 and p17 subunits, and DFF45 p17 and p11 cleavage products in these cell lines after exposure to 100 ng/ml of TRAIL for 6 h (Fig. 2 D). Collectively, the results indicate that TRAIL-induced apoptosis in the sensitive melanoma cells occurs through a caspase-8-initiated caspase cascade.

Cisplatin Sensitizes Resistant Melanoma Cells to TRAIL-induced Apoptosis.

A conventional chemotherapeutic drug, cisplatin, was reported recently to augment TRAIL-induced cytotoxicity in tumor cells from various origins (41, 42, 43, 44). To explore this potential in melanomas, we first examined the ability of cisplatin to sensitize TRAIL-resistant melanoma cell lines to TRAIL-induced cell death. Cisplatin alone showed a limited cytotoxic effect in the TRAIL-resistant WM164 cell line at doses ranging from 0.4 to 100 μg/ml (Fig. 3,A). In contrast, in the presence of TRAIL (100 ng/ml), cisplatin killed WM164 cells in a dose-dependent manner (Fig. 3,A). Pretreatment of WM164 with a pan-caspase inhibitor, z-VAD-fmk, abolished the cisplatin-sensitized TRAIL-induced cell death (Fig. 3 A), implicating involvement of caspases in cisplatin-sensitized TRAIL-induced cell death.

To demonstrate activation of the caspase-8-initiated cascade, we treated WM164 cells with a low dose of cisplatin (10 μg/ml) and a low dose of TRAIL (100 ng/ml) alone or in combination, and, thereafter, examined the cells for cleavage of caspase-8, caspase-3, and DFF45. Western blots failed to detect cleavage products of caspase-8, caspse-3, and DFF45 in the cells treated with either cisplatin or TRAIL alone (Fig. 3,B). In contrast, caspase-8 p43, p41, and p18, caspase-3 p20 and p17, and DFF45 p17 and p11 cleavage products were detected in the cells exposed to a combined treatment of cisplatin and TRAIL (Fig. 3,B). The combined cisplatin and TRAIL treatment induced cellular apoptosis as exhibited by cell-surface blebbing, which was visualized under phase-contrast microscopy in the WN164 cells (Fig. 3,F). The cellular apoptosis was abolished by pretreatment of the cells with z-VAD-fmk (Fig. 3 H), indicating that cisplatin-sensitized TRAIL-induced apoptosis occurs through caspase-8-initiated caspase cascade.

To additionally support this hypothesis, we examined 3 additional TRAIL-resistant cell lines for cisplatin-sensitized and TRAIL-induced cell death, and caspase-8, caspase-3, and DFF45 cleavage. Cell death analysis showed that higher doses of cisplatin (33–100 μg/ml) triggered some cell death in WM35, but not in WM115 and WM852 (Fig. 4,A). However, in the presence of TRAIL (100 ng/ml), cisplatin induced significant cell death in all of the 3 lines in a dose-dependent manner (Fig. 4,A). We additionally treated these cell lines with cisplatin (10 μg/ml) and TRAIL (100 ng/ml) for 6 h and examined cell lysates for cleavage of caspases. Western blots detected caspase-8 (p43, p42, and p18), caspase-3 (p20 and p17), and DFF45 (p17 and p11) cleavage products (Fig. 4,B). In contrast, these cell lines were resistant to either cisplatin or TRAIL, as evidenced by lack of the cleavage products from caspase-8, caspase-3, and DFF45 (Fig. 4,B). Collectively, the results indicate that cisplatin sensitizes resistant melanoma cells to TRAIL-induced apoptosis through a caspase-8-initiated caspase cascade, similar to the apoptotic signaling observed in TRAIL-sensitive melanoma cells (Fig. 2).

Cisplatin Does Not Up-regulate TRAIL DRs in TRAIL-resistant Melanoma Cells.

Cisplatin was reported to up-regulate DR4 and DR5 mRNA expression in glioma cells, corroborating its synergistic cytotoxicity with TRAIL (41). To test this hypothesis in melanoma cells, we treated three TRAIL-resistant melanoma cell lines (WM164, WM115, and WM35) with cisplatin (10 μg/ml) for 16 h and then examined the cells for their expression of cell surface TRAIL-Rs, DR4 and DR5. Flow cytometry analysis showed that all of the cell lines expressed cell surface DR5 (Fig. 5). In contrast, WM164 and WM35 lacked surface DR4, whereas WM115 expressed a limited amount DR4 (Fig. 5). Treatment of WM164, WM115, and WM35 cells with cisplatin neither altered cell surface DR5 expression, nor induced DR4 expression on the cell surface (Fig. 5). These results suggest that alternative mechanisms are involved in the control of cisplatin-sensitized TRAIL-induced apoptosis in melanoma cells.

Cisplatin Down-Regulates c-FLIPS and Inhibits c-FLIPL Phosphorylation in Resistant Melanoma Cells.

The fact that cisplatin restores TRAIL-induced apoptosis through caspase-8-initiated caspase cascade suggests that cisplatin may regulate proteins that are involved in TRAIL-induced DISC. To test this hypothesis, we first compared TRAIL-sensitive and -resistant melanoma cells for their expression of the DISC proteins FADD, caspase-8, c-FLIPL, and c-FLIPS by Western blot analysis. Thereafter, TRAIL-resistant cell lines were exposed to cisplatin (10 μg/ml) for 16 h to determine whether cisplatin alters the expression levels of these proteins. FADD was expressed at lower levels in 3 sensitive (WM793, WM9, and SBC12) and 1 resistant cell line (WM115), but at higher levels in 2 resistant cell lines (WM164 and WM35). Treatment of the resistant cell lines with cisplatin did not alter FADD protein expression in the resistant cell lines (Fig. 6,A). Caspase-8 p55/p53 precursors proteins were detected at constant levels in both sensitive and resistant cell lines tested, and cisplatin treatment did not affect their expression levels (Fig. 6,A). In contrast, Western blot analysis showed that c-FLIPL and c-FLIPS were expressed in lower levels in the TRAIL-sensitive (WM793, WM9, and SBC12), as compared with the TRAIL-resistant cell lines (WM164, WM115, and WM35). Furthermore, cisplatin treatment down-regulated c-FLIPS protein expression in the resistant cell lines, whereas c-FLIPL protein expression remained unaffected (Fig. 6 A).

