CCNU-dependent potentiation of TRAIL/Apo2L-induced apoptosis in human glioma cells is p53-independent but may involve enhanced cytochrome c release.

Death ligands such as CD95 ligand (CD95L) or tumor necrosis factor-related apoptosis-inducing ligand/Apo2 ligand (TRAIL/Apo2L) induce apoptosis in radiochemotherapy-resistant human malignant glioma cell lines. The death-signaling TRAIL receptors 2 (TRAIL-R2/death receptor (DR) 5) and TRAIL-R1/DR4 were expressed more abundantly than the non-death-inducing (decoy) receptors TRAIL-R3/DcR1 and TRAIL-R4/DcR2 in 12 human glioma cell lines. Four of the 12 cell lines were TRAIL/Apo2L-sensitive in the absence of a protein synthesis inhibitor, cycloheximide (CHX). Three of the 12 cell lines were still TRAIL/Apo2L-resistant in the presence of CHX. TRAIL-R2 expression predicted sensitivity to apoptosis. Coexposure to TRAIL/Apo2L and cytotoxic drugs such as topotecan, lomustine (1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, CCNU) or temozolomide resulted in synergistic killing. Synergistic killing was more often observed in cell lines retaining wild-type p53 activity (U87MG, LN-229) than in p53 mutant cell lines (LN-18, T98G, U373MG). Drug exposure resulted in enhanced TRAIL-R2 expression, but decreased TRAIL-R4 expression in U87MG cells. Ectopic expression of dominant-negative p53(V135A) abrogated the drug-induced changes in TRAIL-R2 and TRAIL-R4 expression, but had no effect on synergy. Thus, neither wild-type p53 function nor changes in TRAIL receptor expression were required for synergy. In contrast, synergy resulted possibly from drug-induced cytochrome c release from mitochondria, serving as an amplifier of the TRAIL/Apo2L-mediated cascade of caspase activation. These data provide novel insights into the role of the TRAIL/Apo2L system in malignant gliomas and illustrate that TRAIL/Apo2L-based immunochemotherapy may be an effective therapeutic strategy for these lethal neoplasms.     

Sensitization of multidrug resistant human ostesarcoma cells to Apo2 Ligand/TRAIL-induced apoptosis by inhibition of the Akt/PKB kinase

Chemotherapeutic agents have been used for the treatment of patients with osteosarcoma (OS). However, inherent or acquired resistance to these agents is a serious problem in the management of OS patients. The emergence of the multidrug resistance (MDR) phenotype in cancer cells is often associated with the overexpression of P-glycoprotein, encoded by the multidrug resistance gene MDR-1. The administration of some of the most common chemotherapeutic agents to these cells becomes ineffective because of their P-gp-driven efflux from the cell. Apo2L/TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines that is considered to induce death of cancer cells but not normal cells. Its powerful apoptotic activity is mediated through its cell surface death domain-containing receptors, TRAIL-R1/DR4 and TRAIL-R2/DR5, which in turn spread the signal in the cytosol through the activation of the caspase cascade. The Akt/PKB kinase is an important cell survival protein which is regulated by D3-phosphoinositides. High Akt expression and activity levels are well documented in many types of tumors, which very often show an altered PI3-K/Akt/PTEN pathway. In this study the U2OS human osteosarcoma cell line and its multidrug resistant (MDR) subline that overexpresses MDR-1 gene, MDR-U2OS, have been analyzed for their responsiveness to TRAIL. In conflict with the presence of active DR4 and DR5 receptors in both clones, U2OS cells exhibited only a low responsiveness to TRAIL, while the MDR-U2OS subline did exhibit a marked TRAIL sensitivity. An analysis of the post-receptor events showed that TRAIL responsiveness correlates with a reduced expression of endogenous Akt. In fact, expression in MDR-U2OS cells of a constitutively active Akt strongly decreased their sensitivity to TRAIL. The identification of Akt as a key modulator of TRAIL responsiveness could help to design TRAIL-based combinations for treatment of osteosarcoma. Moreover, the discovery that multidrug resistant osteosarcomas are highly sensitive to TRAIL-induced apoptosis indicates TRAIL as a new candidate for the treatment of multidrug resistant bone malignancies.     

