Interferon-alpha-induced apoptosis via tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-dependent and -independent manner

IFN-alpha regulates tumor cell growth at least through induction of apoptosis. We have recently demonstrated that IFN-alpha causes apoptosis through upregulation of TNF-related apoptosis-inducing ligand (TRAIL) in Daudi B lymphoma and U266 myeloma cells. However, other cell lines such as Ramos and RPMI 8226 underwent apoptosis without any apparent involvement of TRAIL following IFN-alpha stimulation. In this study, we examined whether the IFN-alpha-induced upregulation of TRAIL is essential for the induction of apoptosis. IFN-alpha-induced early phase (48 h) of loss of DeltaPsim was substantially prevented in Daudi B lymphoma cells overexpressing the dominant-negative form of Fas-associated death domain (dnFADD) compared with vector control, whereas a late phase (72 h) of DeltaPsim was comparable to the control. The IFN-alpha-induced early phase of apoptosis was also reduced in the dnFADD-expressing cells, while the late phase of apoptosis was unaffected. IFN-alpha-induced upregulation of TRAIL protein in the dnFADD-expressing Daudi or U266 cells was comparable to their control cells, suggesting that FADD is not involved in the IFN-alpha-induced upregulation of TRAIL. Moreover, the early phase of mitochondrial depolarization was severely prevented by the presence of fusion protein of TRAIL receptor 1 and Fc portion of immunoglobulin (TRAIL-R1:Fc) and TRAIL-R2:Fc. Together, IFN-alpha induces apoptosis in a TRAIL-dependent or -independent manner, depending on the course of the apoptotic process.     

Hyperthermia enhances tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in human cancer cells

There is growing body of evidence linking the cellular response to heat stress with the response of the immune system to cancer. The anti-tumor immune response can be markedly enhanced by treatment with hyperthermia particularly in the fever range. In addition, the heat shock proteins (hsp) which are produced in abundant quantities in cells exposed to heat are potent immune modulators and can lead to stimulation of both the innate and adaptive immune responses to tumors. Immunostimulation by hyperthermia involves both direct effects of heat on the behavior of immune cells as well as indirect effects mediated through hsp release. In addition, the hsp can be deployed as components of antitumor vaccines in protocols that do not include hyperthermia. Understanding these process may permit the effective deployment of hyperthermia and hsp based vaccines in tumor treatment.     

Hyperthermia enhances tumour necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in human cancer cells.

PURPOSE: This study investigated whether hyperthermia can enhance TRAIL-induced apoptotic death. METHODS: Human prostate adenocarcinoma DU-145, human pancreatic carcinoma MIA PaCa-2 and BxPC-3, human colon fibroblast CCD-33Co and rat prostate endothelial YPEN-1 cells were treated with various concentrations of TRAIL (0-200 ngml(-1)) with hyperthermia (40-42 degrees C). RESULTS: It was observed in human cancer cells, but not in normal cells, that TRAIL induced apoptotic death and also that hyperthermia (40-42 degrees C) promoted TRAIL-induced apoptotic death. Enhancement of TRAIL-mediated apoptosis by hyperthermia was detected by an increase in PARP cleavage, the hallmark feature of apoptosis, as well as by activation of caspases. There were no significant changes in the intra-cellular levels of death receptors (DRs), decoy receptors (DcRs) and anti-apoptotic proteins. Interestingly, data from in vitro enzyme kinetics assay demonstrated that hyperthermia promoted caspase enzyme activity. CONCLUSIONS: These data suggest that cancer cells are more susceptible to TRAIL in the condition of hyperthermia (40-42 degrees C). The promotion of caspase enzyme activity by hyperthermia may be responsible for enhancement of TRAIL-induced apoptotic death.     

Etoposide-mediated sensitization of squamous cell carcinoma cells to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced loss in mitochondrial membrane potential