The chemotherapeutic drug CPT was reported to cause synergistic cytotoxicity with FasL in human prostate carcinoma through down-regulation of c-FLIPS(45). In addition, VP16 was reported to enhance TRAIL-induced cell death in glioma cells (41). Here, we examined CPT and VP16 to compare these drugs with cisplatin in their synergistic effects on TRAIL-induced apoptosis in melanoma cells. WM164 cells were treated with a low dose of CPT (100 ng/ml) or VP16 (10 μg/ml) for 16 h and then either left untreated or additionally treated with TRAIL (100 ng/ml) for an additional 16 h. The results showed that pretreatment of the melanoma cells with CPT, but not VP16, rendered the resistant cells sensitive to TRAIL-induced apoptosis, as evidenced by the appearance of the caspase-3 p17 cleavage product and ∼60% cell death (Fig. 6, B and C). Furthermore, treatment of WM164 cells with CPT (100 ng/ml) significantly down-regulated c-FLIPS proteins (Fig. 6,D). In contrast, VP16 treatment did not affect c-FLIPS protein expression (Fig. 6 D). The results suggest that CPT, but not VP16, sensitizes melanoma cells to TRAIL-induced apoptosis through down-regulation of c-FLIPS.

The long form c-FLIP (c-FLIPL), together with caspase-8, is recruited to TRAIL-induced DISC to inhibit the caspase-8 second step cleavage (23). We detected recently three c-FLIPL isoforms: c-FLIPLa, c-FLIPLb, and c-FLIPLc in glioma cells. c-FLIPLc is phosphorylated and recruited to TRAIL-induced DISC to inhibit TRAIL-induced apoptosis in glioma cells (32). To explore the possibility that cisplatin may also regulate c-FLIPL and, thus, facilitate the caspase-8 cleavage in the DISC in melanoma cells, we analyzed TRAIL-resistant melanoma cell lines for their expression of c-FLIPL isoforms. Two-dimensional-PAGE immunoblotting revealed c-FLIPLa, c-FLIPLb, and c-FLIPLc in TRAIL-resistant WM164, WM115, and WM35 cell lines, but treatment of these cell lines with cisplatin eliminated the phosphorylated isoform c-FLIPLc (Fig. 6 E). These results indicate that cisplatin inhibits c-FLIPL phosphorylation through unknown mechanisms, facilitating caspase-8 cleavage and TRAIL-induced apoptosis in TRAIL-resistant melanoma cells.

Cisplatin Sensitizes Primary Melanoma Cultures to TRAIL-induced Apoptosis.

It was reported recently that fresh isolates of early passage melanomas are resistant to TRAIL (46), raising the question of TRAIL effectiveness in clinical melanoma therapy. To examine this issue additionally, we prepared primary melanoma cultures from surgically removed metastatic melanoma tissue. First, we characterized these primary melanoma (early 3–5 passage) cultures (ED343MEL, ED343BMEL, and ED194MEL). Flow cytometry analysis revealed DR5 expression on the cell surface of all three of the primary melanoma cultures (Fig. 7,A). In contrast, DR4 cell surface expression was only detected in one primary culture (ED194MEL). Western blot analysis showed that intracellular form of DR4 was expressed in all of the three cultures, but membranous forms of DR4 were detected only in ED194MEL culture (Fig. 7,B). In contrast, the intracellular and membranous forms of DR5 were detected in all of the three cultures (Fig. 7 B). Caspase-8 and FADD were detected in all of the primary cultures. The results indicate that isolated melanoma cells in primary cultures express death machinery proteins for TRAIL.

We then tested these primary melanoma cultures for their sensitivity to TRAIL and cisplatin. The primary melanoma cultures (ED343MEL, ED343BMEL, and ED194MEL) were treated with TRAIL (100 ng/ml) and cisplatin (10 μg/ml) for 16 h alone or in combination. All three of the primary cultures were resistant to either TRAIL or cisplatin treatment, as shown by the lack of cleavage of caspase-8, caspase-3, and DFF45 on Western blots (Fig. 8,A), and a limited cell death as determined by crystal violet assay (Fig. 8,B). However, a combined treatment with TRAIL and cisplatin achieved a 50–75% cell death rate in all of the three primary cultures (Fig. 8,B). TRAIL-induced melanoma cell death occurred through caspase-8-initiated caspase cascade, as evidenced by the appearance of caspase-8, caspase-3, and DFF45 cleavage products on Western blots (Fig. 8 A). These results suggest that cisplatin sensitizes freshly isolated melanoma cells to TRAIL-induced apoptosis and supports the efficacy of this combined approach in the treatment of clinical melanomas.

Melanoma is the most lethal skin malignancy and, once the tumor has spread beyond its primary location, it becomes refractory to conventional chemotherapeutic agents and radiation therapy. Human recombinant soluble TRAIL has been reported recently to induce apoptosis in melanoma cell lines in vitro and suppresses melanoma growth in vivo, suggesting that recombinant soluble human TRAIL may be a future candidate in melanoma therapy (35, 36). However, TRAIL-induced apoptosis is limited, because many melanoma cell lines (8, 35) and primary melanoma cultures (46) are resistant to TRAIL. Here, we show that a combined treatment of TRAIL and chemotherapeutic drugs can overcome the resistance in melanoma cell lines and primary melanoma cultures. Furthermore, our studies provide several new insights into the molecular mechanisms that regulate TRAIL-induced apoptosis and its modulation by chemotherapeutic drugs in melanoma cells, thus providing new combined therapeutic approaches that target the mechanisms of tumor resistance.

Melanoma cells express TRAIL DRs, which correlate well with the sensitivity of melanoma cells to TRAIL (8, 36). Recent studies have shown that chemotherapeutic drugs such as cisplatin, doxorubicin, and VP16 up-regulate DR4 and DR5 mRNA expression, resulting in a synergistic cytotoxicity of TRAIL in several tumor cell lines (41, 44, 47). Contrary to this view, a recent analysis by flow cytometry and immunoblotting demonstrated that neither cisplatin nor doxorubicin influenced DR4 and DR5 protein expression in colon cancer (43) and mesothelioma cells (48). In this study, we showed that melanoma cells express intracellular forms of DR4 and DR5 proteins, but only DR5 is expressed on the cell surface. Cisplatin has no effect on the cell surface expression of DR5 in melanoma cells. The results prompted us to search for alternative mechanisms involved in cisplatin modulation of TRAIL-induced apoptosis in melanoma cells.

