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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Mol Cancer Ther. Author manuscript; available in PMC Jan 1, 2012.
Published in final edited form as:
PMCID: PMC3075591
NIHMSID: NIHMS253836
Bortezomib Sensitizes Malignant Human Glioma Cells to TRAIL, Mediated by Inhibition of the NF-κB signaling pathway
Esther P. Jane,1,2 Daniel R. Premkumar,1,2 and Ian F. Pollack1,2,3*
1Department of Neurosurgery, Pittsburgh, Pennsylvania
2University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
3University of Pittsburgh Cancer Institute Brain Tumor Center, Pittsburgh, Pennsylvania
* Address correspondence to: Ian F. Pollack, MD. FACS, FAAP., Department of Neurosurgery, Children's Hospital of Pittsburgh, 3705 Fifth Ave., Pittsburgh, PA 15213. Tel: 412-692-5881, Fax: 412-692-5921, ian.pollack/at/chp.edu
Previous studies have demonstrated that the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has significant apoptosis-inducing activity in some glioma cell lines, although many lines are either moderately or completely resistant, which has limited the therapeutic applicability of this agent. Because our recent studies demonstrated that inhibition of proteasomal function may be independently active as an apoptosis-inducing stimulus in these tumors, we investigated the sensitivity of a panel of glioma cell lines (U87, T98G, U373, A172, LN18, LN229, LNZ308, and LNZ428) to TRAIL alone and in combination with the proteasome inhibitor bortezomib. Analysis of these cell lines revealed marked differences in their sensitivity to these treatments, with two (LNZ308 and U373) of the eight cell lines revealing no significant induction of cell death in response to TRAIL alone. No correlation was found between sensitivity of cells to TRAIL and expression of TRAIL receptors DR4, DR5, and decoy receptor DcR1, caspase 8, apoptosis inhibitory proteins XIAP, survivin, Mcl-1, Bcl-2, Bcl-xL and cFLIP. However, TRAIL-resistant cell lines exhibited a high level of basal NF-κB activity. Bortezomib was capable of potentiating TRAIL-induced apoptosis in TRAIL-resistant cells in a caspase-dependent fashion. Bortezomib abolished p65-NF-κB DNA-binding activity, supporting the hypothesis that inhibition of the NF-κB pathway is critical for the enhancement of TRAIL sensitization in glioma cells. Moreover, knockdown of p65-NFκB by shRNA also enhanced TRAIL-induced apoptosis, indicating that p65-NFκB may be important in mediating TRAIL sensitivity and the effect of bortezomib in promoting TRAIL sensitization and apoptosis induction.
Keywords: Glioma, TRAIL resistance, bortezomib, apoptosis
Glioblastomas are highly invasive primary tumors with a poor prognosis despite current surgical, radiation and chemotherapies (1). Targeted therapies have failed to offer a long-term survival benefit in contrast to the improvements in outcome that have been achieved with new treatment approaches in many other cancer types. Genetic heterogeneity and a complex molecular pathology contribute to this lack of success, which highlights the need for novel therapies that target signaling pathways that underlie abnormal cellular growth. Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF/death receptor (DR) gene superfamily, is a promising anticancer agent because it induces apoptosis preferentially in cancer cells (2). TRAIL can bind to four plasma membrane receptors and one soluble receptor, i.e., TRAIL-R1 (DR4), TRAIL-R2 (DR5/KILLER), TRAIL-R3 [decoy receptor (DcR) 1], TRAIL-R4 (DcR2), and osteoprotegerin (3). TRAIL ligation with its receptors leads to formation of the death-inducing signaling complex (DISC), which recruits the adaptor molecule, FADD, and activates caspases-8 and -10. These activated caspases cleave and activate caspase-3, which in turn cleaves substrates that commit cells to undergo apoptosis (4). Alternatively, caspase-8 can cleave the pro-apoptotic Bcl-2 family member Bid, which translocates to the mitochondria and promotes mitochondrial dysfunction leading to apoptosis. This amplifies the apoptotic signal following death receptor activation (5).