Squamous cell carcinoma (SCC) cell lines (MIT7-x(L), MIT8, and MIT16) that overexpress Bcl-x(L) have been demonstrated to show resistance to multiple chemotherapeutic drugs. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), which belongs to the TNF family of proteins, induces apoptosis in tumor, but not in normal, cells. In the present study, we examined whether etoposide sensitizes tumor cells with multiple-drug-resistance to TRAIL-induced apoptosis. Sequential treatment with etoposide and TRAIL resulted in a synergistically induced cell death in the two resistant lines (MIT7-x(L) and MIT16) but not MIT8, as assessed by WST-8 assay. As expected, MIT7 cells (a drug-sensitive line) were sensitive to the combined treatment. The cell death caused by both etoposide and TRAIL appears to involve apoptosis, since the combined treatment caused a loss in mitochondrial membrane potential (DeltaPsim), which is closely associated with apoptosis induction. The density of the TRAIL-receptors (TRAIL-Rs) was not appreciably modulated by the etoposide treatment, suggesting that etoposide targets molecule(s) downstream of the TRAIL-Rs. Regardless of the molecular mechanisms underlying the cell death, sequential treatment with etoposide and TRAIL could be useful in the design of treatment modalities for patients with SCC, especially those with elevated levels of Bcl-x(L).     

Curcumin sensitizes tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis through CHOP-independent DR5 upregulation

Death receptor DR5 (DR5/TRAIL-R2) is an apoptosis-inducing membrane receptor for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). In this study, we showed that curcumin, a plant product containing the phenolic phytochemical, is a potent enhancer of TRAIL-induced apoptosis through upregulation of DR5 expression. Both treatment with DR5/Fc chimeric protein and silencing of DR5 expression using small interfering RNA (siRNA) attenuated curcumin plus TRAIL-induced apoptosis, showing that the critical role of DR5 in this cell death. Curcumin also induced the expression of a potential pro-apoptotic gene, C/EBP homologous protein (CHOP), both at its mRNA and protein levels. However, suppression of CHOP expression by small interfering RNA did not abrogate the curcumin-mediated induction of DR5 and the cell death induced by curcumin plus TRAIL, demonstrating that CHOP is not involved in curcumin-induced DR5 upregulation. Taken together, the present study demonstrates that curcumin enhances TRAIL-induced apoptosis by CHOP-independent upregulation of DR5.     

Resistance to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in neuroblastoma cells correlates with a loss of caspase-8 expression

Disruption of apoptotic pathways may be involved in tumor formation, regression, and treatment resistance of neuroblastoma (NB). Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in cancer cell lines, whereas normal cells are not sensitive to TRAIL-mediated apoptosis. In this study we analyzed the expression and function of TRAIL and its agonistic and antagonistic receptors as well as expression of cellular FLICE-like inhibitory protein and caspase-2, -3, -8, -9, and -10 in 18 NB cell lines. Semiquantitative RT-PCR revealed that TRAIL-R2 and TRAIL-R3 are the main TRAIL-receptors used by NB cells. Sensitivity to TRAIL-induced apoptosis did not correlate with mRNA expression of TRAIL receptors or cellular FLICE-like inhibitory protein. Surprisingly, caspase-8 and caspase-10 mRNA expression was detected in only 5 of 18 NB cell lines. Interestingly, only these five NB cell lines were susceptible to TRAIL-induced apoptosis in a time- and dose-dependent manner. Treatment with 5-aza-2'-deoxycytidine restored mRNA and protein expression of caspase-8 and TRAIL sensitivity of resistant cell lines, suggesting that gene methylation is involved in caspase inactivation. The TRAIL system seems to be functional in NB cells expressing caspase-8 and/or caspase-10. Because many cytotoxic drugs induce caspase-dependent apoptosis, failure to express caspase-8 and/or caspase-10 might be an important mechanism of resistance to chemotherapy in NB.     

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers apoptosis in normal prostate epithelial cells

TRAIL is a pro-apoptotic cytokine believed to selectively kill cancer cells without harming normal ones. However, we found that in normal human prostate epithelial cells (PrEC) TRAIL is capable of inducing apoptosis as efficiently as in some tumor cell lines. At the same time, TRAIL did not cause apoptosis in several other human primary cell lines: aorta smooth muscle cells, foreskin fibroblasts, and umbilical vein endothelial cells. Compared to these primary cells, PrEC were found to contain significantly fewer TRAIL receptors DcR1 and DcR2 which are not capable of conducting the apoptotic signal. This result suggests that the unusual sensitivity of PrEC to TRAIL may result from their deficiency in anti-apoptotic decoy receptors. The protein synthesis inhibitor cycloheximide significantly enhanced TRAIL toxicity toward PrEC as measured by tetrazolium conversion but had little or no effect on other TRAIL-induced apoptotic responses. Although cycloheximide did not further accelerate the processing of caspases 3 and 8, it significantly enhanced cleavage of the caspase 3 substrate gelsolin, indicating that in PrEC a protein(s) with a short half-life may inhibit the activity of the executioner caspases toward specific substrates. As the majority of prostate cancers are derived from epithelial cells, our data suggest the possibility that TRAIL could be a useful treatment for the early stages of prostate cancer.     

