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Bortezomib Sensitizes Primary Human Astrocytoma Cells of WHO Grades I to IV for Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand–Induced Apoptosis

Author's Affiliations: ¹ Division of Apoptosis Regulation (D040), German Cancer Research Center (DKFZ); ² Department of Internal Medicine, University of Heidelberg, Heidelberg, Germany; ³ Biotechnical-Biomedical Centre (BBZ), Faculty of Medicine, ⁴ Clinic of Neurosurgery, ⁵ Institute of Neuropathology, and ⁶ Institute for Clinical Immunology and Transfusion Medicine, University of Leipzig, Leipzig, Germany; and ⁷ Laboratory for Experimental Oncology and Radiobiology, Academic Medical Center, Amsterdam, the Netherlands

Requests for reprints: Henning Walczak, Division for Apoptosis Regulation, D040, Germany Cancer Research Center (DKFZ), Im Neuenheimer Feld 580, Heidelberg D-69120, Germany. Phone: 49-6221-423700; Fax: 49-6221-423699; E-mail: h.walczak{at}dkfz.de.

	Abstract

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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 bortezomibenhanced 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.

Despite highly sophisticated surgical approaches and improved chemotherapeutic and radiotherapeutic options, the survival rates of patients with diffuse astrocytoma of, e.g., 12 months for glioblastoma multiforme is still disappointing (2). Therefore, there is an urgent need for new therapeutic strategies.

Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) efficiently induces apoptosis in glioma cell lines (3) but not in the majority of normal cells (4, 5) thereby representing a novel and promising anticancer cytokine. However, the therapeutic potential of TRAIL for gliomas has only been thoroughly investigated in cancer cell lines. Only very little is known about TRAIL sensitivity of primary glioma cells. Furthermore, many tumor cell lines (6, 7) and especially most primary tumor cells of hematologic origin (8) are TRAIL resistant and require sensitization for TRAIL-induced apoptosis. Bortezomib, the first proteasome inhibitor which is currently used as an anticancer drug in clinical trials (reviewed in ref. 9), sensitized some but not all TRAIL-resistant tumor cell lines, including glioma cell lines, for TRAIL-induced apoptosis (10, 11). However, no data exist thus far on the sensitization of primary glioma cells for TRAIL-induced apoptosis by bortezomib.

Here we assessed the apoptosis-inducing potential of bortezomib/TRAIL cotreatment in primary human glioma cells. We found that primary tumor cells from all 13 investigated glioma patients were TRAIL resistant but could be efficiently sensitized by bortezomib cotreatment. The concomitant activation of different levels of the TRAIL signaling pathway by bortezomib/TRAIL cotreatment seems to be responsible for its highly synergistic antitumor effect. Here, we provide the first native TRAIL death-inducing signaling complex (DISC) analysis of freshly isolated primary cells from solid tumors.

	Materials and Methods

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Cell lines
The human glioma cell line LN215 was maintained in DMEM containing 10% FCS (Invitrogen).

Isolation and cell culture of primary glioma cells
Surgical glioma specimens were obtained under the guidance of a neuronavigation system and intraoperative magnetic resonance imaging, which allowed for a selection of vital tumor tissue for consecutive analyses. For tumor cell isolation, fresh nonnecrotic surgical specimens were washed in PBS and mechanically disaggregated into small pieces, which were evenly distributed in a cell culture flask (Sarstedt) coated with AmnioMax medium (Invitrogen) and incubated at 37°C with 5% CO₂. After tumor cell outgrowth, tumor pieces were removed and cells were covered with supplemented AmnioMax medium. The study was approved by the Ethics Committee, Medical Faculty, University of Leipzig. Detailed information on glioma patients is given in Table 1 .

Western blot and fluorescence-activated cell sorting analysis
Preparation of cell lysates, immunoblotting, and fluorescence-activated cell sorting analysis of tumor cell lines and primary tumor cells were done as described (5, 12).

Cell viability assay, quantification of apoptosis, and clonogenicity assays
Cell viability was quantified by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Late apoptotic cell death was flow cytometrically quantified according to propidium iodide uptake in combination with characterization of cell shrinkage (decrease in forward scatter) and cellular fragmentation (increase in side scatter). Each measurement was done in triplicate as described before (5, 6). To verify apoptotic cell death, we checked for characteristic cell morphology (light microscopy), nuclear condensation/fragmentation (Hoechst 33342 staining), and caspase inhibition by z-VAD-fmk as described (5).