TRAIL is a prime candidate for cancer therapy because it predominantly kills cancer cells while sparing normal cells. This tumor-selective cytotoxicity has been shown for glioma cells in comparison to non-transformed astrocytes in vitro (6). However, many glioma cell lines are resistant to TRAIL-induced apoptosis. The exact mechanism responsible for the resistance is not clear. Although the cell surface expression of DR4 or DR5 is required for TRAIL-induced apoptosis, tumor cells expressing these death receptors are not always sensitive to TRAIL due to intracellular aberrant activation of survival pathways (7). Recent reports suggest that TRAIL resistance may result from a combination of increased Akt activity (8), overexpression of Bcl-2 family members and other anti-apoptotic molecules (9), and deficient expression of caspases. Accordingly, TRAIL alone may not be sufficient to efficiently activate apoptosis in many solid tumors, including gliomas (8, 10). Although resistance of some cancer cells to TRAIL can be reversed by treatment with protein synthesis inhibitors (11) or chemotherapeutic agents (12), these approaches have not been successful in more highly resistant glioma cell lines. Hence, much effort has been made to identify new approaches to improve the efficacy of TRAIL-based apoptosis-inducing therapy.
In this regard, an siRNA-based screen by our group demonstrated that inhibition of proteasome function was a particularly potent independent strategy for apoptosis promotion in gliomas (13). Proteasome inhibitors (PIs) represent a promising novel class of anticancer agents (14) that is already in clinical use, as bortezomib (PS-341/Velcade) has been approved for the treatment of multiple myeloma (15). Moreover, PIs have been observed to induce apoptosis in solid tumors, such as lung and prostate cancer (16, 17), and several reports have shown that the combination of bortezomib and TRAIL overcomes the resistance to TRAIL in various types of cancer (18-22), although the primary mechanistic basis for this effect remains uncertain.
To address the potential contributors to TRAIL sensitivity in gliomas, we profiled the cytotoxic effects of this agent on eight malignant human glioma cell lines, demonstrating variable responses, with some cell lines being extremely sensitive and others highly resistant. We demonstrated that this differential sensitivity correlated with activation status of NF-κB. In addition, bortezomib exhibited potent anti-glioma activity and dramatically sensitized even highly resistant glioma cells to TRAIL cytotoxicity, suggesting that this may be a promising combination strategy for glioma therapeutics.
Cell lines
The established malignant glioma cell lines U87, T98G, U373, LN229, and A172 were obtained from the American Type Culture Collection (Manassas, VA). LN18, LNZ428, and LNZ308 were generously provided by Dr. Nicolas de Tribolet. U87, T98G, and U373 were cultured in growth medium composed of minimum essential medium; LN18, LN229, A172, LNZ428, and LNZ308 were cultured in α-minimal essential medium. All growth media contained 10% fetal calf serum, L-glutamine, 100 IU/ml penicillin, and 100 mg/ml streptomycin (Invitrogen, Carlsbad, CA) supplemented with sodium pyruvate and nonessential amino acids. Human astrocytes (HA) and human cerebellar astrocytes (HAC) were obtained from ScienCell Research Laboratories (San Diego, CA). Human umbilical vein endothelial cells (HUVEC) were purchased from Cell Applications Inc (San Diego, CA). Astrocytes and HUVEC growth media were obtained from respective vendors.
Reagents
Soluble human recombinant SuperKillerTRAIL™ (referred as TRAIL in this manuscript) was purchased from Enzo Biochemicals (Enzo Life Sciences, Plymouth Meeting, PA) diluted and stored in KillerTRAIL storage and dilution buffer (Enzo Life Sciences). Caspase inhibitors (Z-IETD-fmk, Z-LEHD-fmk, Z-DEVD-fmk, and Z-VAD-fmk) were purchased from R & D Systems (Minneapolis, MN). Bortezomib was purchased from Chemie Tek (Indianapolis, IN).
