Upregulation of DR5 by proteasome inhibitors potently sensitizes glioma cells to TRAIL-induced apoptosis Holger Hetschko 1 , Valerie Voss 1 , Volker Seifert 1 , Jochen H. M. Prehn 2 and Donat Ko ¨ gel 1 1 Department of Neurosurgery, Centre for Neurology and Neurosurgery, Johann Wolfgang Goethe University Clinics, Frankfurt ⁄ Main, Germany 2 Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, Dublin, Ireland Gliomas are the most common and malignant primary brain tumors in humans. Glioblastoma multiforme is the highest-grade as well as the most aggressive and frequent glioma [1]. Because gliomas are characterized by a diffuse infiltrative growth into the surrounding brain tissue, complete surgical resection of glioblas- toma multiforme tumors is virtually impossible [2]. In addition, high-grade gliomas exhibit only limited sensi- tivity to ensuing multimodal treatment with radio- therapy and chemotherapy [2], which in large part is Keywords apoptosis; astrocytoma; death receptor; proteasome; stress kinase Correspondence D. Ko ¨ gel, Experimental Neurosurgery, Johann Wolfgang Goethe University Clinics, Theodor-Stern-Kai 7, Neuroscience Centre, D-60590 Frankfurt am Main, Germany Fax: +49 69 6301 5575 Tel: +49 69 6301 6940 E-mail: koegel@em.uni-frankfurt.de (Received 28 January 2008, revised 19 February 2008, accepted 21 February 2008) doi:10.1111/j.1742-4658.2008.06351.x This study was undertaken to explore the potential of new therapeutic approaches designed to reactivate cell death pathways in apoptosis-refrac- tory gliomas and to characterize the underlying molecular mechanisms of this reactivation. Here we investigated the sensitivity of a panel of glioma cell lines (U87, U251, U343, U373, MZ-54, and MZ-18) to apoptosis induced by the death receptor ligand tumor necrosis factor-related apopto- sis-inducing ligand (TRAIL), TRAIL in combination with gamma irradia- tion, and TRAIL in combination with proteasome inhibitors (MG132 and epoxomicin). Analysis of these six glioma cell lines revealed drastic differ- ences in their sensitivity to these treatments, with two of the six cell lines revealing no significant induction of cell death in response to TRAIL alone. Interestingly, the proteasome inhibitors MG132 and epoxomicin were capable of potentiating TRAIL-induced apoptosis in TRAIL-sensitive U87 and U251 cells and of reactivating apoptosis in TRAIL-resistant U343 and U373 cells. In contrast, gamma irradiation had no synergistic effects with TRAIL in the two TRAIL-resistant cell lines. RNA interference against death receptor 5 (DR5) revealed that reactivation of TRAIL- induced apoptosis by proteasome inhibitors depended on enhanced tran- scription and surface expression of DR5. Transient knockdown of the transcription factor GADD153⁄ C ⁄ EBP homologous protein and applica- tion of the synthetic c-Jun N-terminal kinase inhibitor SP600125 indicated that enhanced DR5 expression occurred independently of GADD153 ⁄ C ⁄ EBP homologous protein, but required activation of the c-Jun N-termi- nal kinase ⁄ c-Jun signaling pathway. Novel therapeutic approaches using TRAIL or agonistic TRAIL receptor antibodies in combination with pro- teasome inhibitors may represent a promising approach to reactivate apop- tosis in therapy-resistant high-grade gliomas. Abbreviations Ac-DEVD-AMC, acetyl-DEVD-7-amido-4-methylcoumarin; CHOP, C ⁄ EBP homologous protein; DR4, death receptor 4; DR5, death receptor 5; FACS, fluorescence-activated cell sorting; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; JNK, c-Jun N-terminal kinase; NF-jB, nuclear factor kappa B; PI, proteasome inhibitor; siRNA, small interfering RNA; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand. FEBS Journal 275 (2008) 1925–1936 ª 2008 The Authors Journal compilation ª 2008 FEBS 1925 caused by inherent and potent apoptosis resistance [3]. Clearly, overcoming this resistance by approaches designed to reactivate apoptosis in malignant glioma has important implications for the development of novel glioma therapies. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of the tumor necrosis factor superfamily, has been shown to be a promising candi- date for novel anticancer therapies [4]. Interestingly, TRAIL is capable of inducing apoptosis in a wide range of tumor cells, but not in normal tissue. This tumor-selective cytotoxicity has been shown for glioma cells in comparison to non-transformed astrocytes in vitro [5,6]. The physiological function of TRAIL remains unclear, although studies suggest that TRAIL plays a key role in tumor surveillance by the immune system [7]. TRAIL induces apoptosis by binding to its agonistic cognate cell surface receptors death receptor 4 (DR4) ⁄ TRAIL R1 and death receptor 5 (DR5) ⁄ TRAIL R2, and this then leads to recruitment of the adaptor protein Fas-associated death domain and the initiator caspases procaspase-8 and procaspase- 10, and formation of a membrane-bound multiprotein complex called death-inducing signaling complex [4]. Death-inducing signaling complex formation then leads to autoproteolytic cleavage of procaspase-8 and procaspase-10, and subsequent downstream activa- tion of the extrinsic and intrinsic pathways of apoptosis [4]. Despite the considerable interest raised by the poten- tial of TRAIL in novel anticancer therapies, accumu- lating evidence suggests that TRAIL alone may not be sufficient to efficiently activate apoptosis in many types of cancers, inluding gliomas [8]. Hence, much effort has been made to establish new modalities for com- bined treatments with TRAIL and other antineoplastic agents or apoptosis inducers to improve the potency of TRAIL-based therapeutic approaches. Proteasome inhibitors (PIs) represent a highly prom- ising novel class of anticancer agents [9]. PIs are already in clinical use, as bortezomib (PS-341 ⁄ Velcade) has been approved for the treatment of multi- ple myeloma [10]. Recent evidence suggests that PIs also might be efficient agents for the treatment of solid tumors, such as lung and prostate cancer [11,12]. This study reveals that TRAIL-induced apoptosis can be efficiently reactivated in TRAIL-resistant malig- nant glioma cell lines by combined treatment with PIs. Furthermore, we show that reactivation of TRAIL- induced apoptosis by proteasome inhibition induces the c-Jun N-terminal kinase (JKN) ⁄ c-Jun stress signal- ing pathway and requires enhanced JNK ⁄ c-Jun-depen- dent surface expression of DR5. Results Synergistic effects of combined treatment with TRAIL and gamma irradiation To analyze the sensitivity of high-grade gliomas to cell death induced by the death ligand TRAIL, we employed a panel of six grade III–IV glioma cell lines (U87, U251, U343, U373, MZ-18, and MZ-54). In an initial experiment, the cells were treated for 48 h at a final concentration of 250 ng TRAILÆmL )1 (Fig. 1A). Surprisingly, only two of six cell lines (U87 and U251) significantly responded to TRAIL treatment as measured by annexin-V–FLUOS ⁄ propidium iodide staining and flow cytometry (Fig. 1A). Prolonged incu- bation for up to 96 h also did not induce detectable cell death in the four TRAIL-resistant cell lines (data not shown). As gamma irradiation is an existing component of current glioma therapies, and as activation of TRAIL receptors and DNA damage were shown to have syner- gistic death-inducing effects in other types of cancer [13], we investigated whether similar effects could also be observed in glioma cells. Cell lines U87, U343 and U373 were subjected to gamma irradiation with single doses of 10 Gy and 20 Gy respectively. Twenty-four hours postirradiation, the cultures were treated with 250 ngÆmL )1 TRAIL for an additional 24 h, after which cell death was measured by fluorescence-activated cell sorting (FACS) analysis (Fig. 1B). In comparison to the controls, a dose of 10 Gy had no effect on cell viability, either alone or in combination with TRAIL (data not shown). The higher dose of 20 Gy induced significant cell death in all three cell lines after 48 h ( 20–30%), and was capable of potentiating TRAIL-induced cell death in the TRAIL-sensitive cell line U87. However, in U343 and U373 cells, there was no significant synergis- tic effect of TRAIL and gamma irradiation, indicating that the activation of DNA damage-induced signaling pathways was not sufficient to reactivate TRAIL sensi- tivity in the TRAIL-resistant cell lines (Fig. 1B). It is of note that this lack of apoptosis reactivation was inde- pendent of the p53 status of these cells, as U343 cells, but not U373 cells, have been shown to express func- tional p53 [14,15]. PIs potently reactivate TRAIL-induced apoptosis in glioma cells Next, we investigated the synergistic effects of TRAIL and PIs in our glioma cell lines. For that purpose, we employed two different PIs in combination with TRAIL: MG132, a potent reversible inhibitor targeting Apoptosis by TRAIL and proteasome inihibitors H. Hetschko et al. 1926 FEBS Journal 275 (2008) 1925–1936 ª 2008 The Authors Journal compilation