Clonogenicity assay. Primary tumor cells were cotreated with bortezomib and TRAIL. Dead cells were washed off with PBS after 16 h. Surviving cells were cultured for additional 14 days with medium being replaced once per week without any further death stimulus. At the indicated time points, cell viability was quantified by 3-[4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium] bromide assay.

Quantification of TRAIL death receptor contribution to bortezomib-mediated sensitization for TRAIL-induced apoptosis
Primary tumor cell lines were seeded and pretreated at 37°C with medium or 1 µg/mL TRAIL. Unbound TRAIL was removed after 90 min by five washing steps with PBS. Cells were subsequently sensitized overnight with 25 nmol/L bortezomib either alone or in combination with additional TRAIL (1 µg/mL). Control cells were left untreated, pretreated with TRAIL, or incubated with bortezomib overnight as indicated. Cell death was quantified by propidium iodide exclusion assay.

Ligand affinity precipitation
Biotinylated iz-TRAIL (Bio-iz-TRAIL) was generated for ligand affinity purification experiments essentially as described before for a leucine zipper–tagged form of TRAIL (6).

Lentiviral/retroviral plasmid construction, virus preparation, and transduction of target cells
The Bcl-2 open reading frame was excised from pMIG-Bcl-2 (ref. 13; obtained from Addgene, Inc.) and inserted into pIRES2 (Clontech) to yield pIRES2-Bcl-2. Subsequently, a CMV-Bcl-2-IRES-GFP–containing cassette was excised from pIRES2-Bcl-2 and inserted into pWPTS-eGFP (kindly provided by D. Trono, University of Geneva, Geneva, Switzerland) to replace the EF1--IRES-GFP cassette, yielding pLVIE-Bcl-2.

Lentiviral particles were prepared according to published protocols (14, 15) using pLVIE-Bcl-2, the packaging vector psPAX2, and the envelope plasmid pMD2G. Target cells were transduced by diluting the virus-containing supernatant 1:2 onto the target cells in the presence of 2 µg/mL polybrene (Sigma).

For generation of the retroviral cFLIP small interfering RNA (siRNA) expression vector pSRNG-FLIP911, sense and antisense oligomers containing the cFLIP targeting sequence 5'-gatccccGGAGCAGGGACAAGTTACAttcaagagaTGTAACTTGTCCCTGCTCCtttttggaaa-3' (sense strand) starting at position +911 were annealed and cloned into the pSuper.retro.neo.GFP retroviral vector using HindIII and BglII restriction sites. Viral particles were produced by transient transfection of the producer cell line Phoenix-Ampho together with the vesicular stomatitis virus G protein–expressing plasmid pMD2G. Virus-containing supernatant was harvested after 48 h and used to infect target cells in the presence of 2 µg/mL polybrene.

Transduced cells were sorted by green fluorescent protein (GFP) expression using a fluorescence-activated cell sorter. Bcl-2 overexpression and cFLIP down-regulation were verified by Western blot analysis.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Primary human glioma cells are TRAIL resistant but can be sensitized for TRAIL-induced apoptosis by bortezomib. Primary tumor cells were isolated from surgical specimens of 13 astrocytoma and oligoastrocytoma patients (Table 1) comprising all four WHO grades of malignancy. We established cell culture conditions for primary tumor cells, which provided constant proliferation rates and preserved the heterogeneous tumor cell phenotype for up to eight passages of in vitro culture. In contrast to most glioma cell lines, primary glioma cells from all investigated astrocytoma and oligoastrocytoma patients were highly TRAIL resistant but could be very efficiently sensitized for TRAIL-induced apoptosis by cotreatment with low concentrations of bortezomib (Fig. 1A ). Apoptotic cell death was confirmed by Hoechst 33342 staining and inhibition of cell death by the pan-caspase inhibitor z-VAD-fmk (data not shown). Longer exposure with bortezomib or TRAIL alone or higher concentrations of bortezomib (50 nmol/L) neither induced significantly more cell death in primary glioma samples nor increased the sensitization to TRAIL (see below and data not shown).

View larger version (49K): [in this window] [in a new window]   Fig. 1. Primary human glioma cells are TRAIL resistant but can be sensitized for TRAIL-induced apoptosis by bortezomib. A, primary human astrocytoma cells and oligoastrocytoma cells at passage 1 were treated with TRAIL together with 0 nmol/L (), 12.5 nmol/L (), or 25 nmol/L () bortezomib for 16 h. B, primary astrocytoma cells (patient #14) at passages 1, 2, 4, and 8, respectively, were treated for 16 h with TRAIL together with 0 nmol/L (), 12.5 nmol/L (), or 25 nmol/L () bortezomib. Late apoptotic cell death was quantified by flow cytometry. C, primary tumor cells of WHO grade II, III, and IV gliomas were left untreated or pretreated with 25 nmol/L bortezomib for 16 h and subsequently incubated with 0.1 µg/mL TRAIL for the indicated time periods. Cellular lysates were analyzed for the indicated proteins by Western blot. Molecular weights (kDa) are indicated. *, unspecific bands.