TRAIL-induced apoptosis in tumor cells occurs through incorporation of TRAIL-Rs, adaptor protein FADD, and apoptosis-initiating protease caspase-8 into a DISC whereby caspase-8 is cleaved to initiate apoptosis through a systematic cleavage of downstream effector caspases such as caspase-3 (20, 21, 22, 23). In TRAIL-resistant tumor cells, c-FLIPL and c-FLIPS are also recruited to the TRAIL-induced DISC, leading to the inhibition of caspase-8 cleavage and caspase-8-initiated apoptosis (23). The fact that cisplatin sensitizes the resistant melanoma cells to TRAIL-induced apoptosis through caspase-8-initiated caspase cascade suggests that cisplatin may regulate c-FLIP proteins and, thus, release their inhibition of caspase-8 cleavage in the DISC. Indeed, recent studies have shown that overexpression of c-FLIP in transfected tumor cells promotes tumor growth and facilitates immune escape of these tumors (49, 50), indicating that c-FLIP up-regulation may be implicated in oncogenesis and tumor growth.

Studies of c-FLIP expression using anti-c-FLIP rabbit serum that recognizes p55 c-FLIPL protein (27) have produced controversial results. One study reported a correlation of p55 c-FLIPL protein expression with TRAIL sensitivity in melanoma cell lines (35), whereas other studies fail to demonstrate a correlation in melanoma (8) and glioma cells (9). In this study, we used anti-c-FLIP monoclonal antibody NF6 that recognizes both c-FLIPL and c-FLIPS(30, 31), and demonstrated that c-FLIPL and c-FLIPS were highly expressed in TRAIL-resistant melanoma cell lines, a finding consistent with our previous observation in glioma cell lines (32). Furthermore, we showed that cisplatin and CPT down-regulated c-FLIPS, causing a synergistic effect on TRAIL-induced apoptosis in resistant melanoma cells. Cisplatin did not influence c-FLIPL protein expression in resistant melanoma cells. However, additional analysis by two-dimensional-PAGE immunoblots showed that cisplatin inhibited c-FLIPL phosphorylation in resistant melanoma cells. In an earlier study we showed that glioma cells express three isoforms of c-FLIPL, but only phosphorylated c-FLIPL was recruited to the DISC to inhibit caspase-8 cleavage (32). Here, we demonstrate that the three isoforms of c-FLIPL are also expressed in TRAIL-resistant melanoma cells and that cisplatin down-regulates phosphorylated c-FLIPL isoform to prevent its inhibition of caspse-8 cleavage in TRAIL-induced DISC, thus restoring TRAIL sensitivity in resistant melanoma cells.

Recent studies have provided additional evidence for combined approaches to restore TRAIL sensitivity through mitochondrial signal transduction pathways. CPT, for instance, was reported to up-regulate Bak (51), whereas cisplatin increased mitochondrial membrane potential facilitating cytochrome c release (42), thus resulting in a synergistic effect on TRAIL-induced apoptosis. Additional investigations into the molecular mechanisms involved in the regulation of TRAIL-induced and chemotherapeutic drug modulated tumor cell apoptosis are warranted. Through these studies, insights into molecular antiapoptotic and tumor cell resistance pathways will be better elucidated, facilitating the generation of novel therapeutic strategies required to combat malignant melanomas and other aggressive cancers.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

Supported by grants from the Canadian Institutes of Health Research (MOP49621). C. H. is the Clinical Investigator of Alberta Heritage Foundation for Medical Research.

3

The abbreviations used are: TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; ERK, extracellular signal-regulated kinase; FADD, Fas-associated death domain; c-FLIP, cellular Fas-associated death domain-like interleukin-1β-converting enzyme-like inhibitory protein; c-FLIPL, cellular Fas-associated death domain-like interleukin-1β-converting enzyme-like inhibitory protein long form; c-FLIPS, cellular Fas-associated death domain-like interleukin-1β-converting enzyme-like inhibitory protein short form; TNF, tumor necrosis factor; TRAIL-R, tumor necrosis factor-related apoptosis-inducing ligand receptor; FasL, Fas ligand; DR, death receptor; DD, death domain; DFF45, DNA fragmentation factor 45; DED, death effector domain; DISC, death-inducing signaling complex; CPT, chemptothecin; VP16, etoposide; IPG, immobilized pH gradient; z-VAD-fmk, carbobenzyloxy-Val-Ala-Asp(OMe)fluoromethyl ketone; HRP, horseradish peroxidase.

Fig. 1.

Cell death effect of TRAIL on human melanoma cell lines. Each of 21human melanoma cell lines was grown in 96-well plates (3 × 104 cells/well) overnight and then treated with TRAIL (100 ng/ml) for 16 h. Cell death was determined by crystal violet assay. The results were presented as the percentage cell death: 1 – (absorbance at 550 nm of cells treated/absorbance of cells treated) ×100. Data represent the mean (n = 6); bars, ±SD.

Fig. 1.

Cell death effect of TRAIL on human melanoma cell lines. Each of 21human melanoma cell lines was grown in 96-well plates (3 × 104 cells/well) overnight and then treated with TRAIL (100 ng/ml) for 16 h. Cell death was determined by crystal violet assay. The results were presented as the percentage cell death: 1 – (absorbance at 550 nm of cells treated/absorbance of cells treated) ×100. Data represent the mean (n = 6); bars, ±SD.

Close modal
Fig. 2.