Radioresponsive tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene therapy for malignant brain tumors

Patients with malignant gliomas have a very poor prognosis. To explore a novel and more effective approach for the treatment of malignant gliomas, a strategy that combined tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene therapy and radiation treatment (RT) was designed in this study. Plasmid pE4-GFP was constructed by including the radioinducible early growth response gene 1 (Egr-1) promoter, and it yielded the best response with fractionated RT. Plasmid pE4-TRAIL was constructed by including the Egr-1 promoter and evaluated using U251 and U87 glioma cells. In the assay of apoptosis and killing activities, pE4-TRAIL exhibited radioresponse. pE4-TRAIL combined with RT is capable of inducing cell death synergistically. The expression of TRAIL death receptors was evaluated; which may be influenced by RT. Glioma cells with wild-type p53 showed upregulated expression of death receptors, and more synergistic effects on killing activities are expected. pE4-TRAIL was transfected into the subcutaneous U251 glioma cells in nude mice by the in vivo electroporation method. In the mice treated with pE4-TRAIL and RT, apoptotic cells were detected in pathological sections, and a significant difference of tumor volumes was observed when compared with the other groups (P<0.001). Our results indicate that radioresponsive gene therapy may have great potential as a novel therapy because this therapeutic system can be spatially or temporally controlled by exogenous RT and provides specificity and safety.     

Curcumin sensitizes tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis through reactive oxygen species-mediated upregulation of death receptor 5 (DR5)

Curcumin exhibits anti-inflammatory and antitumor activities.Although its functional mechanism has not been elucidated sofar, numerous studies have shown that curcumin induces apoptosisin cancer cells. In the present study, we show that subtoxicconcentrations of curcumin sensitize human renal cancer cellsto the tumor necrosis factor-related apoptosis inducing ligand(TRAIL)-mediated apoptosis. This apoptosis induced by the combinationof curcumin and TRAIL is not interrupted by Bcl-2 overexpression.We found that treatment with curcumin significantly inducesdeath receptor 5 (DR5) expression both at its mRNA and proteinlevels, accompanying the generation of the reactive oxygen species(ROS). Not only the pretreatment with N-acetylcystine but alsothe ectopic expression of peroxiredoxin II, an antioxidativeprotein, dramatically inhibited the apoptosis induced by curcuminand TRAIL in combination, blocking the curcumin-mediated DR5upregulation. Taken together, the present study demonstratesthat curcumin enhances TRAIL-induced apoptosis by ROS-mediatedDR5 upregulation.     

Epidermal growth factor protects epithelial-derived cells from tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis by inhibiting cytochrome c release.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in combination with chemotherapeutic drugs induces a synergistic apoptotic response in cancer cells. TRAIL death receptors have also been implicated in chemotherapeutic drug-induced apoptosis. This has lead to TRAIL being proposed as a potential cancer treatment. In nude mice injected with human tumors, TRAIL reduces the size of these tumors without toxic side effects. We demonstrate that the epidermal growth factor (EGF) stimulation of human embryonic kidney cells HEK 293 and breast cancer cell line MDA MB 231 effectively protects these cells from TRAIL-induced apoptosis in a dose-dependent manner. This stimulation blocks apoptosis by inhibiting TRAIL-mediated cytochrome c release from the mitochondria and caspase 3-like activation. EGF survival response involves the activation of AKT. Expression of activated AKT was sufficient to block TRAIL-induced apoptosis, and kinase-inactive AKT expression blocked EGF-protective response. In contrast, inhibition of EGF stimulation of extracellular-regulated kinase (ERK) activity did not affect EGF protection. These findings indicate that EGF receptor activation provides a survival response against TRAIL-induced apoptosis by inhibiting mitochondrial cytochrome c release that is mediated by AKT activation in epithelial-derived cells.     