TRAIL, caspases and maturation of normal and leukemic myeloid precursors

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/Apo2L) is a membrane-bound cytokine molecule that belongs to the family of tumor necrosis factor (TNF). Members of this family share diverse biological effects, including induction of apoptosis and/or promotion of cell survival. Identification of TRAIL has generated considerable enthusiasm for its ability to induce apoptotic cell death in a variety of tumor cells, by engaging the death receptors TRAIL-R1/DR4 and TRAIL-R2/DR5, while sparing most normal cells. Beside its anticancer activity, several studies have suggested a role for endogenously expressed TRAIL in hemopoiesis. In this review, we summarize the knowledge about the different lineage-specific roles of TRAIL and its receptors in hemopoiesis regulation. Moreover, the complex interplay among the signaling pathways triggered by TRAIL/TRAIL-receptors in myeloid cells is discussed in some detail.     

Direct stimulation of apoptotic signaling by soluble Apo2l/tumor necrosis factor-related apoptosis-inducing ligand leads to selective killing of glioma cells.

Apo2 ligand tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) is a member of the tumor necrosis factor family that interacts with cell surface "death receptors" (DR4 and DR5) to initiate programmed cell death. Apo2L/TRAIL also binds to "decoy" receptors (DcR1 and DcR2) that can antagonize its interaction with DR4 and DR5. In recent studies, Apo2L/TRAIL has been noted to produce selective toxicity toward certain neoplastic cells versus normal cells. The decoy receptors may in part contribute to this selectivity, because they are expressed in various normal tissues but are present at low or undetectable levels in certain types of neoplastic cells. In the current study, we examined the potential therapeutic applicability of recombinant soluble Apo2L/TRAIL by investigating its effects in vitro and in vivo against a series of cell lines derived from malignant gliomas, which are often resistant to conventional treatment modalities. In cell proliferation assays, Apo2L/TRAIL produced a striking decrease in cell numbers, with a median inhibitory concentration of 30-100 ng/ml, in the TP53 wild-type high-grade glioma cell lines U87 and A172, the TP53-mutated T98G, and the TP53-deleted LN-Z308. In contrast, no significant effects were observed in non-neoplastic astrocytes at concentrations up to 3000 ng/ml. Clonogenic assays showed that exposure to Apo2L produced a time-dependent decrease in the viability of glioma-derived cell lines. This correlated with the induction of apoptosis as assessed by a terminal transferase-catalyzed in situ end-labeling assay. Pretreatment of the cells with the caspase inhibitors Acetyl-Asp-Glu-Val-L-aspartic acid aldehyde or Acetyl-Tyr-Val-Ala-Asp-chlormethylketone (200 microM) largely eliminated the effects of Apo2L/TRAIL. Administration of Apo2L/TRAIL (0.3, 1, 3, 10, and 30 mg/kg/day for 7 days via i.p. infusion) to nude mice harboring established intracranial U87 xenografts produced a significant, dose-dependent prolongation of survival versus control animals. Survival in the control group was 27 +/- 1.7 days, compared with more than 50 days in each of the treatment groups (P < 0.001). At the 30 mg/kg dose level, 100% of animals survived for 120 days without evidence of tumor, a substantial improvement in comparison with lower dose levels (P < 0.01). No overt toxicity was apparent even at the highest Apo2L dose. We conclude that soluble Apo2L/TRAIL is effective in inducing apoptosis in high-grade glioma cells in vitro. Because this ligand appears to exhibit selective cytotoxicity for glioma cells versus non-neoplastic cells in vitro and demonstrates significant activity in vivo when administered systemically in an otherwise uniformly fatal central nervous system glioma model system, Apo2L may constitute a useful therapeutic agent for these challenging tumors.     