TRAIL-induced apoptosis in TRAIL-sensitive melanoma cells. A, TRAIL-induced cell death in WM793. WM793 cells were grown in 96-well plates overnight, treated with z-VAD-fmk (20 μm) or medium for 2 h, and then with TRAIL (100 ng/ml) for 16 h. Cell death was determined by crystal violet assay. The data represent the mean (n = 6); bars, ±SD. B, kinetics of caspase-8, caspase-3, and DFF45 cleavage in WM793 cells after exposure to TRAIL. One × 106 WM793 cells grown in a 75 cm2 culture dish were treated with TRAIL (100 ng/ml) for the times indicated or, together with z-VAD-fmk (20 μm), for 5 h. Caspase-8, caspase-3, and DFF45 cleavage products were examined by Western blotting using anti-caspase-8, caspase-3, and DFF45 antibodies. Proteins detected are indicated to the right. C, TRAIL-induced cell death in SBC12, WM1366, WM278, and WM9 cell lines. Each of the cell lines was treated with various doses of TRAIL, and cell death was quantified by crystal violet assay. Data represent the mean (n = 6); bars, ±SD. D, cleavage of caspase-8, caspase-3, and DFF45 in SBC12, WM1366, WM278, and WM9 cell lines. Cells were incubated with TRAIL (100 ng/ml) for 4 h. Cell lysates were analyzed by Western blotting with caspase-8, caspase-3, and DFF45 antibodies to detect caspase-8 (p43, p41, and p18), caspase-3 (p20 and p17), and DFF45 (p25, p17, and p11) cleavage products.

Fig. 2.

TRAIL-induced apoptosis in TRAIL-sensitive melanoma cells. A, TRAIL-induced cell death in WM793. WM793 cells were grown in 96-well plates overnight, treated with z-VAD-fmk (20 μm) or medium for 2 h, and then with TRAIL (100 ng/ml) for 16 h. Cell death was determined by crystal violet assay. The data represent the mean (n = 6); bars, ±SD. B, kinetics of caspase-8, caspase-3, and DFF45 cleavage in WM793 cells after exposure to TRAIL. One × 106 WM793 cells grown in a 75 cm2 culture dish were treated with TRAIL (100 ng/ml) for the times indicated or, together with z-VAD-fmk (20 μm), for 5 h. Caspase-8, caspase-3, and DFF45 cleavage products were examined by Western blotting using anti-caspase-8, caspase-3, and DFF45 antibodies. Proteins detected are indicated to the right. C, TRAIL-induced cell death in SBC12, WM1366, WM278, and WM9 cell lines. Each of the cell lines was treated with various doses of TRAIL, and cell death was quantified by crystal violet assay. Data represent the mean (n = 6); bars, ±SD. D, cleavage of caspase-8, caspase-3, and DFF45 in SBC12, WM1366, WM278, and WM9 cell lines. Cells were incubated with TRAIL (100 ng/ml) for 4 h. Cell lysates were analyzed by Western blotting with caspase-8, caspase-3, and DFF45 antibodies to detect caspase-8 (p43, p41, and p18), caspase-3 (p20 and p17), and DFF45 (p25, p17, and p11) cleavage products.

Close modal
Fig. 3.

Cisplatin enhancement of TRAIL-induced apoptosis in the cell line WM164. A, cell death analysis. Cells were seeded in 96-well plates and pretreated with indicated doses of cisplatin for 16 h. Cells were additionally incubated either in the presence or absence of TRAIL (100 ng/ml) for 16 h. Cell death was determined by crystal violet assay. The data represent the mean (n = 6); bars, ±SD. B, cleavage of caspase-8, caspase-3, and DFF45. Cells were pretreated with a low dose of cisplatin (10 μg/ml) for 16 h, and then additionally incubated with TRAIL (100 ng/ml) for the times indicated. Cell lysates were subjected to Western blotting to detect caspase-8 (p43, p41, and p18), caspase 3 (p20 and p17), and DFF45 (p25, p17, and p11) cleavage products. C–H, cellular apoptosis. WM164 cells grown in 24-well plates (5 × 105 cells/well) were untreated (C), treated with 100 ng/ml TRAIL for 4 h (D), treated with 10 μg/ml cisplatin alone for 20 h (E), or treated 10 μg/ml cisplatin alone for 16 h and then with TRAIL for 4 h (F). Cells were also treated with 20 μm z-VAD-fmk (G) alone or pretreated with 20 μm z-VAD-fmk and cisplatin for 16 h and followed by TRAIL treatment for 6 h (H). Cells were examined under phase contrast microscopy for cellular apoptosis.

Fig. 3.

Cisplatin enhancement of TRAIL-induced apoptosis in the cell line WM164. A, cell death analysis. Cells were seeded in 96-well plates and pretreated with indicated doses of cisplatin for 16 h. Cells were additionally incubated either in the presence or absence of TRAIL (100 ng/ml) for 16 h. Cell death was determined by crystal violet assay. The data represent the mean (n = 6); bars, ±SD. B, cleavage of caspase-8, caspase-3, and DFF45. Cells were pretreated with a low dose of cisplatin (10 μg/ml) for 16 h, and then additionally incubated with TRAIL (100 ng/ml) for the times indicated. Cell lysates were subjected to Western blotting to detect caspase-8 (p43, p41, and p18), caspase 3 (p20 and p17), and DFF45 (p25, p17, and p11) cleavage products. C–H, cellular apoptosis. WM164 cells grown in 24-well plates (5 × 105 cells/well) were untreated (C), treated with 100 ng/ml TRAIL for 4 h (D), treated with 10 μg/ml cisplatin alone for 20 h (E), or treated 10 μg/ml cisplatin alone for 16 h and then with TRAIL for 4 h (F). Cells were also treated with 20 μm z-VAD-fmk (G) alone or pretreated with 20 μm z-VAD-fmk and cisplatin for 16 h and followed by TRAIL treatment for 6 h (H). Cells were examined under phase contrast microscopy for cellular apoptosis.

Close modal
Fig. 4.

TRAIL-induced apoptosis in resistant melanoma cells pretreated with cisplatin. A, cell death analysis. Cells growth in 96-well plates (3 × 104 cells/well) were treated with various doses of cisplatin for 16 h and then incubated with or without TRAIL (100 ng/ml) for 16 h. Cell death was determined by crystal violet assay. Data represent the mean (n = 6); bars, ±SD. B, cleavage of caspases and DFF45. TRAIL-resistant melanoma cell lines (WM115, WM35, and WM852) were pretreated with cisplatin (10 μg/ml) for 16 h and additionally incubated with TRAIL (100 ng/ml) for 8 h or left untreated as control. Caspase-8, caspase-3, and DFF45 cleavage was examined by Western blot analysis.