Cell Proliferation and Cytotoxicity Assay
Cells (5 × 103/well) were plated in 96-well microtiter plates (Costar, Cambridge, MA) in 100 μl of growth medium, and after overnight attachment, were exposed for 3 days to a range of concentrations of inhibitors, alone and in combination. Control cells received vehicle alone (DMSO). After the treatment interval, cells were washed in inhibitor-free medium, and the number of viable cells was determined using a colorimetric cell proliferation assay (CellTiter96 Aqueous NonRadioactive Cell Proliferation Assay; Promega, Madison, WI). All studies were conducted in triplicate and repeated at least three times independently. Morphological changes such as cell shrinkage, rounding, and membrane blebbing were evaluated by microscopic inspection of cells. Images were taken using an Olympus FluoView 1000 microscope. Images were assembled using Adobe Photoshop CS2 software (Adobe Systems).
Clonogenic Growth Assay
The effect of different inhibitor concentrations on cell viability was also assessed using a clonogenic assay. For this analysis, 250 cells were plated in six-well trays in growth medium, and after overnight attachment, cells were exposed to selected inhibitor concentrations or vehicle for 1 day. Cells were then washed with inhibitor-free medium and allowed to grow for 2 weeks under inhibitor-free conditions. Cells were then fixed and stained according to the manufacturer's protocol (Hema 3 Manual Staining Systems; Fisher Scientific, Pittsburgh, PA). After staining, six well plates were scanned and images were assembled using Adobe Photoshop CS2 software (Adobe Systems).
Annexin V Apoptosis Assay
Apoptosis induction in control (DMSO-treated) or inhibitor-treated cells was assayed by the detection of membrane externalization of phosphatidylserine with Annexin V-FITC conjugate using an Annexin V assay kit according to the manufacturer's protocol (Invitrogen). In brief, 2 × 105 cells were harvested at various intervals after treatment and washed twice with ice-cold phosphate-buffered saline (PBS) and resuspended in 200 μl of binding buffer. Annexin V-FITC and 1 μg/ml propidium iodide were added and cells were incubated for 15 min in a dark environment. The reaction was stopped by adding 300 μl of 1 × binding buffer, and labeling was analyzed by flow cytometry with a FACSCalibur flow cytometer (BD Biosciences, San Jose, CA).
Antibodies and Western blot analysis
The following antibodies were used: p21 Waf1/ Cip 1 (#2946), Bim (#2819), Bcl-2 (#2872), Bcl-xL (#2764), DcR3 (#4758), DcR1 (#4756), FLIP (# 3210), XIAP (#2042), Survivin (#2808), FADD (#2782), Mcl-1 (#4572), Bik (#4592), Bid (#2002), Cleaved PARP (#9546), Cleaved Caspase 3 (#9664), Cleaved Caspase 8 (9496), DR3 (#3254), NF-κB p65 (#3034), IκB-α (#4814), phospho- IκB-α -Ser32/36 (#9246), β-Actin (#4970) were from Cell Signaling Technology Inc., (Beverly, MA). DR4 (#IMG-141A) and DR5 (#IMG-120A) were from IMGENEX (San Diego, CA). Western blot analysis was performed as described previously (23). Scanning densitometry was performed on Western blots using acquisition into Adobe Photoshop (Adobe Systems, Inc., San Jose, CA) followed by image analysis (UN-SCAN-IT gel™, version 6.1, Silk Scientific, Utah, UT).
shRNA transfection
Four optimal 29mer-pRS-shRNA constructs were obtained from Origene (Rockville, MD). Sequences specific for human p65/NF-κB knockdown: GAT GAA GAC TTC TCC TCC ATT GCG GAC AT (shRNA-1); GCT GTG TTC ACA GAC CTG GCA TCC GTC GA (shRNA-2); CCA CCA TCA AGA TCA ATG GCT ACA CAG GA (shRNA-3); GAT CGT CAC CGG ATT GAG GAG AAA CGT AA (shRNA-4); non-targeting (control) sequences: TGA CCA CCC TGA CCT ACG GCG TGC AGT GC (shRNA-non-target) were used for this study. Glioma cells were seeded in a six-well plate (for Western analysis) or 96 well plate (cell proliferation assay) and maintained in complete media containing 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin and 100 mg/mL streptomycin at 37°C in a humidified atmosphere of 5% CO2. Transfection of p65/NF-κB shRNA was performed by using FuGene 6 according to the manufacturer's recommendations (Roche Applied Science, Indianapolis, IN). For control experiments, cells were transfected with a nontargeting (scrambled) shRNA construct. Cells were plated in culture plates and reached 70% to 80% confluence before transfection. For six well plates, two μg of p65/NF-κB B shRNA or non-targeting shRNA in 250 μL Opti-MEM medium was mixed with 6 μL of FuGene 6 in 250 μL Opti-MEM medium by vortexing. After the mixture was incubated at room temperature for 20 min, 1.5-mL of culture medium was added to make the total volume up to 2 mL. For 96 well plates, 25 ng of shRNA or non-targeting shRNA in 25 μl Opti-MEM medium was mixed with 0.6 μl of FuGene 6 in 25 μl Opti-MEM medium. After the mixture was incubated at room temperature for 20 min, complete medium was added to make the total volume up to 150 μl. Cell proliferation and Western blot analysis was performed as described above.