TRAIL death receptor-4 expression positively correlates with the tumor grade in breast cancer patients with invasive ductal carcinoma

PURPOSE: Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) selectively induces apoptosis in cancer cells but not in normal cells, and a number of clinical trials have recently been initiated to test the safety and antitumoral potential of TRAIL in cancer patients. Four different receptors have been identified to interact with TRAIL: two are death-inducing receptors (TRAIL-R1 [DR4] and TRAIL-R2 [DR5]), whereas the other two (TRAIL-R3 [DcR1] and TRAIL-R4 [DcR2]) do not induce death upon ligation and are believed to counteract TRAIL-induced cytotoxicity. Because high levels of DcR2 expression have recently been correlated with carcinogenesis in the prostate and lung, this study investigated the importance of TRAIL and TRAIL receptor expression in breast cancer patients with invasive ductal carcinoma, taking various prognostic markers into consideration. METHODS AND MATERIALS: Immunohistochemical analyses were performed on 90 breast cancer patients with invasive ductal carcinoma using TRAIL and TRAIL receptor-specific antibodies. Age, menopausal status, tumor size, lymph node status, tumor grade, lymphovascular invasion, perineural invasion, extracapsular tumor extension, presence of an extensive intraductal component, multicentricity, estrogen and progesterone receptor status, and CerbB2 expression levels were analyzed with respect to TRAIL/TRAIL receptor expression patterns. RESULTS: The highest TRAIL receptor expressed in patients with invasive ductal carcinoma was DR4. Although progesterone receptor-positive patients exhibited lower DR5 expression, CerbB2-positive tissues displayed higher levels of both DR5 and TRAIL expressions. CONCLUSIONS: DR4 expression positively correlates with the tumor grade in breast cancer patients with invasive ductal carcinoma.     

Pharmacological inhibition of Bcl-2 family members reactivates TRAIL-induced apoptosis in malignant glioma

The major aim of this study was to develop novel therapeutic approaches to potentiate and reactivate apoptosis induced by TNF-Related Apoptosis Inducing Ligand (TRAIL) in malignant glioma. Analysis of five glioma cell lines (U87, U251, U373, MZ-54 and MZ-18) indicated that only two of the cell lines were sensitive to apoptosis induced by TRAIL alone. TRAIL resistance was not correlated to expression levels of the death receptors DR4 and DR5 or the decoy receptors DcR1 and DcR2, suggesting that it was mediated by inactivation of TRAIL-induced downstream signalling. Activation of the BH3 only protein Bid and subsequent activation of the mitochondrial apoptosis pathway are known to play a pivotal role in TRAIL-induced apoptosis. Since this process is blocked by overexpression of anti-apoptotic Bcl-2 family members, we analyzed the therapeutic potential of BH3 mimetics in potentiating TRAIL-induced apoptosis. Treatment with TRAIL in combination with the specific Bcl-2 inhibitor HA14-1 and the Bcl-2/Bcl-xL inhibitor BH3I-2′ potently enhanced apoptosis in TRAIL-sensitive U87 cells in a dose-dependent fashion. TRAIL-induced apoptosis was significantly reactivated by HA14-1 and BH3I-2′ in one (U343) and two (MZ-54 and MZ-18) of three investigated TRAIL-insensitive cell lines, respectively. Knockdown of the anti-apoptotic Bcl-2 family member Mcl-1 by RNA interference had no additional effect on apoptosis induced by TRAIL and HA14-1 in U87 and U343 cells. Our data indicate that Bcl-2 and Bcl-xL play fundamental roles in TRAIL resistance of malignant glioma and suggest that using TRAIL or agonistic TRAIL receptor antibodies in combination with BH3 mimetics may represent a promising approach to reactivate apoptosis in therapy-resistant high grade gliomas.     