Fig. 4.

TRAIL-induced apoptosis in resistant melanoma cells pretreated with cisplatin. A, cell death analysis. Cells growth in 96-well plates (3 × 104 cells/well) were treated with various doses of cisplatin for 16 h and then incubated with or without TRAIL (100 ng/ml) for 16 h. Cell death was determined by crystal violet assay. Data represent the mean (n = 6); bars, ±SD. B, cleavage of caspases and DFF45. TRAIL-resistant melanoma cell lines (WM115, WM35, and WM852) were pretreated with cisplatin (10 μg/ml) for 16 h and additionally incubated with TRAIL (100 ng/ml) for 8 h or left untreated as control. Caspase-8, caspase-3, and DFF45 cleavage was examined by Western blot analysis.

Close modal
Fig. 5.

DR4 and DR5 expression on the surface of WM164, WM115, and WM35 cell lines. Cells (1 × 106 cells) were treated with or without 10 μg/ml cisplatin for 16 h and analyzed for cell surface DR4 and DR5 expression by flow cytometric analysis. Bolded lines in histograms represent the results using anti-DR4 or anti-DR5 mouse IgG1 antibody, whereas dotted lines represent results from mouse IgG1 control antibody. Histograms represent 104 gated tumor cells.

Fig. 5.

DR4 and DR5 expression on the surface of WM164, WM115, and WM35 cell lines. Cells (1 × 106 cells) were treated with or without 10 μg/ml cisplatin for 16 h and analyzed for cell surface DR4 and DR5 expression by flow cytometric analysis. Bolded lines in histograms represent the results using anti-DR4 or anti-DR5 mouse IgG1 antibody, whereas dotted lines represent results from mouse IgG1 control antibody. Histograms represent 104 gated tumor cells.

Close modal
Fig. 6.

Cisplatin regulation of c-FLIPS and c-FLIPL. A, cisplatin down-regulation of c-FLIPS. TRAIL-sensitive (WM793, WM9, and SBC12) and resistant cell lines (WM164, WM115, and WM35) were either left untreated or treated with 10 μg/ml cisplatin for 16 h. Cell lysates were subjected to Western blot analysis using anti-FADD, capsase-8, and c-FLIP antibody as indicated to the left. Proteins detected were indicated to the right. ERK1/2 antibody was used as a loading control. B and C, differential effect of CPT and VP16 on TRAIL-induced apoptosis. WM164 cells were treated with either 50 ng/ml CPT or 10 μg/ml VP16 for 16 h and then incubated with 100 ng/ml TRAIL for additional 6 h. Caspase-3 cleavage products were examined by Western blotting (B) and cell death was evaluated by crystal violet assay (C). D, effect of CPT and VP16 on c-FLIP expression. Protein expression of c-FLIPS and c-FLIPL was examined by Western blotting in CPT and VP16 treated or untreated WM164 cells. E, effect of cisplatin on c-FLIPL. TRAIL-resistant WM164, WM115, and WM35 cell lines were left untreated as control and treated with 10 μg/ml cisplatin for 16 h. Cell lysates were subjected to two-dimensional-PAGE immunoblotting using rabbit anti-c-FLIP antibody. Isoforms of c-FLIPL were detected as c-FLIPLa (left dot), c-FLIPLb (right dot), and c-FLIPLc (top dot; indicated by arrows).

Fig. 6.

Cisplatin regulation of c-FLIPS and c-FLIPL. A, cisplatin down-regulation of c-FLIPS. TRAIL-sensitive (WM793, WM9, and SBC12) and resistant cell lines (WM164, WM115, and WM35) were either left untreated or treated with 10 μg/ml cisplatin for 16 h. Cell lysates were subjected to Western blot analysis using anti-FADD, capsase-8, and c-FLIP antibody as indicated to the left. Proteins detected were indicated to the right. ERK1/2 antibody was used as a loading control. B and C, differential effect of CPT and VP16 on TRAIL-induced apoptosis. WM164 cells were treated with either 50 ng/ml CPT or 10 μg/ml VP16 for 16 h and then incubated with 100 ng/ml TRAIL for additional 6 h. Caspase-3 cleavage products were examined by Western blotting (B) and cell death was evaluated by crystal violet assay (C). D, effect of CPT and VP16 on c-FLIP expression. Protein expression of c-FLIPS and c-FLIPL was examined by Western blotting in CPT and VP16 treated or untreated WM164 cells. E, effect of cisplatin on c-FLIPL. TRAIL-resistant WM164, WM115, and WM35 cell lines were left untreated as control and treated with 10 μg/ml cisplatin for 16 h. Cell lysates were subjected to two-dimensional-PAGE immunoblotting using rabbit anti-c-FLIP antibody. Isoforms of c-FLIPL were detected as c-FLIPLa (left dot), c-FLIPLb (right dot), and c-FLIPLc (top dot; indicated by arrows).

Close modal
Fig. 7.

Characterization of primary melanoma cultures. A, flow cytometric analysis. One × 106 cells from each of the primary melanoma cultures (ED343MEL, ED194MEL, and ED343BMEL) were analyzed for cell surface DR4 and DR5 expression. Bolded lines in histograms represent the results using anti-DR4 or anti-DR5 mouse IgG1 antibody, and dotted lines show results from mouse IgG1 control. Histograms represent 104 gated cells. B, Western blot analysis. Subconfluent cultures were treated with (+) or without (−) 10 μg/ml cisplatin for 16 h and subjected to Western blot analysis using anti-DR4, anti-DR5, anti-FADD, and anti-caspase-8, respectively. The antibodies were indicated to the left and proteins detected to the right. Intracellular forms of DR4 (p57) and DR5 (p60) are indicated by arrows, whereas the membranous forms of DR4 and DR5 are indicated by arrowheads.

Fig. 7.