Induction and regulation of tumor necrosis factor-related apoptosis-inducing ligand/Apo-2 ligand-mediated apoptosis in renal cell carcinoma

The lack of effective therapy for disseminated renal cell carcinoma (RCC) has stimulated the search for novel treatments including immunotherapeutic strategies. However, poor therapeutic responses and marked toxicity associated with immunological agents has limited their use. The tumor necrosis factor family member tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/Apo-2 ligand induces apoptosis in a variety of tumor cell types, while having little cytotoxic activity against normal cells. In this study the activation and regulation of TRAIL-induced apoptosis and TRAIL receptor expression in human RCC cell lines and pathologic specimens was examined. TRAIL induced caspase-mediated apoptotic death of RCC cells with variable sensitivities among the cell lines tested. Compared with TRAIL-sensitive RCC cell lines (A-498, ACHN, and 769-P), the TRAIL-resistant RCC cell line (786-O) expressed lesser amounts of the death-inducing TRAIL receptors, and greater amounts of survivin, an inhibitor of apoptosis. Incubation of 786-O with actinomycin D increased the expression of the death-inducing TRAIL receptors and, concomitantly, decreased the intracellular levels of survivin, resulting in TRAIL-induced apoptotic death. The link between survivin and TRAIL regulation was confirmed when an increase in TRAIL resistance was observed after overexpression of survivin in the TRAIL-sensitive, survivin-negative RCC line A-498. These findings, along with our observation that TRAIL receptors are expressed in RCC tumor tissue, suggest that TRAIL may be useful as a therapeutic agent for RCC and that survivin may partially regulate TRAIL-induced cell death.     

Antitumor activity and bystander effects of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) gene

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has been reported to specifically kill malignant cells but to be relatively nontoxic to normal cells. To evaluate the antitumor activity and therapeutic value of the TRAIL gene, we constructed adenoviral vectors expressing the human TRAIL gene and transferred them into malignant cells in vitro and tumors in vivo. The in vitro transfer elicited apoptosis, as demonstrated by the quantification of viable or apoptotic cells and by the analysis of activation of pro-caspase-8 and cleavage of poly(ADP-ribose) polymerase. The intratumoral delivery elicited tumor cell apoptosis and suppressed tumor growth. In comparison with Bax gene treatment, which is toxic to normal cells, TRAIL gene treatment caused no detectable toxicity in cultured normal fibroblasts nor in mouse hepatocytes after systemic gene delivery. Furthermore, coculture of cancer cells expressing TRAIL with those expressing green fluorescent protein (GFP) resulted in apoptosis of both cells, whereas coculture of Bax-expressing cells with GFP-expressing cells resulted in the cell death of the Bax-expressing cells only, which suggested that the transfer of the TRAIL gene resulted in bystander effects. Moreover, culture of cells with medium from TRAIL-expressing cells showed the proapoptotic activity and bystander effect of the TRAIL gene to be not transferable with medium. To further demonstrate the bystander effect of the TRAIL gene, we constructed plasmid vectors encoding GFP-TRAIL or GFP-Bik chimeric proteins. Transfection of the GFP-TRAIL gene into cancer cells resulted in the death of GFP-positive cells and their neighbors, whereas transfection of the GFP-Bik gene killed GFP-positive cells only. Finally, GFP-TRAIL genes, transfected into normal human fibroblasts or bronchial epithelial cells, did not kill such cells, whereas transfected GFP-Bik genes did. Thus, the direct transfer of the TRAIL gene led to selective killing of malignant cells with bystander effect, which suggests that the TRAIL gene could be valuable for treatment for cancers. Together, these results suggest that delivering the TRAIL gene to cancerous cells may be an alternative approach to cancer treatment.     

Induction and intracellular regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mediated apotosis in human malignant glioma cells

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) preferentially triggers apoptosis in tumor cells versus normal cells, thus providing a therapeutic potential. In this study, we examined a large panel of human malignant glioma cell lines and primary cultures of normal human astrocytes for their sensitivity to TRAIL. Of 13 glioma cell lines, 3 were sensitive (80-100% death), 4 were partially resistant (30-79% death), and 6 were resistant (< 30% death). Normal astrocytes were also resistant. TRAIL-induced cell death was characterized by activation of caspase-8 and -3, poly(ADP-ribose) polymerase cleavage, and DNA fragmentation. Decoy receptor (DcR1 and DcR2) expression was limited in the glioma cell lines and did not correlate with TRAIL sensitivity. Both sensitive and resistant cell lines expressed TRAIL death receptor (DR5), adapter protein Fas-associated death domain (FADD), and caspase-8; but resistant cell lines expressed 2-fold higher levels of the apoptosis inhibitor phosphoprotein enriched in diabetes/phosphoprotein enriched in astrocytes-15 kDa (PED/PEA-15). In contrast, cellular FADD-like IL-1beta-converting enzyme-like inhibitory protein (cFLIP) expression was similar in sensitive and resistant cells. Transfection of sense PED/PEA-15 cDNA in sensitive cells resulted in cell resistance, whereas transfection of antisense in resistant cells rendered them sensitive. Inhibition of protein kinase C (PKC) activity restored TRAIL sensitivity in resistant cells, suggesting that PED/ PEA-15 function might be dependent on PKC-mediated phosphorylation. In summary, TRAIL induces apoptosis in > 50% of glioma cell lines, and this killing occurs through activation of the DR pathway. This caspase-8-induced apoptotic cascade is regulated by intracellular PED/PEA-15, but not by cFLIP or decoy receptors. This pathway may be exploitable for glioma and possibly for other cancer therapies.     