Predominant antitumor effects by fully human anti-TRAIL-receptor2 (DR5) monoclonal antibodies in human glioma cells in vitro and in vivo

Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL/Apo2 L) preferentially induces apoptosis in human tumor cells through its cognate death receptors DR4 or DR5, thereby being investigated as a potential agent for cancer therapy. Here, we applied fully human anti-human TRAIL receptor monoclonal antibodies (mAbs) to specifically target one of death receptors for TRAIL in human glioma cells, which could also reduce potential TRAIL-induced toxicity in humans. Twelve human glioma cell lines treated with several fully human anti-human TRAIL receptor mAbs were sensitive to only anti-DR5 mAbs, whereas they were totally insensitive to anti-DR4 mAb. Treatment with anti-DR5 mAbs exerted rapid cytotoxicity and lead to apoptosis induction. The cellular sensitivity was closely associated with cell-surface expression of DR5. Expression of c-FLIP_L, Akt, and Cyclin D1 significantly correlated with sensitivity to anti-DR5 mAbs. Primary cultures of glioma cells were also relatively resistant to anti-DR5 mAbs, exhibiting both lower DR5 and higher c-FLIP_L expression. Downregulation of c-FLIP_L expression resulted in the sensitization of human glioma cells to anti-DR5 mAbs, whereas overexpression of c-FLIP_L conferred resistance to anti-DR5 mAb. Treatment of tumor-burden nude mice with the direct agonist anti-DR5 mAb KMTR2 significantly suppressed growth of subcutaneous glioma xenografts leading to complete regression. Similarly, treatment of nude mice bearing intracerebral glioma xenografts with KMTR2 significantly elongated lifespan without tumor recurrence. These results suggest that DR5 is the predominant TRAIL receptor mediating apoptotic signals in human glioma cells, and sensitivity to anti-DR5 mAbs was determined at least in part by the expression level of c-FLIP_L and Akt. Specific targeting of death receptor pathway through DR5 using fully human mAbs might provide a novel therapeutic strategy for intractable malignant gliomas.     

A mechanism of resistance to TRAIL/Apo2L-induced apoptosis of newly established glioma cell line and sensitisation to TRAIL by genotoxic agents

Most tumour cells are sensitive to TRAIL-induced apoptosis, but not normal cells; thus, cancer therapy using TRAIL is expected clinically. Several tumour cells are resistant to TRAIL-induced apoptosis, and various mechanisms of such resistance were reported in individual cases. In this study, we established a TRAIL-resistant glioma cell line, which completely lacked TRAIL receptors. In addition, this tumour cell line had wild-type p53 tumour-suppressive gene, suggesting new mechanisms for tumour cells to expand and escape from immune surveillance. The present study further explored the mechanisms that determine the sensitivity to TRAIL. We show that genotoxic agents such as cisplatin, doxorubicin and camptothecin, in addition to UV radiation, can induce TRAIL-R2 on the cell surface of TRAIL receptor-negative tumour cells. Newly synthesised TRAIL-R2 is functional, so apoptosis is effectively induced by TRAIL, but it is significantly inhibited by constitutive expression of dominant-negative p53. In addition, apoptosis induced by pretreatment of genotoxic agents and additional stimulation of TRAIL is efficiently inhibited by either antagonistic anti-TRAIL-R2 antibody or pan-caspase inhibitor z-VAD-FMK. Taken together, these findings suggest that resistance to TRAIL by lack of TRAIL receptors on glioma is restored by genotoxic agents, which support the new strategies for tumour killing by TRAIL-bearing cytotoxic cells in combination with genotoxic treatment.     

Relation of TNF-related apoptosis-inducing ligand (TRAIL) receptor and FLICE-inhibitory protein expression to TRAIL-induced apoptosis of melanoma.