Characterization of primary melanoma cultures. A, flow cytometric analysis. One × 106 cells from each of the primary melanoma cultures (ED343MEL, ED194MEL, and ED343BMEL) were analyzed for cell surface DR4 and DR5 expression. Bolded lines in histograms represent the results using anti-DR4 or anti-DR5 mouse IgG1 antibody, and dotted lines show results from mouse IgG1 control. Histograms represent 104 gated cells. B, Western blot analysis. Subconfluent cultures were treated with (+) or without (−) 10 μg/ml cisplatin for 16 h and subjected to Western blot analysis using anti-DR4, anti-DR5, anti-FADD, and anti-caspase-8, respectively. The antibodies were indicated to the left and proteins detected to the right. Intracellular forms of DR4 (p57) and DR5 (p60) are indicated by arrows, whereas the membranous forms of DR4 and DR5 are indicated by arrowheads.

Close modal
Fig. 8.

Synergistic effect of cisplatin on TRAIL-induced apoptosis in primary melanoma cultures. A, primary melanoma cultures were pretreated with 10 μg/ml cisplatin for 16 h and additionally incubated either with medium or TRAIL (100 ng/ml) for 8 h. Caspase-8, caspase-3, and DFF45 cleavage products were examined on Western blots. B, cells were grown in 96-well plates (3 × 104 cells/well) overnight and treated with 10 μg/ml cisplatin for 16 h. Some of the cells were left untreated and others were treated with 100 ng/ml TRAIL for 16 h. Cell deaths were determined by crystal violet assay. The data represent the mean (n = 6); bars, ±SD.

Fig. 8.

Synergistic effect of cisplatin on TRAIL-induced apoptosis in primary melanoma cultures. A, primary melanoma cultures were pretreated with 10 μg/ml cisplatin for 16 h and additionally incubated either with medium or TRAIL (100 ng/ml) for 8 h. Caspase-8, caspase-3, and DFF45 cleavage products were examined on Western blots. B, cells were grown in 96-well plates (3 × 104 cells/well) overnight and treated with 10 μg/ml cisplatin for 16 h. Some of the cells were left untreated and others were treated with 100 ng/ml TRAIL for 16 h. Cell deaths were determined by crystal violet assay. The data represent the mean (n = 6); bars, ±SD.

Close modal

We thank Urosh Vilimanovich for his valuable comments on the manuscript.