NF-kB DNA Binding Activity by ELISA and Electrophoretic mobility-shift (EMSA) assay
To detect p65/NF-kB binding activity, cell nuclear extracts were obtained using Nuclear Extraction kit (Active Motif, Carlsbad, CA). Nuclear extracts (15 μg) were used in each reaction in triplicate. The binding activity was evaluated using TransAM kits for NF-kB family members according to manufacturer's protocol (Active Motif). The absorbance was measured at wavelength of 450 nm by a microplate reader (Gene Mate-UniRead 800).
For NF-κB binding activity, a nonradioactive electrophoresis mobility-shift assay kit was used according to the manufacturer's instructions (Panomics, Fremont, CA). Briefly, malignant human glioma cell lines were incubated with or without inhibitors for the indicated period of time. Equal amounts of nuclear extracts was incubated at 15 °C for 30 minutes with a biotinylated oligonucleotide containing the p65/NF-κB binding site, and then the samples were separated on 6.0% non-denaturing polyacrylamide gel in 0.5 × TBE buffer for 90 minutes at 120 V at 4 °C. To confirm specificity, NFκB DNA binding was competitively blocked by an NFκB cold probe. The gel contents were transferred onto Biodyne B membrane (Pall, Ann Arbor, MI) for 45 minutes at 300 mA. The protein/DNA complexes were visualized after exposure to film.
DiOC6 labeling and detection of mitochondrial membrane depolarization
Mitochondrial membrane depolarization was measured as described (24). In brief, floating cells were collected, and attached cells were trypsinized and resuspended in PBS. Then the cells were loaded with 50 nM 3′,3′-dihexyloxacarbo-cyanine iodide (DiOC6, Invitrogen) at 37°C for 15 min. The positively charged DiOC6 accumulates in the intact mitochondria, whereas mitochondria with depolarized membranes accumulate less DiOC6. Cells were spun at 3,000 × g, and rinsed with PBS twice and resuspended in 1 ml of PBS. Fluorescence intensity was detected by flow cytometry (FACScan, Becton-Dickinson) and analyzed with the CellQuest software (Becton Dickinson). Percentage of cells with decreased fluorescence was determined.
Statistical analysis
Unless otherwise stated, data are expressed as mean ± S.D. The significance of differences between experimental conditions was determined using a two-tailed Student's t test. Differences were considered significant at p values <0.05.
Effect of Soluble TRAIL on Malignant Human Glioma Cell Lines
The cytotoxic effects of TRAIL were tested on a panel of eight glioma cell lines. Cell proliferation assays demonstrated that three of these cell lines, LN18, T98G, and LNZ428 were very sensitive (IC50 ranging between 2.7 to 10.1 ng/ml); LN229, A172 and U87 revealed moderate sensitivity (IC50 ranging between 31.3 to 41.8 ng/ml), whereas LNZ308 and U373 cells were resistant to TRAIL (IC50 >250 ng/ml) (Fig. 1A, top). As shown in Fig. 1A (bottom), treatment with TRAIL for 24 h induces cell death with characteristic apoptotic features, including cell detachment, shrinkage, and generation of apoptotic bodies as observed by phase contrast microscopy in TRAIL sensitive (LN18, T98G and LNZ428) and moderately sensitive (A172, LN229 and U87) cells but not in TRAIL-resistant cell lines (LNZ308 and U373). Western blot (Fig. 1B, top) and clonogenic growth assay (Fig. 1B, bottom) studies revealed that TRAIL treatment resulted in the significant PARP activation and cytotoxicity (colony formation) in TRAIL sensitive and moderately sensitive cells, but minimal or no activation in TRAIL-resistant cell lines.