Past studies have shown that apoptosis mediated by TNF-related apoptosis-inducing ligand (TRAIL) is regulated by the expression of two death receptors [TRAIL receptor 1 (TRAIL-R1) and TRAIL-R2] and two decoy receptors (TRAIL-R3 and TRAIL-R4) that inhibit apoptosis. In previous studies, we have shown that TRAIL but not other members of the tumor necrosis factor family induce apoptosis in approximately two-thirds of melanoma cell lines. Here, we examined whether the expression of TRAIL-R at the mRNA and protein level in a panel of 28 melanoma cell lines and melanocytes correlated with their sensitivity to TRAIL-induced apoptosis. We report that at least three factors appear to underlie the variability in TRAIL-induced apoptosis. (a) Four of nine cell lines that were insensitive to TRAIL-induced apoptosis failed to express death receptors, and in two instances, lines were devoid of all TRAIL-Rs. Southern analysis suggested this was due to loss of the genes for the death receptors. (b) Despite the presence of mRNA for the TRAIL-R, some of the lines failed to express TRAIL-R protein on their surface. This was evident for TRAIL-R1 and more so for the TRAIL decoy receptors TRAIL-R3 and -R4. Studies on permeabilized cells revealed that the receptors were located within the cytoplasm and redistribution from the cytoplasm may represent a posttranslational control mechanism. (c) Surface expression of TRAIL-R1 and -R2 (but not TRAIL-R3 and -R4) showed an overall correlation with TRAIL-induced apoptosis. However, certain melanoma cell lines and clones were relatively resistant to TRAIL-induced apoptosis despite the absence of decoy receptors and moderate levels of TRAIL-R1 and -R2 expression. This may indicate the presence of inhibitors within the cells, but resistance to apoptosis could not be correlated with expression of the caspase inhibitor FLICE-inhibitory protein. mRNA for another TRAIL receptor, osteoprotegerin, was expressed in 22 of the melanoma lines but not on melanocytes. Its role in induction of apoptosis remains to be studied. These results appear to have important implications for future clinical studies on TRAIL.     

Anticancer agents sensitize tumor cells to tumor necrosis factor-related apoptosis-inducing ligand-mediated caspase-8 activation and apoptosis

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a new cytokine that was proposed to specifically induce apoptosis of cancer cells. In tumor cells that are resistant to the cytokine, subtoxic concentrations of chemotherapeutic drugs can restore the response to TRAIL. The present study further explores the mechanisms that determine tumor cell sensitivity to TRAIL by comparing four human colon carcinoma cell lines We show that colon cancer cell sensitivity to TRAIL-induced apoptosis and cytotoxicity correlates with the expression of the death receptors TRAIL-R1 and TRAIL-R2 at the cell surface, as determined by now cytometry, whereas the two decoy receptors TRAIL-R3 and TRAIL-R4 can be detected only in permeabilized cells. Clinically relevant concentrations of cisplatin and doxorubicin sensitize the most resistant colon cancer cell lines to TRAIL-induced cell death without modifying the expression nor the localization of TRAIL receptors in these cells. TRAIL induces the activation of procaspase-8 and triggers caspase-dependent apoptosis off colon cancer cells. Cytotoxic drugs lower the signaling threshold required for TRAIL-induced procaspase-8 activation. In turn, caspase-8 cleaves Bid, a BH3 domain-containing proapoptotic molecule of the Bcl-2 family and activates effector caspases. Together, these data indicate that chemotherapeutic drugs sensitize colon tumor cells to TRAIL-mediated caspase-8 activation and apoptosis.     

The topoisomerase I inhibitor topotecan increases the sensitivity of prostate tumor cells to TRAIL/Apo-2L-induced apoptosis