1
Ashkenazi A., Dixit V. M. Death receptors: signaling and modulation.
Science (Wash. DC)
,
281
:
1305
-1308,  
1998
.
2
Wiley S. R., Schooley K., Smolak P. J., Din W. S., Huang C. P., Nicholl J. K., Sutherland G. R., Smith T. D., Rauch C., Smith C. A., et al Identification and characterization of a new member of the TNF family that induces apoptosis.
Immunity
,
3
:
673
-682,  
1995
.
3
Pitti R. M., Marsters S. A., Ruppert S., Donahue C. J., Moore A., Ashkenazi A. Induction of apoptosis by Apo-2 ligand, a new member of the tumor necrosis factor cytokine family.
J. Biol. Chem.
,
271
:
12687
-12690,  
1996
.
4
Takeda K., Hayakawa Y., Smyth M. J., Kayagaki N., Yamaguchi N., Kakuta S., Iwakura Y., Yagita H., Okumura K. Involvement of tumor necrosis factor-related apoptosis-inducing ligand in surveillance of tumor metastasis by liver natural killer cells.
Nat. Med.
,
7
:
94
-100,  
2001
.
5
Walczak H., Miller R. E., Ariail K., Gliniak B., Griffith T. S., Kubin M., Chin W., Jones J., Woodward A., Le T., Smith C., Smolak P., Goodwin R. G., Rauch C. T., Schuh J. C., Lynch D. H. Tumoricidal activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo.
Nat. Med.
,
5
:
157
-163,  
1999
.
6
Ashkenazi A., Pai R. C., Fong S., Leung S., Lawrence D. A., Marsters S. A., Blackie C., Chang L., McMurtrey A. E., Hebert A., DeForge L., Koumenis I. L., Lewis D., Harris L., Bussiere J., Koeppen H., Shahrokh Z., Schwall R. H. Safety and antitumor activity of recombinant soluble Apo2 ligand.
J. Clin. Investig.
,
104
:
155
-162,  
1999
.
7
Leverkus M., Neumann M., Mengling T., Rauch C. T., Brocker E. B., Krammer P. H., Walczak H. Regulation of tumor necrosis factor-related apoptosis-inducing ligand sensitivity in primary and transformed human keratinocytes.
Cancer Res.
,
60
:
553
-559,  
2000
.
8
Zhang X. D., Franco A., Myers K., Gray C., Nguyen T., Hersey P. Relation of TNF-related apoptosis-inducing ligand (TRAIL) receptor and FLICE-inhibitory protein expression to TRAIL-induced apoptosis of melanoma.
Cancer Res.
,
59
:
2747
-2753,  
1999
.
9
Hao C., Beguinot F., Condorelli G., Trencia A., Van Meir E. G., Yong V. W., Parney I. F., Roa W. H., Petruk K. C. Induction and intracellular regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mediated apotosis in human malignant glioma cells.
Cancer Res.
,
61
:
1162
-1170,  
2001
.
10
Lawrence D., Shahrokh Z., Marsters S., Achilles K., Shih D., Mounho B., Hillan K., Totpal K., DeForge L., Schow P., Hooley J., Sherwood S., Pai R., Leung S., Khan L., Gliniak B., Bussiere J., Smith C. A., Strom S. S., Kelley S., Fox J. A., Thomas D., Ashkenazi A. Differential hepatocyte toxicity of recombinant Apo2L/TRAIL versions.
Nat. Med.
,
7
:
383
-385,  
2001
.
11
Pan G., O’Rourke K., Chinnaiyan A. M., Gentz R., Ebner R., Ni J., Dixit V. M. The receptor for the cytotoxic ligand TRAIL.
Science (Wash. DC)
,
276
:
111
-113,  
1997
.
12
Pan G., Ni J., Wei Y. F., Yu G., Gentz R., Dixit V. M. An antagonist decoy receptor and a death domain-containing receptor for TRAIL.
Science (Wash. DC)
,
277
:
815
-818,  
1997
.
13
Schneider P., Bodmer J. L., Thome M., Hofmann K., Holler N., Tschopp J. Characterization of two receptors for TRAIL.
FEBS Lett.
,
416
:
329
-334,  
1997
.
14
Sheridan J. P., Marsters S. A., Pitti R. M., Gurney A., Skubatch M., Baldwin D., Ramakrishnan L., Gray C. L., Baker K., Wood W. I., Goddard A. D., Godowski P., Ashkenazi A. Control of TRAIL-induced apoptosis by a family of signaling and decoy receptors.
Science (Wash. DC)
,
277
:
818
-821,  
1997
.
15
Wu G. S., Burns T. F., McDonald E. R., 3rd, Jiang W., Meng R., Krantz I. D., Kao G., Gan D. D., Zhou J. Y., Muschel R., Hamilton S. R., Spinner N. B., Markowitz S., Wu G., el-Deiry W. S. KILLER/DR5 is a DNA damage-inducible p53-regulated death receptor gene.
Nat. Genet.
,
17
:
141
-143,  
1997
.
16
Chaudhary P. M., Eby M., Jasmin A., Bookwalter A., Murray J., Hood L. Death receptor 5, a new member of the TNFR family, and DR4 induce FADD-dependent apoptosis and activate the NF-κB pathway.
Immunity
,
7
:
821
-830,  
1997
.
17
Walczak H., Degli-Esposti M. A., Johnson R. S., Smolak P. J., Waugh J. Y., Boiani N., Timour M. S., Gerhart M. J., Schooley K. A., Smith C. A., Goodwin R. G., Rauch C. T. TRAIL-R2: a novel apoptosis-mediating receptor for TRAIL.
EMBO J.
,
16
:
5386
-5397,  
1997
.
18
Boldin M. P., Varfolomeev E. E., Pancer Z., Mett I. L., Camonis J. H., Wallach D. A novel protein that interacts with the death domain of Fas/APO1 contains a sequence motif related to the death domain.
J. Biol. Chem.
,
270
:
7795
-7798,  
1995
.
19
Chinnaiyan A. M., O’Rourke K., Tewari M., Dixit V. M. FADD, a novel death domain-containing protein, interacts with the death domain of Fas and initiates apoptosis.
Cell
,
81
:
505
-512,  
1995
.
20
Bodmer J. L., Holler N., Reynard S., Vinciguerra P., Schneider P., Juo P., Blenis J., Tschopp J. TRAIL receptor-2 signals apoptosis through FADD and caspase-8.
Nat. Cell Biol.
,
2
:
241
-243,  
2000
.
21
Kischkel F. C., Lawrence D. A., Chuntharapai A., Schow P., Kim K. J., Ashkenazi A. Apo2L/TRAIL-dependent recruitment of endogenous FADD and caspase-8 to death receptors 4 and 5.
Immunity
,
12
:
611
-620,  
2000
.
22
Kuang A. A., Diehl G. E., Zhang J., Winoto A. FADD is required for DR4- and DR5-mediated apoptosis: lack of trail-induced apoptosis in FADD-deficient mouse embryonic fibroblasts.
J. Biol. Chem.
,
275
:
25065
-25068,  
2000
.
23
Xiao C., Yang B. F., Asadi N., Beguinot F., Hao C. Tumor necrosis factor-related apoptosis-inducing ligand-induced death-inducing signaling complex and its modulation by c-FLIP and PED/PEA-15 in glioma cells.
J. Biol. Chem.
,
277
:
25020
-25025,  
2002
.
24
Muzio M., Stockwell B. R., Stennicke H. R., Salvesen G. S., Dixit V. M. An induced proximity model for caspase-8 activation.
J. Biol. Chem.
,
273
:
2926
-2930,  
1998
.
25
Thornberry N. A., Rano T. A., Peterson E. P., Rasper D. M., Timkey T., Garcia-Calvo M., Houtzager V. M., Nordstrom P. A., Roy S., Vaillancourt J. P., Chapman K. T., Nicholson D. W. A combinatorial approach defines specificities of members of the caspase family and granzyme B. Functional relationships established for key mediators of apoptosis.
J. Biol. Chem.
,
272
:
17907
-17911,  
1997
.
26
Liu X., Zou H., Slaughter C., Wang X. DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis.
Cell