Figure 1
Figure 1
Figure 1
Figure 1
TRAIL inhibits cell proliferation and induces apoptosis in malignant human glioma cell lines
To investigate the mechanisms controlling the resistance of glioma cells to the cytotoxic effect of TRAIL, a series of Western blot experiments were done to compare the expression of various components of the TRAIL signaling pathway among the eight glioma cell lines. No correlation was found between the sensitivity of cells to TRAIL and the expression of TRAIL receptors DR4, DR5 or decoy receptor for TRAIL, DcR1, initiator caspase 8, and apoptosis inhibitory proteins XIAP, survivin, Mcl-1, Bcl-2, Bcl-xL and cFLIP. All glioma cells showed high levels of Bcl-xL (Fig. 1C). Western blot analysis of LN18 (TRAIL-sensitive), U87 (moderately resistant), LNZ308, and U373 (TRAIL-resistant) cells did not show any significant changes in the levels of DR4, DR5, FLIP, and FADD after TRAIL treatment (Fig. 1D).
Bortezomib Sensitized TRAIL-resistant Glioma Cells
To investigate the potential of bortezomib to sensitize glioma to TRAIL cytotoxicity, we first examined the independent effect of bortezomib on cell proliferation in our panel of eight established glioma cell lines by MTS assay. Bortezomib resulted in a dose-dependent inhibition of cell proliferation with median effective concentrations of approximately 10 nM (Supplementary Figure 1, left). In order to confirm the specificity of bortezomib toward tumor cells, we compared the effect on normal cells (human astrocytes, HA; human cerebellar astrocytes, HAC; Human umbilical vein endothelial cells, HUVEC). At concentrations as low as 10 nM, bortezomib eliminated ≥75% of glioma cells, but had little or no effect on non-neoplastic cells (at 10nM), suggesting that bortezomib acts selectively against tumor cells compared with non-neoplastic cells (Supplementary Figure 1, right).
We then examined the ability of bortezomib to promote an apoptotic effect of TRAIL in the TRAIL-resistant LNZ308 and U373 cell lines, at clinically relevant concentrations. Apoptotic cells were determined by flow cytometry after 48 h treatment. Our results showed that both LNZ308 (Supplementary Figure 2, top) and U373 (Supplementary Figure 2, bottom) cells were resistant to TRAIL, as TRAIL alone was unable to induce apoptosis. However, combining bortezomib at 5 nM with TRAIL reversed the resistance and induced significant apoptosis in a dose-dependent manner starting with TRAIL concentrations as low as 1 ng/ml. In addition to short-term apoptosis assays, we also assessed the effect of bortezomib and TRAIL on long-term clonogenic survival of glioma cells. Colony-forming assays were used to determine whether inhibitor-treated cells re-enter the cell cycle and divide. Cells were cultured in the presence of bortezomib (5nM) or in combination with indicated concentrations of TRAIL for 1 day. After 1 day, inhibitor was removed and then cells were cultured in inhibitor-free medium for 14 additional days. Neither bortezomib (Fig. 2, top) nor TRAIL (Fig. 1B, bottom) alone resulted in a significant reduction of viable cells. In contrast, combinatorial treatment with bortezomib and TRAIL reduced cell viability significantly at day 14 after treatment (Fig. 2, top). In addition, simultaneous treatment with bortezomib and TRAIL resulted in a significant increase of cleaved caspase 3 and PARP, as determined by Western blot analysis (Fig. 2, bottom). These findings demonstrate that proteasomal inhibition cooperates with TRAIL to inhibit clonogenicity by preventing cells from re-entering the cell cycle and dividing and by inducing apoptosis.