PURPOSE.:TRAIL/Apo-2L is cytotoxic against numerous prostate tumor cell lines; however, some lines are more resistant than others. Identification of an agent that increases prostate tumor cell sensitivity to TRAIL/Apo-2L would prove valuable for TRAIL/Apo-2L-mediated tumor therapy. Thus, we examined the effect of combining five clinically approved chemotherapeutic agents with TRAIL/Apo-2L for treating prostate tumor cells. METHODS: Four human prostate tumor cell lines were initially tested for TRAIL/Apo-2L sensitivity. Subsequent studies examined whether the TRAIL/Apo-2L-induced killing of DU-145 cells was augmented in the presence of the chemotherapeutic molecules, as measured by annexin V-FITC/propidium iodide staining. Furthermore, caspase 8 activation and BID cleavage were examined by immunoblotting. RT-PCR and flow cytometry were performed to monitor TRAIL-R1 and TRAIL-R2 levels after chemotherapeutic treatment. RESULTS: DU-145 cells were the least responsive of the prostate tumor cell lines tested to TRAIL/Apo-2L-induced death. Surprisingly, only topotecan, a topoisomerase I inhibitor, when used in combination with rTRAIL/Apo-2L led to significant apoptosis of DU-145 cells, as measured by caspase 8 activation, BID cleavage, and annexin V-FITC/PI staining. Topotecan alone had little to no toxicity on the DU-145 cells. Furthermore, the increase in TRAIL/Apo-2L sensitivity following topotecan treatment correlated with increased expression of TRAIL-R1 and TRAIL-R2 and decreased intracellular levels of the antiapoptotic protein survivin. CONCLUSIONS: Our results define a promising direction for alternative therapies against androgen-independent prostate cancers. The sensitivity of DU-145 cells to TRAIL/Apo-2L was dramatically increased when combined with topotecan, suggesting that low-dose topotecan treatment to upregulate TRAIL-R1 and TRAIL-R2 and downregulate survivin, followed by TRAIL/Apo-2L administration, may be a viable therapy for treating cancer of the prostate.     

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.     

TRAIL is a key target in S-phase slowing-dependent apoptosis induced by interferon-beta in cervical carcinoma cells

Interferon (IFN)-beta induces S-phase slowing and apoptosis in human papilloma virus (HPV)-positive cervical carcinoma cell line ME-180. Here, we show that apoptosis is a consequence of the S-phase lengthening imposed by IFN-beta, demonstrating the functional correlation between S-phase alteration and apoptosis induction. In ME-180 cells, where p53 function is inhibited by HPV E6 oncoprotein, IFN-beta effects on cell cycle and apoptosis occur independently of p53. The apoptosis due to IFN-beta is mediated by the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in a manner dependent on the S-phase deregulation. IFN-beta appears to increase TRAIL expression both directly at the mRNA level and indirectly by augmenting surface protein levels as a consequence of the induced S-phase cell accumulation. Moreover, the alteration of the S-phase due to IFN-beta promotes TRAIL-dependent apoptosis by potentiating cell sensitivity to TRAIL, possibly through induction of a proapoptotic NF-kappaB activity and TRAIL-R2 receptor expression. Interestingly, IFN-beta-induced TRAIL-dependent apoptotic events strongly differ in the requirement of caspase activity. These results show that IFN-beta may induce an apoptotic response by deregulating cell cycle. Understanding the linkage between these mechanisms appears to be of primary importance in the search for new IFN-based therapeutic strategies to circumvent cancer disease or improve clinical outcome.     

TRAIL receptor-selective mutants signal to apoptosis via TRAIL-R1 in primary lymphoid malignancies

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its agonistic antibodies, which are currently in early clinical trials for treating various malignancies, induce apoptosis through triggering of either TRAIL-R1 or TRAIL-R2. Based on studies using agonistic monoclonal antibodies, we recently proposed that primary chronic lymphocytic leukemic cells seem to signal apoptosis primarily through TRAIL-R1. We have now synthesized mutant forms of TRAIL specific for TRAIL-R1 or TRAIL-R2. The selectivity of these mutants to induce apoptosis in cell lines is due to selective binding to their cognate receptors resulting in apoptosis via formation of a death-inducing signaling complex. Using these mutants, we now unequivocally show that primary cells from patients with chronic lymphocytic leukemia and mantle cell lymphoma signal to apoptosis almost exclusively through TRAIL-R1. Thus, no significant therapeutic benefit can be anticipated from treating such patients with agents currently in clinical trials that signal predominantly through TRAIL-R2, such as HGS-ETR2 or Apo2L/TRAIL. Our study highlights the necessity to determine whether primary cells from a particular tumor signal via TRAIL-R1 or TRAIL-R2. Such information will provide a rational approach to optimize TRAIL therapy.     

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.