Reversal of methylation silencing of Apo2L/TRAIL receptor 1 (DR4) expression overcomes resistance of SK-MEL-3 and SK-MEL-28 melanoma cells to interferons (IFNs) or Apo2L/TRAIL

Human melanoma cell lines, SK-MEL-3 and SK-MEL-28, despite induction of the proapoptotic cytokine, Apo2L/TRAIL, did not undergo apoptosis in response to interferons (IFN-alpha2b or IFN-beta). Postulating that genes important for apoptosis induction by IFNs might be silenced by methylation, the DNA demethylating agent 5-aza-2'-deoxycytidine (5-AZAdC) was assessed. DR4 (TRAIL-R1) was identified as one of the genes reactivated by 5-AZAdC with a >3-fold increase in 8 of 10 melanoma cell lines. Pretreatment with 5-AZAdC sensitized SK-MEL-3 and SK-MEL-28 cells to apoptosis induced by IFN-alpha2b and IFN-beta; methylation-specific PCR and bisulfite sequencing confirmed demethylation of 5'CpG islands of DR4 and flow cytometry showed an increase in DR4 protein on the cell surface. In cells with reactivated DR4, neutralizing mAB to TRAIL reduced apoptosis in response to IFN-beta or Apo2L/TRAIL. To further confirm the role of DR4, it was expressed by retroviral vector in SK-MEL-3 and SK-MEL-28 cells with reversal of resistance to IFN-beta and Apo2L/TRAIL. Thus, reexpressing DR4 by 5-AZAdC or retroviral transfection in melanoma cell in which promoter methylation had suppressed its expression, potentiated apoptosis by IFN-alpha2b, IFN-beta and Apo2L/TRAIL. Reactivation of silenced proapoptotic genes by inhibitors of DNA methylation may enhance clinical response to IFNs or Apo2L/TRAIL.     

Interferon (IFN)-beta induces apoptotic cell death in DHL-4 diffuse large B cell lymphoma cells through tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)

The effect of interferon (IFN)-alpha, beta and gamma on the growth of DHL-4 diffuse large B cell lymphoma cells was studied. IFN-beta significantly inhibited the cell growth, and the effect was stronger than that of IFN-alpha. IFN-gamma did not inhibit the cell growth because of lack of IFN-gamma receptors. IFN-beta caused apoptotic cell death which was accompanied by DNA fragmentation, caspase 3 activation and annexin V binding. IFN-beta lead to the expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) mRNA. Anti-TRAIL antibody significantly prevented IFN-beta-induced apoptosis. It was suggested that IFN-beta might cause apoptosis in DHL-4 cells through TRAIL.     

Role of Apo2L/TRAIL and Bcl-2-family proteins in apoptosis of multiple myeloma

Apo2 Ligand or Tumour Necrosis Factor (TNF)-Related Apoptosis-Inducing Ligand (Apo2L/TRAIL) is a member of the TNF gene superfamily that selectively induces apoptosis in tumor cells of diverse origins through engagement of death receptors. We have recently demonstrated that Type I interferons (IFN-alpha and beta) induce apoptosis in multiple myeloma (MM) cell lines and in plasma cells from MM patients. Moreover, Apo2L selectively induces apoptosis of patient MM tumor cells while sparing non-malignant cells. Apo2L induction is one of the earliest events following IFN administration in these cells. IFNs activate Caspases and the mitochondrial-dependent apoptotic pathway mediated by Apo2L production. Cell death induced by IFNs and Apo2L can be blocked by a dominant-negative Apo2L receptor, DRS, and is regulated by members of the Bcl-2 family of proteins. This review is focused on the apoptotic signaling pathways regulated by Apo2L and Bcl-2-family proteins and summarizes what is known about their clinical role.     

IFN-alpha and bortezomib overcome Bcl-2 and Mcl-1 overexpression in melanoma cells by stimulating the extrinsic pathway of apoptosis

We hypothesized that IFN-alpha would enhance the apoptotic activity of bortezomib on melanoma cells. Combined treatment with bortezomib and IFN-alpha induced synergistic apoptosis in melanoma and other solid tumor cell lines. Apoptosis was associated with processing of procaspase-3, procaspase-7, procaspase-8, and procaspase-9 and with cleavage of Bid and poly(ADP-ribose) polymerase. Bortezomib plus IFN-alpha was effective at inducing apoptosis in melanoma cells that overexpressed Bcl-2 or Mcl-1, suggesting that this treatment combination can overcome mitochondrial pathways of cell survival and resistance to apoptosis. The proapoptotic effects of this treatment combination were abrogated by a caspase-8 inhibitor, led to increased association of Fas and FADD before the onset of cell death, and were significantly reduced in cells transfected with a dominant-negative FADD construct or small interfering RNA targeting Fas. These data suggest that bortezomib and IFN-alpha act through the extrinsic pathway of apoptosis via FADD-induced caspase-8 activation to initiate cell death. Finally, bortezomib and IFN-alpha displayed statistically significant antitumor activity compared with either agent alone in both the B16 murine model of melanoma and in athymic mice bearing human A375 xenografts. These data support the future clinical development of bortezomib and IFN-alpha for malignant melanoma.     