,
89
:
175
-184,  
1997
.
27
Irmler M., Thome M., Hahne M., Schneider P., Hofmann K., Steiner V., Bodmer J. L., Schroter M., Burns K., Mattmann C., Rimoldi D., French L. E., Tschopp J. Inhibition of death receptor signals by cellular FLIP.
Nature (Lond.)
,
388
:
190
-195,  
1997
.
28
Shu H. B., Halpin D. R., Goeddel D. V. Casper is a FADD- and caspase-related inducer of apoptosis.
Immunity
,
6
:
751
-763,  
1997
.
29
Rasper D. M., Vaillancourt J. P., Hadano S., Houtzager V. M., Seiden I., Keen S. L., Tawa P., Xanthoudakis S., Nasir J., Martindale D., Koop B. F., Peterson E. P., Thornberry N. A., Huang J., MacPherson D. P., Black S. C., Hornung F., Lenardo M. J., Hayden M. R., Roy S., Nicholson D. W. Cell death attenuation by ’Usurpin’, a mammalian DED-caspase homologue that precludes caspase-8 recruitment and activation by the CD-95 (Fas, APO-1) receptor complex.
Cell Death Differ.
,
5
:
271
-288,  
1998
.
30
Scaffidi C., Schmitz I., Krammer P. H., Peter M. E. The role of c-FLIP in modulation of CD95-induced apoptosis.
J. Biol. Chem.
,
274
:
1541
-1548,  
1999
.
31
Krueger A., Schmitz I., Baumann S., Krammer P. H., Kirchhoff S. Cellular FLICE-inhibitory protein splice variants inhibit different steps of caspase-8 activation at the CD95 death-inducing signaling complex.
J. Biol. Chem.
,
276
:
20633
-20640,  
2001
.
32
Yang B. F., Xiao C., Roa W. H., Krammer P. H., Hao C. Calcium/calmodulin-dependent protein kinase II regulation of c-FLIP expression and phosphorylation in modulation of Fas-mediated signaling in malignant glioma cells.
J. Biol. Chem.
,
278
:
7043
-7050,  
2003
.
33
Hsu M-Y., Elder D. E., Herlyn M. Melanoma: the Wistar (WM) melanoma cell lines Masters J. R. W. Palsson B. eds. .
Human Cell Culture
,
Vol. 3
:
259
-274, Kluwer Acad. Publ. London  
1999
.
34
Parney I. F., Petruk K. C., Zhang C., Farr-Jones M., Sykes D. B., Chang L. J. Granulocyte-macrophage colony-stimulating factor and B7-2 combination immunogene therapy in an allogeneic Hu-PBL-SCID/beige mouse-human glioblastoma multiforme model.
Hum. Gene. Ther.
,
8
:
1073
-1085,  
1997
.
35
Griffith T. S., Chin W. A., Jackson G. C., Lynch D. H., Kubin M. Z. Intracellular regulation of TRAIL-induced apoptosis in human melanoma cells.
J. Immunol.
,
161
:
2833
-2840,  
1998
.
36
Zhang X. D., Zhang X. Y., Gray C. P., Nguyen T., Hersey P. Tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis of human melanoma is regulated by smac/DIABLO release from mitochondria.
Cancer Res.
,
61
:
7339
-7348,  
2001
.
37
Muzio M., Chinnaiyan A. M., Kischkel F. C., O’Rourke K., Shevchenko A., Ni J., Scaffidi C., Bretz J. D., Zhang M., Gentz R., Mann M., Krammer P. H., Peter M. E., Dixit V. M. FLICE, a novel FADD-homologous ICE/CED-3-like protease, is recruited to the CD95 (Fas/APO-1) death–inducing signaling complex.
Cell
,
85
:
817
-827,  
1996
.
38
Medema J. P., Scaffidi C., Kischkel F. C., Shevchenko A., Mann M., Krammer P. H., Peter M. E. FLICE is activated by association with the CD95 death-inducing signaling complex (DISC).
EMBO J.
,
16
:
2794
-2804,  
1997
.
39
Samali A., Cai J., Zhivotovsky B., Jones D. P., Orrenius S. Presence of a pre-apoptotic complex of pro-caspase-3, Hsp60 and Hsp10 in the mitochondrial fraction of jurkat cells.
EMBO J.
,
18
:
2040
-2048,  
1999
.
40
Gu J., Dong R. P., Zhang C., McLaughlin D. F., Wu M. X., Schlossman S. F. Functional interaction of DFF35 and DFF45 with caspase-activated DNA fragmentation nuclease DFF40.
J. Biol. Chem.
,
274
:
20759
-20762,  
1999
.
41
Nagane M., Pan G., Weddle J. J., Dixit V. M., Cavenee W. K., Huang H. J. Increased death receptor 5 expression by chemotherapeutic agents in human gliomas causes synergistic cytotoxicity with tumor necrosis factor-related apoptosis-inducing ligand in vitro and in vivo.
Cancer Res.
,
60
:
847
-853,  
2000
.
42
Ferreira C. G., Span S. W., Peters G. J., Kruyt F. A., Giaccone G. Chemotherapy triggers apoptosis in a caspase-8-dependent and mitochondria-controlled manner in the non-small cell lung cancer cell line NCI-H460.
Cancer Res.
,
60
:
7133
-7141,  
2000
.
43
Lacour S., Hammann A., Wotawa A., Corcos L., Solary E., Dimanche-Boitrel M. T. Anticancer agents sensitize tumor cells to tumor necrosis factor-related apoptosis-inducing ligand-mediated caspase-8 activation and apoptosis.
Cancer Res.
,
61
:
1645
-1651,  
2001
.
44
Evdokiou A., Bouralexis S., Atkins G. J., Chai F., Hay S., Clayer M., Findlay D. M. Chemotherapeutic agents sensitize osteogenic sarcoma cells, but not normal human bone cells, to Apo2L/TRAIL-induced apoptosis.
Int. J. Cancer
,
99
:
491
-504,  
2002
.
45
Chatterjee D., Schmitz I., Krueger A., Yeung K., Kirchhoff S., Krammer P. H., Peter M. E., Wyche J. H., Pantazis P. Induction of apoptosis in 9-nitrocamptothecin-treated DU145 human prostate carcinoma cells correlates with de novo synthesis of CD95 and CD95 ligand and down-regulation of c-FLIP(short).
Cancer Res.
,
61
:
7148
-7154,  
2001
.
46
Nguyen T., Zhang X. D., Hersey P. Relative resistance of fresh isolates of melanoma to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis.
Clin. Cancer Res.
,
7
:
966s
-973s,  
2001
.
47
Gibson S. B., Oyer R., Spalding A. C., Anderson S. M., Johnson G. L. Increased expression of death receptors 4 and 5 synergizes the apoptosis response to combined treatment with etoposide and TRAIL.
Mol. Cell Biol.
,
20
:
205
-212,  
2000
.
48
Liu W., Bodle E., Chen J. Y., Gao M., Rosen G. D., Broaddus V. C. Tumor necrosis factor-related apoptosis-inducing ligand and chemotherapy cooperate to induce apoptosis in mesothelioma cell lines.
Am. J. Respir. Cell Mol. Biol.
,
25
:
111
-118,  
2001
.
49
Djerbi M., Screpanti V., Catrina A. I., Bogen B., Biberfeld P., Grandien A. The inhibitor of death receptor signaling, FLICE-inhibitory protein defines a new class of tumor progression factors.
J. Exp. Med.
,
190
:
1025
-1032,  
1999
.
50
Medema J. P., de Jong J., van Hall T., Melief C. J., Offringa R. Immune escape of tumors in vivo by expression of cellular FLICE-inhibitory protein.
J. Exp. Med.
,
190
:
1033
-1038,  
1999
.
51
LeBlanc H., Lawrence D., Varfolomeev E., Totpal K., Morlan J., Schow P., Fong S., Schwall R., Sinicropi D., Ashkenazi A. Tumor-cell resistance to death receptor–induced apoptosis through mutational inactivation of the proapoptotic Bcl-2 homolog Bax.
Nat. Med.
,
8
:
274
-281,  
2002
.