ª 2008 FEBS the 26S complex of the proteasome [16], and epoxomi- cin, a highly specific and irreversible inhibitor of several hydrolyzing activities of the proteasome [17], at concentrations of 2.5 lm and 50 nm respectively (Fig. 2). Whereas treatment with MG132 alone had a moderate cytotoxic effect in three cell lines (U87, U373, and MZ-54), treatment with epoxomicin signifi- cantly induced cell death in only two of six cell lines (U373 and MZ-54) (Fig. 2). Interestingly, both PIs potently enhanced TRAIL-induced apoptosis in the two TRAIL-sensitive cell lines (U87 and U251), and were able to reactivate apoptosis in four TRAIL-resis- tant cell lines (U343, U373, MZ-54, and MZ-18). It is of note that all six investigated cell lines were previ- ously shown to express DR5, whereas DR4 expression was undetectable in U251, U373 and MZ-54 cells [18]. Furthermore, reactivation of TRAIL-induced cell death did not depend on functional p53, as it was also observed in mutant p53 expressing U373 cells. PIs potently induce expression of DR5 in glioma cells To elucidate the underlying molecular mechanisms of the observed synergistic effects of PIs, we focused on the transcriptional activation of pro-apoptotic genes after proteasome inhibition with MG132 and epoxo- micin. Samples of cells exposed to gamma irradiation served as a control for p53-dependent gene activation. Although we found markedly enhanced DR5 trans- criptional activation after proteasome inhibition and gamma irradiation in all four cell lines investigated (Fig. 3A), there was no evidence for activation of DR4 by any stimulus. However the stress-activated pro-apoptotic transcription factors C ⁄ EBP homolo- gous protein (CHOP) and c-Jun were transcriptionally activated after proteasome inhibition in all four cell lines, whereas induction of CHOP and c-Jun was less prominent or even absent after gamma irradiation (Fig. 3A). Western blot analysis confirmed that DR4 protein levels were not elevated after proteasome inhibition or gamma irradiation (Fig. 3B). Although we found a strong elevation of DR5 protein levels after protea- some inhibition, we could detect only moderately enhanced protein levels after gamma irradiation. Simi- larly, the amount of CHOP protein was markedly increased by proteasome inhibition in all three cell lines, but in only one cell line (U87) after gamma irradiation. B A Fig. 1. Synergistic effects of combined treatment with TRAIL and gamma irradiation. (A) TRAIL resistance is commonly observed in glioma cell lines. U87, U251, U343, U373, MZ-54 and MZ-18 cells were treated with 250 ngÆmL )1 TRAIL for 48 h. Cells were stained with annexin- V–FLUOS ⁄ propidium iodide, and cell death was measured with flow cytometry. Data are means ± SEM from four independent cultures. *P < 0.05 as compared to untreated control. (B) TRAIL-sensitive cells (U87) and TRAIL-resistant cells (U343, U373) were subjected to gamma irradiation (20 Gy) and subsequently were treated with 250 ngÆmL )1 TRAIL or were left untreated for an additional 24 h. Cells were irradiated once in an Elekta SL75 ⁄ 5 linear accelerator (6 MeV). Apoptosis was quantified with annexin-V–FLUOS ⁄ propidium iodide staining and flow cytometry. Data are means ± SEM from four independent cultures. *P < 0.05 as compared to untreated control. # P < 0.05 as com- pared to treatment of the same cell line with gamma irradiation alone. Similar results were obtained in three separate experiments. H. Hetschko et al. Apoptosis by TRAIL and proteasome inihibitors FEBS Journal 275 (2008) 1925–1936 ª 2008 The Authors Journal compilation ª 2008 FEBS 1927 Upregulation of DR5 by PIs contributes to the reactivation of TRAIL-induced cell death in glioma cells Although the PIs prominently upregulated DR5 (Fig. 3), it was not clear whether this elevated expres- sion was responsible for the dramatic reactivation of TRAIL-induced apoptosis observed in malignant gli- oma cells. To address this question, we knocked down expression of DR5 by small interfering RNA (siRNA) duplexes targeted against DR5 mRNA and studied the effect on caspase activation after treatment of the cells with TRAIL, MG132, and TRAIL in combination with MG132. In initial experiments, U343 cells were transfected with DR5 siRNA, treated with or without 2.5 lm MG132, and subjected to western blot analysis (Fig. 4A). Transfection with siRNA against DR5 resulted in a robust reduction of DR5 activation after MG132 treatment as compared to MG132-treated con- trol cells