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.     

Stat1-dependent induction of tumor necrosis factor-related apoptosis-inducing ligand and the cell-surface death signaling pathway by interferon beta in human cancer cells

Type I IFNs are known to inhibit tumor cell growth and stimulate the immune system. However, little is known of the mechanism of type I IFN-induced apoptosis in human cancer cells. In this study, we have IFN-beta treatment of a human colorectal cell line (KM12L4) and a resistant clone of this cell line, L4RIFN. We demonstrate the induction of apoptosis in the parent cell line. This process was associated with the induction of the Jak-Stat signaling pathway, induction of the proapoptotic mediator tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), and activation of procaspase-3, -8, -9, and -10. Additionally, we evaluated the role of Stat1 in mediating IFN-beta induction of these proapoptotic signals in a fibrosarcoma cell line (2ftgh) and a Stat1-deficient clone (U3A). Our results demonstrate that IFN-beta induction of apoptosis and the induction of proapoptotic mediator TRAIL is Stat1 dependent. Evaluation of a stable transfectant of the KM12L4 cell line expressing c-FLIP supports the role of TRAIL and the cell-surface death signaling pathways in IFN-beta induction of apoptosis. Studies evaluating the TRAIL promoter indicate induction of TRAIL promoter activity by IFN-beta. These results may represent a novel pathway by which IFN-beta may induce therapeutic effects.     

Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand cooperates with chemotherapy to inhibit orthotopic lung tumor growth and improve survival

Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) is a tumor necrosis factor superfamily member that induces apoptosis through the death receptors DR4 and/or DR5 in various cancer cell types but not in most normal cells. Several lung cancer cell lines express DR4 and DR5 and undergo apoptosis in vitro in response to Apo2L/TRAIL. We investigated the efficacy of recombinant soluble human Apo2L/TRAIL and its interaction with chemotherapy in xenograft models based on human NCI-H460 non-small cell lung carcinoma cells. In vitro, Taxol enhanced caspase activation and apoptosis induction by Apo2L/TRAIL. In vivo, Apo2L/TRAIL or Taxol plus carboplatin chemotherapy partially delayed progression of established subcutaneous tumor xenografts, whereas combined treatment caused tumor regression and a substantially longer growth delay. Apo2L/TRAIL, chemotherapy, or the combination of both inhibited growth of preformed orthotopic lung parenchymal tumors versus control by 60%, 57%, or 97%, respectively (all P < 0.01; n = 8-10). Furthermore, combination treatment improved day-90 survival relative to control (7 of 15 versus 1 of 15; P = 0.0003 by Mantel-Cox) as well as to Apo2L/TRAIL (3 of 14; P = 0.031) or chemotherapy (3 of 15; P = 0.035). These studies provide evidence for in vivo activity of Apo2L/TRAIL against lung tumor xenografts and underscore the potential of this ligand for advancing current lung cancer treatment strategies.     

Enhancement of TRAIL/Apo2L-mediated apoptosis by adriamycin through inducing DR4 and DR5 in renal cell carcinoma cells

Renal cell carcinoma (RCC) is one of the most drug-resistant malignancies in humans. We show that adriamycin (ADR) and TNF-related apoptosis-inducing ligand (TRAIL)/Apo2L have a synergistic cytotoxic effect against RCC cells. This synergistic cytotoxicity was obtained in ACHN, A704, Caki-1 and Caki-2 human RCC cell lines and freshly derived RCC cells from 6 patients. This synergistic effect, however, was not achieved in 5 samples of freshly isolated normal kidney cells. We further explored the mechanisms underlying this synergistic effect and found that the synergistic cytotoxicity of TRAIL/Apo2L and ADR was realized by inducing apoptosis. Sequential treatment with ADR followed by TRAIL/Apo2L induced significantly more cytotoxicity than the reverse treatment. ADR increased the expression of DR4 and DR5 in RCC cells, but not in the normal kidney cells. Furthermore, the synergistic cytotoxicity was significantly inhibited by DR4:Fc and DR5:Fc fusion proteins, which inhibit TRAIL/Apo2L-mediated apoptosis. In addition, caspase activity assays and treatment of caspase inhibitors demonstrated that the combination treatment with ADR and TRAIL/Apo2L activated caspase cascade, including caspase-9, -8, -6 and -3, which were the downstream molecules of death receptors. These findings indicate that ADR sensitizes RCC cells to TRAIL/Apo2L-mediated apoptosis through induction of DR4 and DR5, suggesting that the combination therapy of TRAIL/Apo2L and ADR might be effective for RCC therapy.     