To investigate the occurrence of biochemical apoptotic events induced by TRAIL treatment, bortezomib-sensitized versus nonsensitized primary glioma cells were treated with TRAIL for different periods and cell lysates were analyzed for the cleavage of caspases and caspase substrates (Fig. 1C). Only in bortezomib-treated and not in nonsensitized primary glioma cells did TRAIL induce the rapid cleavage of caspase-8 and, consequently, the processing of Bid, caspase-9, caspase-7, and caspase-3 and the effector caspase substrate poly(ADP-ribose) polymerase. Accordingly, active caspase-3 was immunohistochemically detected only in sensitized but not in nonsensitized primary glioma cells on TRAIL treatment (data not shown).

Bortezomib enhances TRAIL DISC formation upon TRAIL triggering in primary glioma cells. Bortezomib treatment enhanced the surface expression of TRAIL-R1 (patients #14 and #17) or TRAIL-R2 (most prominently in patients #16, #17, and #20) in some primary glioma cells (Fig. 2A ). TRAIL-R3 and TRAIL-R4 could not be detected on the surface of nonsensitized or sensitized glioma cells (data not shown).

View larger version (57K): [in this window] [in a new window]   Fig. 2. Bortezomib enhances TRAIL DISC formation on TRAIL triggering in primary glioma cells. A, primary human astrocytoma cells of WHO grades I, II, III, and IV and oligoastrocytoma cells of WHO grades II and III were left untreated or treated with bortezomib for 16 h and analyzed for TRAIL-R1 and TRAIL-R2 expression by flow cytometry. B, contribution of TRAIL death receptor up-regulation for bortezomib-mediated sensitization for TRAIL-induced apoptosis was determined: Primary tumor cells (patient #13) were pretreated for 90 min with medium or TRAIL. Unbound TRAIL was washed off and cells were left untreated or sensitized with bortezomib overnight either with or without additional TRAIL. One of two independent experiments is shown. *, P < 0.05 (Mann-Whitney U test). C, primary glioma cells of WHO grades 2, 3, and 4 were left untreated or pretreated with 25 nmol/L bortezomib for 16 h. TRAIL receptors were immunoprecipitated from cell surface after 45-min TRAIL stimulation (+) or from total cell lysates without prior stimulation with TRAIL (–). Denatured precipitates were analyzed by Western blot for the indicated proteins. Molecular weights (kDa) and cleavage fragments are indicated.

To investigate the influence of bortezomib on TRAIL DISC formation, the native TRAIL DISC was immunoprecipitated from sensitized versus nonsensitized primary glioma cells of WHO grades I, II, and IV (Fig. 2C, lanes "+"). As a control, TRAIL receptors were precipitated from total cell lysates (when no TRAIL DISC can be formed; Fig. 2C, lanes "–"). Although TRAIL treatment induced TRAIL DISC formation in all nonsensitized primary gliomas, only little procaspase-8 (p55/p53) and cleaved caspase-8 (p43/41) were detectable in the TRAIL DISC. On sensitization with bortezomib, significantly more TRAIL-R1, TRAIL-R2, FADD, procaspase-8, caspase-8 p43/p41, procaspase-10 (only in patient #18), and caspase-10 p47/43 (only in patient #18) could be precipitated from primary tumor cells.

In contrast, full-length cFLIP long form (cFLIP_L) was hardly detectable, whereas the cleaved form of cFLIP_L (cFLIP_L p43) was apparent in all three investigated TRAIL DISCs. A background-normalized quantification of caspase-8 and cFLIP_L/cFLIP_L-cleaved in the TRAIL DISC of glioblastoma multiforme patient #20 was done. It revealed a 10-fold stronger caspase-8 recruitment to the sensitized versus the nonsensitized TRAIL DISC. In contrast, recruitment of cFLIP_L and the cleaved p43 fragment to the TRAIL DISC was only 1.4-fold enhanced by bortezomib. This results in an increase of the caspase-8/cFLIP ratio in the TRAIL DISC of sensitized cells.