transfected with a nontargeting scrambled siRNA (Fig. 4A). In contrast, transfection with the siRNAs had no major effect on expression levels of DR4. To determine whether the changes in the amount of DR5 protein in the protein lysates were also reflected by the levels of DR5 surface expression, we performed a flow cytometric analysis, which indeed confirmed enhanced DR5 surface expression after treatment of the cells with MG132. In analogy to the western blot data, DR5 surface expression was not completely abolished in DR5 siRNA-transfected cells treated with MG132, but was reduced to basal surface expression levels of scrambled siRNA-transfected con- trol cells (Fig. 4B). Under the experimental conditions chosen, TRAIL alone caused only weak induction of caspase-3-like activity in the TRAIL-sensitive cell lines U87 and U251 (Fig. 4C). Nevertheless, we observed a clear trend towards an apoptosis-inhibitory effect of the DR5 knockdown, indicating that basal expression of DR5 is required for the TRAIL sensitivity in these cells. Apoptosis induced by TRAIL in combination Fig. 2. Reactivation of TRAIL-induced apop- tosis after combined treatment with TRAIL and PIs. TRAIL-sensitive cells (U87 and U251) and TRAIL-resistant cells (U343, U373, MZ-54, and MZ-18) were treated with 250 ngÆmL )1 TRAIL and the PIs MG132 (2.5 l M) and epoxomicin (50 nM) or vehicle (dimethylsulfoxide) for 16 h. Apoptosis was quantified with annexin-V–FLUOS ⁄ propidium iodide staining and flow cytometry. Data are means ± SEM from four independent cul- tures. *P < 0.05 as compared to dimethyl- sulfoxide-treated control. # P < 0.05 as compared to treatment of the same cell line with the respective PI alone. Similar results were obtained in three separate experi- ments. Apoptosis by TRAIL and proteasome inihibitors H. Hetschko et al. 1928 FEBS Journal 275 (2008) 1925–1936 ª 2008 The Authors Journal compilation ª 2008 FEBS with MG132 was potently attenuated in TRAIL-resis- tant U343 cells, TRAIL-sensitive U251 cells and TRAIL-sensitive U87 cells after knockdown of DR5 in comparison to the respective controls (Fig. 4C). These results suggest that enhanced surface expression of DR5 plays a critical role in the reactivation of TRAIL-induced apoptosis after proteasome inhibition in glioma cells. CHOP is not involved in DR5 upregulation by PIs and cell death induced by proteasome inhibition plus TRAIL It was recently reported that CHOP, an endoplasmic reticulum stress-inducible member of the CCAAT ⁄ enhancer-binding protein family, can act as an upstream activator of DR5 in certain types of cancer cells [19,20]. In line with our previous findings [21], we found CHOP to be strongly activated after pro- teasome inhibition (Fig. 3), and were therefore inter- ested in whether CHOP was also involved in DR5 upregulation by PIs in glioma cells. To determine this, U343 cells were transfected with siRNA duplexes targeted against CHOP or with nontargeting scram- bled siRNA, and then treated with MG132 (Fig. 5A). Western blot analysis revealed that although CHOP expression was knocked down significantly in the CHOP siRNA-transfected cells as compared to the scrambled siRNA-transfected control cells, the activa- tion of DR5 after MG132 treatment was not affected by the knockdown (Fig. 5A). Furthermore, the knockdown of CHOP did not result in a significant attenuation of apoptosis induced by TRAIL plus MG132 in CHOP siRNA-transfected cells as com- pared to scrambled siRNA-transfected control cells (Fig. 5B). Inhibition of the JNK/c-Jun pathway abrogates PI-mediated DR5 upregulation and cell death Our data so far had suggested that PIs potently induced the expression of DR5 in glioma cells in a CHOP-independent manner. Although the tumor sup- pressor p53 has also been implicated in regulation of DR5 expression [22], the p53-deficient cell line U373 showed a similiar increase in DR5 upregulation as the wild-type p53-expressing cell lines U87 and U343 after treatment with PIs in this study (Fig. 3A,B), indicating that p53 was not responsible for the observed DR5 induction. As a third potential upstream regulator of DR5, we next focused on the JNK ⁄ c-Jun stress signal- ing pathway. It is well established that PIs such as MG132 are capable of inducing the JNK ⁄ c-Jun path- way [23]. To address the issue of whether activation of B A Fig. 3. PIs enhance expression of DR5. (A) DR5, CHOP and c-Jun are transcriptionally induced after proteasome inhibition. Cells were either treated with 2.5 l M MG132, 