Strong inhibition of Ewing tumor xenograft growth by combination of human interferon-alpha or interferon-beta with ifosfamide

Ewing sarcoma is the second most common bone tumor in childhood. Despite aggressive chemotherapy and radiotherapy strategies, the prognosis of patients with metastatic disease remains poor. We have recently reported that Ewing tumor cell proliferation was strongly inhibited by IFN-beta and to a lesser degree by IFN-alpha. Moreover, under IFN-beta treatment, some cell lines undergo apoptosis. Since the possibility of using IFNs for Ewing tumor treatments may be of interest, we have evaluated the efficacy of Hu-IFNs in a nude mice model of Ewing tumor xenografts. The results reported here show that human type I IFNs, Hu-IFN-alpha and Hu-IFN-beta impaired tumor xenograft take and displayed an anti-growth effect toward established xenografts. Furthermore, we have also shown that combined therapy with Hu-IFNs and ifosfamide (IFO), an alkylating agent widely used in high-dose chemotherapy of Ewing tumors, results in a strong antitumor effect. Pathological analysis showed that Hu-IFN-alpha/IFO and Hu-IFN-beta/IFO were characterized by a dramatic decrease in the mitotic index and marked necrosis, as well as extensive fibrosis associated with numerous calcifications. To our knowledge, this is the first demonstration of a potential antitumor effect of human type I IFNs and IFO on Ewing tumors, providing a rational foundation for a promising therapeutic approach to Ewing sarcoma.     

Stronger growth-inhibitory effect of interferon (IFN)-beta compared to IFN-alpha is mediated by IFN signaling pathway in hepatocellular carcinoma cells

Interferon (IFN) is a promising drug for prevention and treatment of hepatocellular carcinoma (HCC) in combination with chemotherapeutic agents. We previously reported that the spectra of antiproliferative activity and synergistic effect of IFN-beta when combined with anticancer drugs are more potent than those of IFN-alpha in HCC cells. However, the mechanism of the diverse antitumor effects of the IFNs is not understood yet. We studied the expression of IFN alpha receptor 2 (IFNAR2), STATs, and IFN-alpha, IFN-beta's growth-inhibitory effect, signal transduction and binding to IFNAR2 on three HCC cell lines and a tumor xenografted mouse model (12 animals/group). From the results, IFN-beta showed a significantly stronger growth-inhibitory effect than IFN-alpha on the HuH7 cell line (expressing low IFNAR2), however it was similarly high on PLC/PRF/5 and weak on HLE. In the nude mouse tumor xenograft model, IFN-beta injection significantly suppressed tumor volume relative to vehicle injection, while IFN-alpha showed weaker growth-inhibition. IFN signal transduction (phosphorylated-STAT1, 3) induced by IFN-beta was higher than that by IFN-alpha in HuH7 and tumor xenografts. Pretreatment of hepatoma cells with anti-IFNAR2 antibody blocked the IFN signaling, more for IFN-alpha. IFN-alpha's antiproliferative effect was reduced by the antibody in lower concentrations compared to that of IFN-beta. Taken together, the HCC cells that express low IFNAR2 and are resistant to IFN-alpha were sensitive to the growth-inhibitory effect of IFN-beta, which might be mediated by stronger IFN signal transduction and distinct binding to IFNAR compared to IFN-alpha.     