Sensitization of human glioblastomas to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) by NF-kappaB inhibitors

Glioblastoma is the most malignant form of primary brain tumor in adults, with no effective therapy and a low survival rate. TRAIL is a member of the TNF family, which selectively induces apoptosis in certain neoplastic cells, but not normal cells. In this study, we investigated the sensitivity of 7 human glioblastoma cell lines to TRAIL and the expression in them of TRAIL receptors. TRAIL exhibited significant cytotoxicity in 5 of 7 glioma cell lines. These glioblastoma cell lines expressed TRAIL-R2, but not TRAIL-R1, R3, or R4. However, no correlation was observed between the TRAIL sensitivity and the TRAIL-R2 expression level, suggesting that there is an additional determinant of TRAIL sensitivity. Treatments with NF-kappaB inhibitors, such as LLnL, MG132, and SN50, significantly increased the sensitivity of glioma cells to TRAIL. These results suggested that activation of NF-kappaB is a protective mechanism against TRAIL-induced cell death in some glioma cells, and thus NF-kappaB inhibitors may be useful to improve the clinical treatment of glioblastoma with TRAIL.     

Osteoprotegerin (OPG) binds with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL): suppression of TRAIL-induced apoptosis in ameloblastomas

Osteoprotegerin (OPG) is a useful receptor in inhibiting Receptor Activator of NFkappaB Ligand (RANKL) in inducing osteoclastogenesis. Tumor Necrosis Factor (TNF)-Related Apoptosis-Inducing Ligand (TRAIL) is a potent apoptosis-inducing ligand in ameloblastomas. Since OPG has been reported to bind to TRAIL as well, the effect of OPG in TRAIL's function in inducing apoptosis should also be investigated. To investigate on the expression of OPG in ameloblastomas, immuhistochemistry, immunofluorescence and Western blot were performed. From the immunohistochemistry results, we found that OPG was expressed in ameloblastoma tissues. Expression of OPG was clearly seen in AM-1 cells by immunofluorescence as well. Additionally, Western blot analysis confirmed OPG expression in ameloblastoma tissues and AM-1 cells. To investigate on the potential of TNFalpha, TRAIL and RANKL in inducing apoptosis, we performed an apoptosis assay. From the apoptosis assay, we found that TRAIL had the highest potential in inducing apoptosis compared to TNFalpha and RANKL. To investigate the binding of OPG with RANKL and TRAIL, we performed a binding assay. We noticed that OPG preferably bind with RANKL than TRAIL. However, at low levels of RANKL, marked binding of OPG with TRAIL was seen. As we suspected that the binding of OPG and TRAIL might cause the effect of TRAIL in inducing apoptosis in ameloblastomas, we combined the treatment of OPG and TRAIL in AM-1 cells. From the apoptosis assay, we found that under treatment of OPG, TRAIL's function in inducing apoptosis was suppressed. These data suggest that by binding with TRAIL, OPG suppressed TRAIL's function in inducing apoptosis in ameloblastomas.     

Tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis of human melanoma is regulated by smac/DIABLO release from mitochondria

In previous studies we have shown that the sensitivity of melanoma cell lines to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis was determined largely by the level of expression of death receptor TRAIL receptor 2 on the cells. However, approximately one-third of melanoma cell lines were resistant to TRAIL, despite expression of high levels of TRAIL receptor 2. The present studies show that these cell lines had similar levels of TRAIL-induced activated caspase-3 as the TRAIL-sensitive lines, but the activated caspase-3 did not degrade substrates downstream of caspase-3 [inhibitor of caspase-activated DNase and poly(ADP-ribose) polymerase]. This appeared to be due to inhibition of caspase-3 by X-linked inhibitor of apoptosis (XIAP) because XIAP was bound to activated caspase-3, and transfection of XIAP into TRAIL-sensitive cell lines resulted in similar inhibition of TRAIL-induced apoptosis. Conversely, reduction of XIAP levels by overexpression of Smac/DIABLO in the TRAIL-resistant melanoma cells was associated with the appearance of catalytic activity by caspase-3 and increased TRAIL-induced apoptosis. TRAIL was shown to cause release of Smac/DIABLO from mitochondria, but this release was greater in TRAIL-sensitive cell lines than in TRAIL-resistant cell lines and was associated with down-regulation of XIAP levels. Furthermore, inhibition of Smac/DIABLO release by overexpression of Bcl-2 inhibited down-regulation of XIAP levels. These results suggest that Smac/DIABLO release from mitochondria and its binding to XIAP are an alternative pathway by which TRAIL induces apoptosis of melanoma, and this pathway is dependent on the release of activated caspase-3 from inhibition by XIAP and possibly other inhibitor of apoptosis family members.     

Role of antiapoptotic proteins in tumor necrosis factor-related apoptosis-inducing ligand and cisplatin-augmented apoptosis.

PURPOSE AND EXPERIMENTAL DESIGN: The purpose of this study was to examine the effect of combined treatment with tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and cisplatin in human head and neck squamous cell carcinoma. HNSCC-6 cells were treated with 0.1-1 micro g/ml TRAIL and/or 1-10 micro g/ml cisplatin for 24 h. RESULTS: TRAIL alone or cisplatin alone caused minimal cytotoxicity. The combination of TRAIL and cisplatin synergistically enhanced apoptotic death, caspase-8 and caspase-3 activation, as well as poly(ADP-ribose) polymerase cleavage. However, the total cellular levels and the surface expression of TRAIL receptor proteins, such as death receptors 4 and 5 and decoy receptors 2 and 1, were not significantly changed by treatment with TRAIL and cisplatin. Interestingly, the level of the short form of Fas-associated death domain-like interleukin-1beta-converting enzyme-inhibitory protein (FLIP(S)) but not the long form of Fas-associated death domain-like interleukin-1beta-converting enzyme-inhibitory protein was reduced through cleavage. Benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone a caspase-3 inhibitor, blocked the cleavage of FLIP(S) and caspase-3 activation. Overexpression of FLIP(S) protected cells from apoptotic death and FLIP(S) cleavage during treatment with TRAIL in combination with cisplatin. CONCLUSIONS: These results suggest that caspase-3 is responsible for FLIP(S) cleavage, and the cleavage of FLIP(S) is one of facilitating factors for TRAIL-induced apoptotic death.     

Combination of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and actinomycin D induces apoptosis even in TRAIL-resistant human pancreatic cancer cells.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a novel member of the tumor necrosis factor superfamily of cytokines that induces cell death by apoptosis. TRAIL has been shown to be effective in almost two-thirds of solid tumors tested thus far, but its effect on pancreatic cancer cells is unknown. We tested the effect of TRAIL on seven human pancreatic cancer cell lines (HPAF, Panc1, Miapaca2, Bxpc3, Panc89, SW979, and Aspc1) in vitro. Of these cell lines, all but Aspc1 showed a significant dose-dependent increase in apoptosis. The apoptotic rate, as detected by a terminal deoxynucleotidyl transferase-mediated nick end labeling assay, was highest in Bxpc3 (71.5%), followed by HPAF (38.0%), Miapaca2 (24.9%), Panc1 (16.1%), Panc89 (15.8%), SW979 (13.9%), and Aspc1 (5.2%). Multiple treatments were more effective than a single treatment and caused a sustained and profound cell death in all but Aspc1 cells. There was no correlation between the effect of TRAIL and the differentiation grade of the cell lines, p53 mutation, or bcl-2 or bax expression. The resistance of Aspc1 cells to TRAIL was not related to the lack of TRAIL receptors. The combination of actinomycin D and TRAIL induced an almost complete lysis of Aspc1 cells, whereas actinomycin D alone had no effect on cell survival but inhibited the expression of the Flice inhibitory protein, which is assumed to play a role in the apoptotic pathway of TRAIL. Thus, the combination of actinomycin D and TRAIL appears to be a promising approach for the therapy of pancreatic cancers resistant to TRAIL.