50 n M epoxomicin or vehicle (dimethylsulf- oxide) for 16 h or subjected to gamma irradi- ation (20 Gy) and subsequently lysed 24 h after exposure. Expression of DR4 (28 cycles), DR5 (25 cycles), CHOP (28 cycles) and GAPDH (25 cycles) was determined by semiquantitative RT-PCR. For detection of c-Jun expression, RT-PCR with GAPDH serving as an internal control (in the same PCR reaction) was performed (30 cycles for c-Jun and 30 cycles for GAPDH). Similar results were obtained in at least three sepa- rate experiments. (B) Western blot analysis of DR4, DR5 and CHOP expression levels. Forty micrograms of protein were loaded onto each lane, with a-tubulin serving as a loading control. Similar results were obtained in two separate experiments. H. Hetschko et al. Apoptosis by TRAIL and proteasome inihibitors FEBS Journal 275 (2008) 1925–1936 ª 2008 The Authors Journal compilation ª 2008 FEBS 1929 JNKs and c-Jun contributes to PI-dependent DR5 upregulation in human glioma cells, we investigated the effect of MG132 on DR5 upregulation in the pres- ence of the JNK-specific inhibitor SP600125 by wes- tern blot analysis. At concentrations of 10–30 lm, SP600125 abrogated the MG132-induced increase in phosphorylated c-Jun levels significantly (Fig. 6A). We also observed that in combination with MG132, SP600125 effectively blocked MG132-induced DR5 expression in a dose-dependent manner in U251 cells, and this was subsequently confirmed for the highest concentration used (30 lm) in U343 cells (Fig. 6B). There was no detectable effect of SP600125 on MG132-triggered induction of CHOP, suggesting that the AP-1 site in the CHOP promoter [24] is not required for enhanced CHOP expression after protea- some inhibition, and lending further support to the notion that the observed DR5 upregulation occured in a CHOP-independent manner. To analyze the rele- vance of JNK ⁄ c-Jun-dependent DR5 upregulation in cell death triggered by TRAIL in combination with MG132, we again performed caspase assays. Blocking the JNK ⁄ c-Jun signaling pathway before treatment of the cells with MG132 led to a significant decrease of caspase-3-like activity induced by TRAIL plus MG132 in both TRAIL-sensitive and TRAIL-resistant glioma cells (Fig. 6C). These results strongly suggest a promi- nent role of the JNK ⁄ c-Jun signaling pathway in A B C Fig. 4. RNA interference against DR5 effi- ciently protects cells from TRAIL-induced apoptosis after proteasome inhibition. Twenty-four hours after transfection with scrambled control siRNA or DR5 siRNA, TRAIL-resistant U343 cells were treated with 2.5 l M MG132 or dimethylsulfoxide for 16 h. (A) Analysis of DR5 and DR4 expres- sion levels by western blotting. a-Tubulin served as a loading control. Similar results were obtained in two separate experiments. (B) Analysis of cell surface expression of DR5 after RNA interference against DR5 (DR5) or treatment with control siRNA (scr), subsequent treatment with 2.5 l M MG132 or dimethylsulfoxide (16 h), and staining with a specific goat anti-DR5 IgG. Unspecific goat IgG served as isotype control. The experiment was repeated twice with similiar results. (C) Knockdown of DR5 inhibits apoptosis after treatment with TRAIL and MG132. Following transfection with DR5 siRNA (DR5) and control siRNA (scr), cells were treated with dimethylsulfoxide (con- trol), 2.5 l M MG132, TRAIL (250 ngÆmL )1 ) or TRAIL in combination with MG-132 for an additional 16 h, after which the cells were harvested and whole cell lysates were moni- tored for caspase-3-like activity by measur- ing cleavage of the fluorogenic substrate Ac-DEVD-AMC (10 l M). Data are means ± SEM from four to eight indepen- dent cultures. *P < 0.05 as compared to dimethylsulfoxide-treated control. Similar results were obtained in two separate experiments. Apoptosis by TRAIL and proteasome inihibitors H. Hetschko et al. 1930 FEBS Journal 275 (2008) 1925–1936 ª 2008 The Authors Journal compilation ª 2008 FEBS upregulation of DR5 and resensitization to apoptosis after proteasome inhibition. Discussion The high resistance of malignant gliomas to the cur- rently used radiation therapy and chemotherapy proto- cols is a persistent obstacle to the improvement of the outcome in glioma patients. Hence, a much effort has been made in recent years to obtain more data on the molecular mechanisms underlying gliomagenesis and therapy resistance of gliomas, with the aim of develop- ing more efficient and target-specific therapies. The death receptor ligand TRAIL and