Chemotherapeutic agents sensitize osteogenic sarcoma cells, but not normal human bone cells, to Apo2L/TRAIL-induced apoptosis

Apo2L/TRAIL is a member of the tumor necrosis factor (TNF) family of cytokines that induces death of cancer cells but not normal cells. Its potent apoptotic activity is mediated through its cell surface death domain-containing receptors, DR4 and DR5. Apo2L/TRAIL interacts also with 3 "decoy" receptors that do not induce apoptosis, DcR1, DcR2, which lack functional death domains, and osteoprotegerin (OPG). The aim of our study was to investigate the cytotoxic activity of Apo2L/TRAIL on established osteogenic sarcoma cell lines (BTK-143, HOS, MG-63, SJSA-1, G-292 and SAOS2) and in primary cultures of normal human bone (NHB) cells. When used alone, Apo2L/TRAIL at 100 ng/ml for 24 hr induced greater than 80% cell death in only 1 (BTK-143) of the 6 osteogenic sarcoma cell lines. In contrast, Apo2L/TRAIL-resistant cells were susceptible to Apo2L/TRAIL-mediated apoptosis in the presence of the anticancer drugs, Doxorubicin (DOX), Cisplatin (CDDP) and Etoposide (ETP) but not Methotrexate (MTX) or Cyclophosphamide (CPM). Importantly, neither Apo2L/TRAIL alone nor in combination with any of these drugs affected primary normal human bone cells under equivalent conditions. Apo2L/TRAIL-induced apoptosis, and its augmentation by chemotherapy in the resistant cell lines was mediated through caspase-8 and caspase-3 activation. Furthermore, Apo2L/TRAIL-induced apoptosis and its augmentation by chemotherapy was effectively inhibited by caspase-8 zIETD-fmk and caspase-3 zDEVD-fmk protease inhibitors and by the pan-caspase inhibitor zVAD-fmk. The pattern of basal Apo2L/TRAIL receptor mRNA expression, or expression of the intracellular caspase inhibitor FLICE-inhibitory protein, FLIP, could not be readily correlated with resistance or sensitivity to Apo2L/TRAIL-induced apoptosis. However, the augmentation of Apo2L/TRAIL effects by chemotherapy was associated with drug-induced up-regulation of death receptors DR4 and DR5 mRNA and protein. No obvious correlation was seen between the expression of OPG mRNA or protein and susceptibility of cells to Apo2L/TRAIL-induced apoptosis. Stable over-expression of a dominant negative form of the Fas-associated death domain protein (FADD) in the Apo2L/TRAIL-sensitive BTK-143 cells completely inhibited Apo2L/TRAIL-induced cell death. Our results indicate that chemotherapy and Apo2L/TRAIL act synergistically to kill cancer cells but not normal bone-derived osteoblast-like cells, which has implications for future therapy of osteosarcoma.     

Simvastatin inhibits growth via apoptosis and the induction of cell cycle arrest in human melanoma cells

Competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (the statins) that inhibit the synthesis of mevalonic acid are in wide use for treatment of hypercholesterolemia. Although antitumor effects on a variety of cell types have been reported for statins, the effect of simvastatin (one of the statins) on human melanoma cell lines is not known. Here, we report antitumor effects of simvastatin on human melanoma cell lines. We treated human melanoma cell lines, A375M, G361, C8161, GAK, and MMAc with simvastatin in various concentrations for 1 to 3 days. To investigate the antitumor effect of simvastatin, we analyzed cell viability, morphologic changes, reversibility of inhibition by geranylgeranyl pyrophosphate and farnesyl pyrophosphate, apoptosis and the cell cycle. Simvastatin treatment reduced cell viability in all five melanoma cell lines. The different melanoma cell lines, however, displayed different sensitivities to simvastatin. The addition of geranylgeranyl pyrophosphate to A375M and G361 cells in the presence of simvastatin completely restored the viability of cells, but the addition of farnesyl pyrophosphate did not. DNA fragmentation assay showed that simvastatin induced apoptosis in A375M and G361 cells. Simvastatin caused a G1 arrest in G361 and MMAc cells. Consistent with the cell cycle arrest, simvastatin caused an increase in the mRNA levels of p21 and p27 on G361 and MMAc cells.We conclude that simvastatin has an antitumor effect on human melanoma cells in vitro via apoptosis and cell cycle arrest. These results suggest that simvastatin may be an effective anticancer drug for malignant melanoma.     

The histone deacetylase inhibitor, suberoylanilide hydroxamic acid, overcomes resistance of human breast cancer cells to Apo2L/TRAIL