Bortezomib influences the expression of intracellular regulators of TRAIL sensitivity. Total cell lysates of bortezomib-treated primary glioma cells of all four WHO grades of malignancy were analyzed by Western blot for the expression of regulators of TRAIL sensitivity (Fig. 3 ). In some tumor samples and to a different extent, bortezomib treatment resulted in accumulation of Bid (patients #27 and #3), Bak (patients #27 and #18), or Bax (patient #27; Fig. 3). Quantification of the cFLIP_L/ß-actin ratio shows a cFLIP_L down-regulation in all four glioma samples, most prominently in patient #3 (cFLIP_L/ß-actin ratio on 0, 12.5, and 25 nmol/L bortezomib, respectively, for patient #27: 1.1, 1.1, and 0.8; patient #17: 0.9, 0.5, and 0.5; patient #18: 0.9, 0.8, and 0.6; and patient #3: 1.1, 0.8, and 0.4). FADD, caspase-8, and caspase-10 were abundantly expressed in all primary glioma cells and were not significantly influenced by bortezomib (data not shown).

View larger version (15K): [in this window] [in a new window]   Fig. 3. Bortezomib influences the expression of intracellular regulators of TRAIL sensitivity. Primary glioma cells of all four WHO grades of malignancy were incubated for 16 h with bortezomib and whole-cell lysates were analyzed by Western blots for expression of regulators of TRAIL sensitivity. Molecular weights (kDa) are indicated. ß-Actin was used as a loading control.

View larger version (15K): [in this window] [in a new window]   Fig. 4. Bcl-2 and cFLIP expressions influence apoptosis induction by bortezomib/TRAIL cotreatment. A, human glioma cell line LN215 was treated for 16 h with TRAIL together with 0 nmol/L (), 12.5 nmol/L (), or 25 nmol/L () bortezomib. Apoptosis was measured by flow cytometry. TRAIL-R1 and TRAIL-R2 expression and whole-cell lysates of LN215 cells incubated with medium, 12.5, or 25 nmol/L bortezomib were analyzed by flow cytometry and Western blot, respectively. Molecular weights (kDa) are indicated. B and C, stably cFLIP siRNA–transfected (B) or Bcl-2–transfected (C) versus control transfected LN215 cells were cotreated with bortezomib and TRAIL and apoptotic cells were quantified after 16 h. Knockdown of cFLIPL/S/R and overexpression of Bcl-2 were verified by Western blot analysis (insets).

View larger version (24K): [in this window] [in a new window]   Fig. 5. Bortezomib/TRAIL cotreatment massively reduces the clonogenic survival of primary glioma cells. Primary glioma cells (#18) were treated for 16 h with medium (), 12.5 nmol/L (), or 25 nmol/L () bortezomib in combination with TRAIL as indicated; dead cells were washed off and fresh medium was added once per week. Cell viability was quantified by 3-[4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium] bromide assay at the indicated time points after initial treatment (top) or visualized by crystal violet at day 14 (bottom).

	Discussion

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Many TRAIL-resistant cancer cell lines could be sensitized for TRAIL-induced apoptosis by chemotherapeutic drugs including proteasome inhibitors (19). Here, we show that primary glioma cells from all investigated patients could be very efficiently sensitized for TRAIL-induced apoptosis by 16-h cotreatment with as little as 12.5 to 25 nmol/L bortezomib (Fig. 1). Furthermore, in combination with 25 nmol/L bortezomib, 100 ng/mL TRAIL massively reduced the clonogenic survival of primary glioma cells in long-term experiments (Fig. 5) but was not toxic to normal cells like primary human hepatocytes (20). Both TRAIL and bortezomib monotherapy are already in clinical trials, which will accelerate future clinical studies on their combination. Our study shows that concentrations of bortezomib far below the measured peak plasma level of bortezomib-treated patients (21) are sufficient to sensitize primary glioma cells for TRAIL. In contrast, applying bortezomib alone for 48 h, 100 nmol/L to 1 µmol/L bortezomib were needed to reduce the viability of ex vivo glioblastoma multiforme cells from two patients by only 60% (11). In contrast to bortezomib, other chemotherapeutic drugs were far less efficient (doxorubicin) or failed (etoposide) to sensitize primary glioma cells for TRAIL-induced apoptosis (data not shown) although they have been described to efficiently sensitize glioma cell lines for TRAIL-induced apoptosis (3, 22).