agonistic TRAIL-R antibodies are being considered as novel and promising therapeutic agents for the treatment of hematological malignancies and solid tumors, includ- ing gliomas. Despite the described tumor-selective pro- apoptotic properties of TRAIL, many cancer cells are innately resistant to TRAIL. In line with previous studies [6,25], all 18 glioma cell lines investigated in this study exhibited expression of DR5, but four of six cell lines showed no significant response to TRAIL. In order to identify synergistic treatments to modu- late TRAIL sensitivity and to reactivate apoptosis in glioma cells, we compared the effects of already exist- ing treatment modalities (gamma irradiation) with PIs (MG132 and epoxomicin), a new class of chemothera- peutic drugs with tremendous therapeutic potential [9]. The correct functioning of the ubiquitin–proteasome pathway is essential for the degradation of the major- ity of intracellular proteins and is implicated in many cellular processes, including regulation of apoptosis. Both epoxomicin and MG132 were able to potently enhance apoptosis in the TRAIL-sensitive cell lines. Both PIs increased apoptosis  5–6-fold in the TRAIL-sensitive cell lines U87 and U251, and even at subtoxic levels they were able to reactivate apoptosis in both TRAIL-resistant cell lines (U343 and U373). It is widely accepted that PIs can act on mutiple cellu- lar targets implicated in regulation of apoptosis [20,21,26,27]. However, despite abundant evidence for the therapeutic potential of PIs in a variety of malig- nancies, the relevant signaling pathways leading to apoptosis triggered by proteasome inhibition seem to vary substantially between different types of cancer. In some types of cancer, such as prostate cancer and leu- kemia, upregulation of DR5 seems to play a critical role in the potentiating effects of PIs on TRAIL- induced cell death [20,28,29]. Our data clearly indicate that treatment with PIs potently elevated DR5 expres- sion at the mRNA and protein levels in glioma cells, whereas gamma irradiation induced only a moderate increase in DR5 protein levels, even in the wild-type p53-expressing cell lines U87 and U343. Enhanced pro- tein levels and surface expression of DR5 after PI treatment might be caused by a cumulative effect of transcriptional induction and decreased degradation of DR5 protein, thus providing an explanation for the high efficiency of PIs in reactivation of TRAIL-depen- dent cell death observed in this study, and also for the reduced potency of gamma irradiation in elevating DR5 protein levels. To assess the importance of DR5 in PI-triggered reactivation of TRAIL-dependent apoptosis, we per- formed transient RNA interference against DR5 expression. Indeed, the knockdown of PI-triggered DR5 induction had a very potent effect on DR5 A B Fig. 5. DR5 protein levels are not affected by RNA interference against CHOP. (A) Four hours after transfection with CHOP siRNA (CHOP) and scrambled control siRNA (scrambled), TRAIL-resistant U343 cells were treated with 2.5 l M MG132 or dimethylsulfoxide for 16 h. CHOP and DR5 expression levels were analyzed by wes- tern blotting. a-Tubulin served as a loading control. (B) Knockdown of CHOP expression has no effect on TRAIL-induced apoptosis after proteasome inhibition. U343 cells were treated as described in (A), and caspase-3-like activity was measured by Ac-DEVD-AMC cleavage. Data are means ± SEM from four to eight independent cultures. *P < 0.05 as compared to dimethylsulfoxide-treated con- trol. Similar results were obtained in two separate experiments. H. Hetschko et al. Apoptosis by TRAIL and proteasome inihibitors FEBS Journal 275 (2008) 1925–1936 ª 2008 The Authors Journal compilation ª 2008 FEBS 1931 surface expression levels and apoptosis induced by TRAIL in combination with MG132 in U343 and U251 cells. These data suggest that, in contrast to other cancer types, such as hepatocellular carcinoma [30], transcriptional upregulation of DR5 plays a piv- otal role in potentiation and reactivation of TRAIL- induced apoptosis in glioma cells. We then addressed the question of which upstream signaling pathways might lead to enhanced DR5 sur- face expression after PI treatment, and in line with previous observations in other types of cancer cells [21], we demonstrated that the pro-apoptotic transcrip- tion factor CHOP was potently activated on the tran- scriptional level by PIs in glioma cells. Although CHOP has been previously described as a putative