While the apoptosis-inducing ligand Apo2L/TRAIL is a promising new agent for the treatment of cancer, the sensitivity of cancer cells for induction of apoptosis by Apo2L/TRAIL varies considerably. Identification of agents that can be used in combination with Apo2L/TRAIL to enhance apoptosis in breast cancer cells would increase the potential utility of this agent as a breast cancer therapeutic. Here, we show that the histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA), can sensitize Apo2L/TRAIL-resistant breast cancer cells to Apo2L/TRAIL-induced apoptosis. Importantly, neither Apo2L/TRAIL alone, nor in combination with SAHA, affected the viability of normal human cells in culture. Apo2L/TRAIL-resistant MDA-MB-231 breast cancer cells, generated by long-term culture in the continuous presence of Apo2L/TRAIL, were resensitized to Apo2L/TRAIL-induced apoptosis by SAHA. The sensitization of these cells by SAHA was accompanied by activation of caspase 8, caspase 9 and caspase 3 and was concomitant with Bid and PARP cleavage. The expression of the proapoptotic protein, Bax, increased significantly with SAHA treatment and high levels of Bax were maintained in the combined treatment with Apo2L/TRAIL. Treatment with SAHA increased cell surface expression of DR5 but not DR4. Interestingly, SAHA treatment also resulted in a significant increase in cell surface expression of DcR1. Taken together, our findings indicate that the use of these 2 agents in combination may be effective for the treatment of breast cancer.     

Cisplatin down-regulation of cellular Fas-associated death domain-like interleukin-1beta-converting enzyme-like inhibitory proteins to restore tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis in human melanoma cells.

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-1beta-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.     

Sensitization of tumor cells to Apo2 ligand/TRAIL-induced apoptosis by inhibition of casein kinase II

Tumor-cell death can be triggered by engagement of specific death receptors with Apo2 ligand/tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL). Apo2L/TRAIL-induced apoptosis involves caspase-8-mediated cleavage of BID. The active truncated form of BID (tBID) triggers the mitochondrial activation of caspase-9 by inducing the activation of BAK or BAX. Although a broad spectrum of human cancer cell lines express death receptors for Apo2L/TRAIL, many remain resistant to TRAIL/Apo2L-induced death. A variety of human cancers exhibit increased activity of casein kinase II (CK2). Here we demonstrate that CK2 is at the nexus of two signaling pathways that protect tumor cells from Apo2L/TRAIL-induced apoptosis. We find that CK2 inhibits Apo2L/TRAIL-induced caspase-8-mediated cleavage of BID, thereby reducing the formation of tBID. In addition, CK2 promotes nuclear factor kappa B (NF-kappa B)-mediated expression of Bcl-x(L), which sequesters tBID and curtails its ability to activate BAX. Tumor cells with constitutive activation of CK2 exhibit a high Bcl-x(L)/tBID ratio and fail to activate caspase-9 or undergo apoptosis in response to Apo2L/TRAIL. Conversely, reduction of the Bcl-x(L)/tBID ratio by inhibition of CK2 renders such cancer cells sensitive to Apo2L/TRAIL-induced activation of caspase-9 and apoptosis. Using isogenic cancer cell lines that differ only in the presence or absence of either the p53 tumor suppressor or the BAX gene, we show that the enhancement of Apo2L/TRAIL-induced tumor-cell death by CK2 inhibitors requires BAX, but not p53. The identification of CK2 as a key survival signal that protects tumor cells from death-receptor-induced apoptosis could aid the design of Apo2L/TRAIL-based combination regimens for treatment of diverse cancers.     

Bortezomib sensitizes primary human astrocytoma cells of WHO grades I to IV for tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis.

PURPOSE: Malignant gliomas are the most aggressive human brain tumors without any curative treatment. The antitumor effect of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in gliomas has thus far only been thoroughly established in tumor cell lines. In the present study, we investigated the therapeutic potential of TRAIL in primary human glioma cells. EXPERIMENTAL DESIGN: We isolated primary tumor cells from 13 astrocytoma and oligoastrocytoma patients of all four WHO grades of malignancy and compared the levels of TRAIL-induced apoptosis induction, long-term tumor cell survival, caspase, and caspase target cleavage. RESULTS: We established a stable culture model for isolated primary human glioma cells. In contrast to cell lines, isolated primary tumor cells from all investigated glioma patients were highly TRAIL resistant. Regardless of the tumor heterogeneity, cotreatment with the proteasome inhibitor bortezomib efficiently sensitized all primary glioma samples for TRAIL-induced apoptosis and tremendously reduced their clonogenic survival. Due to the pleiotropic effect of bortezomib-enhanced TRAIL DISC formation upon TRAIL triggering, down-regulation of cFLIP(L) and activation of the intrinsic apoptosis pathway seem to cooperatively contribute to the antitumor effect of bortezomib/TRAIL cotreatment. CONCLUSION: TRAIL sensitivity of tumor cell lines is not a reliable predictor for the behavior of primary tumor cells. The widespread TRAIL resistance in primary glioma cells described here questions the therapeutic clinical benefit of TRAIL as a monotherapeutic agent. Overcoming TRAIL resistance by bortezomib cotreatment might, however, provide a powerful therapeutic option for glioma patients.