In accordance to other tumor cells (6, 23), some of the investigated primary glioma cells showed an enhanced surface expression of TRAIL-R1 and/or TRAIL-R2 on bortezomib treatment, which contributed to the sensitization for TRAIL-induced apoptosis (Fig. 2). Performing the first native TRAIL DISC analysis of primary glioma cells of WHO grades II, III, and IV, we found that bortezomib treatment resulted in enhanced TRAIL DISC formation on TRAIL triggering (Fig. 2C). Due to a reduction of the cFLIP_L protein level by bortezomib (Fig. 3), less cFLIP_L was recruited to the sensitized TRAIL DISC in comparison with FADD or caspase-8 (Fig. 2C), resulting in a change of the ratio between antiapoptotic and proapoptotic molecules in the TRAIL DISC of sensitized cells. This is in accordance with reports on cancer cell lines in which bortezomib sensitized for TRAIL-induced apoptosis by cFLIP_L down-regulation (24). Accordingly, as reported for other cell lines (6, 25), siRNA-mediated cFLIP down-regulation sensitized LN215 cells for TRAIL-induced apoptosis (Fig. 4B). In a recent report, three primary glioblastoma multiforme samples were TRAIL sensitive (26). TRAIL resistance in another three primary samples correlated with high expression of cFLIP_S (26). In our study, however, all primary tumor cells were TRAIL resistant regardless of their cFLIP_S expression level. PED/PEA-15 plays an important role in the regulation of TRAIL sensitivity in glioma cell lines (27). Representing an important regulatory difference to tumor cell lines, PED/PEA-15 was not recruited to the TRAIL DISC of primary glioma cells (data not shown) and does not seem be involved in the regulation of the TRAIL pathway in primary cells. Bortezomib-induced growth inhibition in glioma cell lines correlated with reduced transcriptional activity of nuclear factor B (11). However, recent data show that TRAIL sensitization of glioma cells by proteasome inhibition is nuclear factor B independent (10).

Bortezomib treatment has been shown to sensitize tumor cells for TRAIL-induced apoptosis by involvement of the mitochondrial pathway (28). Interestingly, some primary gliomas, although to a different extent, showed enhanced Bid, Bax, and/or Bak expression on bortezomib treatment (Fig. 3). The basal levels of these proteins differed considerably in the tumor samples (Fig. 3), underlining the heterogeneity of the investigated gliomas. Investigating the involvement of the intrinsic apoptosis pathway by bortezomib/TRAIL cotreatment, we found that Bcl-2 overexpression in LN215 glioma cells inhibited bortezomib-mediated toxicity but only marginally the sensitization of bortezomib for TRAIL-induced apoptosis (Fig. 4C).

Because all investigated tumor cells were efficiently sensitized for TRAIL-induced apoptosis, bortezomib cotreatment represents a powerful sensitizing tool for TRAIL-based therapies even in a heterogeneous population of cancer patients. We recently showed that bortezomib/TRAIL cotreatment at concentrations similar to those used in the present study are not toxic to primary human hepatocytes (6). It will be interesting to test potential adverse effects of bortezomib/TRAIL cotreatment on nonneoplastic normal brain tissue.

Taken together, having established a stable and reproducible in vitro model for the analysis of TRAIL-induced apoptosis in primary glioma cells, we showed that (a) primary glioma cells, in contrast to most glioma cell lines, are highly TRAIL resistant; (b) cotreatment with low concentrations of bortezomib and TRAIL efficiently sensitized all investigated primary human astrocytoma and oligoastrocytoma cells of all four WHO grades of malignancy for TRAIL-induced apoptosis; (c) this synergy involves regulatory mechanisms at the TRAIL DISC and presumably also at the intrinsic apoptosis pathway in a very heterogeneous cellular background; and, of clinical importance, (d) bortezomib/TRAIL cotreatment was superior to any single treatment in the reduction of clonogenic survival of primary tumor cells in vitro.

Thus, bortezomib/TRAIL cotreatment could represent a novel and effective therapeutic approach for glioma patients. In vivo experiments have to confirm the therapeutic potential of bortezomib/TRAIL cotreatment in primary glioma cells and are subject of our current studies.

	Acknowledgments

 
We thank Bärbel Moos, Jutta Mohr, and Rainer Baran-Schmidt for excellent technical assistance, Till Wenger (DKFZ Heidelberg) for help with lentiviral transduction, and Klaus Hexel for cell sorting.

	Footnotes

 
Grant support: Tumorzentrum Heidelberg/Mannheim (T.M. Ganten and H. Walczak), Deutsche Krebshilfe (H. Walczak), and scholarships from the Deutsche Forschungsgemeinschaft (R. Koschny and T.M. Ganten).

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

Received 1/31/07; revised 3/ 2/07; accepted 3/21/07.

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