upstream regulator of DR5 in different types of cancer cells [19,20], we did not observe any significant CHOP- dependent effects on DR5 expression and cell death, indicating that CHOP is not required for DR5 induc- tion triggered by PIs in glioma cells. Two other apoptosis-regulating transcription fac- tors whose activity is known to be modulated by PIs are p53 and nuclear factor kappa B (NF-jB). Although the majority of reports suggest that PIs induce a p53-dependent form of apoptosis in most cancer cell types, such as leukemia cells [27], as well as melanoma, colon cancer and myeloma cells [21,26,31], PIs have been shown to induce p53-inde- pendent apoptosis in glioma cells [32,33]. In line with these observations, enhanced DR5 expression did not depend on the p53 status (wild-type or mutated) in the three glioma cell lines investigated in this study. As IjBa, the endogenous inhibitor of the transcrip- tion factor NF-jB, is continuously degraded via the proteasome pathway, PIs act as indirect inhibitors of the antiapoptotic transcription factor NF-jB [34]. However, we have previously shown that treatment of neuroblastoma and colon cancer cells with epoxomicin is not associated with noticeable transcriptional A C B Fig. 6. The JNK ⁄ c-Jun signaling pathway contributes to DR5 induction and apoptosis induced by TRAIL plus PIs in glioma cells. (A) Inhibition of JNK ⁄ c-Jun signaling downmodulates DR5 induction in a dose-dependent manner. U251 cells were pretreated with the indicated concen- trations of the JNK inhibitor SP600125 or vehicle (dimethylsulfoxide) for 2 h and treated with 2.5 l M MG132 or dimethylsulfoxide for an addi- tional 16 h. Whole cell lysates were analyzed by western blotting for levels of phosphorylated c-Jun and DR5. a-Tubulin served as a loading control. (B) SP600125 downmodulates PI-triggered DR5 induction in U343 cells. U343 cells were pretreated with 30 l M SP600125 or di- methylsulfoxide for 2 h, and subsequently treated with 2.5 l M MG132 for an additional 16 h in the presence or absence of 30 lM SP600125. Protein levels of phosphorylated c-Jun, DR5 and CHOP were analyzed by western blotting. a-Tubulin served as a loading control. (C) SP600125 inhibits apoptosis induced by TRAIL plus MG132 in three different glioma cell lines. U87, U251 and U343 cells were treated as described in (B). Caspase-3-like activity was measured by Ac-DEVD-AMC cleavage. Data are means ± SEM from four to eight indepen- dent cultures. *P < 0.05 as compared to dimethylsulfoxide-treated control. Similar results were obtained in three separate experiments. Apoptosis by TRAIL and proteasome inihibitors H. Hetschko et al. 1932 FEBS Journal 275 (2008) 1925–1936 ª 2008 The Authors Journal compilation ª 2008 FEBS changes of antiapoptotic NF-jB target genes such as Bcl-2, Bcl-xL, and the inhibitors of apoptosis (IAPs) [21]. In addition, it was recently reported that most glioblastoma cell lines exhibit only low constitutive NF-jB activity, and inhibition of NF-jB did not sig- nificantly influence apoptosis induced by DNA dam- age, TRAIL and PIs in glioma cells in this study [35]. Although inactivation of NF-jB has been suggested to play a major role in the antitumorigenic effect of the PI bortezomib (Velcade ⁄ PS-341) in multiple myeloma [36] and melanoma cells [26], inhibition of NF-kB is not required to sensitize hepatocellular carcinoma cells and lymphoma cells to apoptosis [37,38], again empha- sizing the cancer type-specific effects of PIs. As PIs are also known to trigger the stress-induced JNK ⁄ c-Jun pathway [23,33], we finally asked the question of whether this pathway might be involved in the observed DR5 induction triggered by PIs. RT-PCR analysis revealed a potent upregulation of c-Jun at the transcriptional level, suggesting upstream activation of JNKs, phosphorylation of c-Jun and enhanced c-Jun expression by an autoregulatory feed- forward mechanism [39]. Indeed, application of the synthetic JNK inhibitor SP600125 significantly reduced levels of phosphorylated c-Jun in a dose- dependent manner after PI treatment in glioma cells, whereas it had no discernible effect on CHOP induction. Inhibition of the JNK ⁄ c-Jun pathway with SP600125 also significantly downmodulated PI-induced DR5 induction and cell death triggered by TRAIL in combination with MG132. In conclusion, our data emphasize the importance of the JNK ⁄ c-Jun signaling cascade in transcriptional induction of DR5 and resensitization to TRAIL- induced apoptosis in malignant glioma cells, and sug- gest that new target-specific therapeutic approaches employing PIs in combination with TRAIL may repre- sent a highly promising strategy to reactivate apoptosis in therapy-resistant high-grade astrocytomas. Experimental procedures Materials The caspase substrate acetyl-DEVD-7-amido-4-methyl- coumarin (Ac-DEVD-AMC) was purchased from Bachem (Heidelberg, Germany). MG132 and epoxomicin were pur- chased from Sigma-Aldrich (Deisenhofen, Germany). JNK inhibitor II (SP600125) was from Merck Biosciences (Darmstadt, Germany). Recombinant human TRAIL was from PeproTech Inc. (Rocky Hill, NJ, USA). All other chemicals were of analytic grade purity and were from Sigma-Aldrich (Deisenhofen, Germany). Cell lines and culture Human glioma cell lines U87, U251, U343, and U373, as well as the newly established glioma cell lines, were main- tained in DMEM with 10% heat-inactivated fetal bovine serum, 100 U ÆmL )1 penicillin, and 100 mgÆmL )1 strepto- mycin. Human glioma cell lines MZ-18 and MZ-54 were established from a primary glioblastoma and a recurrent grade IV tumor, respectively. To isolate glioma cells, tumor specimens were homogenized, suspended in NaCl ⁄ P i and centrifuged at 400 g. Pellets were resuspended in 10 mL of medium, and plated into Petri dishes. Cultivation was per- formed under standard conditions at 37 °C and a humidi- fied 5% CO 2 atmosphere. When confluent monolayers had been obtained, tumor-derived cells were trypsinized and replated in new Petri dishes for serial passaging. All newly established cell lines were analyzed for glial fibrillary acidic protein expression by immunostaining with a monoclonal glial fibrillary acidic protein IgG (R&D Systems, Wiesbaden, Germany). Isotypic primary antibody (Serotec, Du ¨ sseldorf, Germany) was used as control. RT-PCR Extraction of total cellular RNA, reverse transcription and PCR were performed as previously described [40]. Primer sequences were as follows: CHOP-sense, 5¢-GGT CCT GTC TTC AGA TGA AAA TG-3¢; CHOP-antisense, 5¢-CCT GGT GCA GAT TCA CCA TTC-3¢; c-Jun-sense, 5¢-TGA CTG CAA AGA TGG AAA CG-3¢; c-Jun-antisense, 5¢-CCT GCT CAT CTG TCA CGT TC-3¢; DR4-sense, 5¢-AGA GAG AAG TCC CTG CAC CA-3¢;DR4-antisense, 5¢-AGA GAG AAG TCC CTG CAC CA-3¢; DR5-sense, 5¢-CAG AGG GAT TGT GTC CAC CT-3¢; DR5-anti- sense, 5¢-TAC GGC TGC AAC TGT GAC TC-3¢; glyceral- dehyde-3-phosphate dehydrogenase (GAPDH)-sense; 5¢-CCT GAC CTG CCG TCT AGA AA-3¢; GAPDH-anti- sense, 5¢-TTA CTC CTT GGA GGC CAT GT-3¢. SDS/PAGE and western blotting SDS ⁄ PAGE and western blotting were performed as described elsewhere [40]. The resulting blots were probed with rabbit polyclonal anti-DR5 serum (Imgenex, San Diego, CA, USA) diluted 1 : 200, rabbit polyclonal anti-DR4 serum (ProSci, Poway, CA, USA) diluted 1 : 500, rabbit polyclonal anti-phospho-c-Jun serum (Cell Signaling Technology, Danvers, MA, USA) diluted 1 : 800, mouse monoclonal anti-CHOP IgG diluted 1 : 200, or mouse monoclonal anti-a-tubulin IgG (clone DM 1A; Sigma), diluted 1 : 5000. Determination of caspase-3-like protease activity DEVDase (caspase-3-like) activity was determined as previ- ously described [40]. H. Hetschko et al. Apoptosis by TRAIL and proteasome inihibitors FEBS Journal 275 (2008) 1925–1936 ª 2008 The Authors Journal compilation ª 2008 FEBS 1933 Flow cytometry For cell death analysis, cells were stained with annexin- V–FLUOS ⁄ propidium iodide (Roche Applied Science, Mannheim, Germany), following the manufacturer’s instructions. For analysis of DR5 surface expression, cells were stained with goat polyclonal anti-DR5 IgG (Axxora, Gru ¨ nberg, Germany) and a goat IgG isotype control (SouthernBiotech, Birmingham, AL, USA), respectively, according to the manufacturer’s instructions. In all cases, a minimum of 10 4 events per sample were acquired. Flow cytometric analyses were performed on a FACScan (BD Biosciences; Heidelberg, Germany) followed by analysis using cellquest and winmdi software. Gene silencing using siRNA The following annealed double-stranded siRNAs from Dharmacon (Chicago, IL, USA) were used: CHOP siGe- nome duplexes D-004819-01-0005 and D-004819-02-0005; and DR5 siGenome duplexes D-004448-01-0005 and D-004448-03-0005. Scrambled siRNA siCONTROL from Dharmacon was used as a negative, nonsilencing control. Cells were transfected with 100 nm siRNAs using siM- PORTER (Biomol, Hamburg, Germany) as described by the manufacturer. Statistics Data are given as means ± SEM. 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