Figure 2
Figure 2
TRAIL-resistant glioma cells can be sensitized by bortezomib
To understand the effect of bortezomib on TRAIL-resistant cell lines (U373 and LNZ308), we analyzed the involvement of caspase pathways using the chemical inhibitors IETD-fmk (caspase 8 inhibitor), LEHD-fmk (caspase 9 inhibitor), DEVD-fmk (caspase 3 inhibitor) and ZVAD-fmk (pan caspase inhibitor) in the culture media 4 h prior to treatment with bortezomib and TRAIL. Apoptotic cells were determined by flow cytometry and Western immunoblot analysis after 48-h treatment. When LNZ308 and U373 cells were pretreated with caspase inhibitors, apoptosis induction was inhibited, indicating that the enhanced apoptosis induction by the combination of bortezomib and TRAIL was caspase-dependent (Supplementary Figure 3).
Combination of TRAIL and bortezomib activates extrinsic and intrinsic pathways
Loss of mitochondrial transmembrane potential is another indicator of mitochondria dysfunction and insufficient activation of the extrinsic pathway has been implicated in contributing to TRAIL resistance at the mitochondrial level (25). Mitochondrial membrane depolarization and release of pro-apoptotic proteins (including cytochrome c, Smac/ DIABLO, AIF and HtrA2/Omi) have been implicated in both TRAIL and proteasome inhibitor-induced apoptosis (26). Because bortezomib induces loss of mitochondrial membrane depolarization and apoptosis (27), and mitochondrial changes are necessary for the activation of downstream caspases, we investigated the effect of bortezomib on mitochondrial membrane depolarization in the LNZ308 (Supplementary Figure 4, top) and U373 (Supplementary Figure 4, bottom) cell lines. Bortezomib induced mitochondrial membrane depolarization in a dose-dependent manner. However, combination of bortezomib and TRAIL induced strong increase in mitochondrial membrane depolarization. These results indicated that the synergistic interaction between the two stimuli is apparent at the level of mitochondria. Then we evaluated the role of caspases on the effects on membrane depolarization observed following treatment with TRAIL and bortezomib, by assessing the effect of pretreatment with various caspase inhibitors. As shown in supplementary figure 5, caspase inhibitors completely abolished the membrane depolarization induced by TRAIL and bortezomib. These results suggest that cotreatment of bortezomib and TRAIL affects the mitochondrial changes that typically occur during apoptosis.
Effects of Bortezomib on Death Receptors on TRAIL-resistant cell lines
To further elucidate the molecular events underlying the observed enhancement of apoptosis by the combination of TRAIL and bortezomib in TRAIL-resistant cell lines (U373 and LNZ308), we examined expression of TRAIL receptors and TRAIL DISC proteins, including FADD and c-FLIP, in bortezomib-treated cells. LNZ308 and U373 cells were exposed to varying concentrations and durations of bortezomib treatment and Western blot analysis was performed. As shown in Fig. 3A, bortezomib did not significantly alter the expression levels of DR4, DR5, FADD, and c-FLIP in U373 and LNZ308 cells. FADD, in particular, plays a key role in DISC formation and mediates TRAIL-induced apoptosis, and we found that the expression of FADD was minimally changed in the presence of bortezomib. We also analyzed the Bcl-2 family and proapoptotic protein levels. Bcl-2, Bcl-xL, Bik, Bid and Bim levels were not changed significantly in cells exposed to bortezomib. In contrast, the expression of Mcl-1 and p21 was significantly enhanced by bortezomib, whereas the expression of XIAP, an inhibitor of caspase-3 and -9, decreased in LNZ308 and U373 cells (Fig. 3B).
Figure 3
Figure 3
Figure 3
Bortezomib does not alter death receptor expression in TRAIL-resistant glioma cell lines
Bortezomib inhibits NF-κB-DNA binding activity and sensitizes glioma cells to TRAIL
It has been shown that bortezomib inhibits tumor cell growth by inhibiting NF-κB activation, particularly in tumors constitutively expressing this pivotal transcription factor (28). NF-κB activation in cancer cells contributes to resistance to TRAIL-induced apoptosis (29). Because NF-κB can modulate glioma cell survival and/or proliferation (30), we examined constitutive DNA-binding activity of NF-κB by electrophoretic mobility shift assay (EMSA) in eight established glioma cell lines. As shown in Fig. 4A, among the eight established glioma cell lines, TRAIL-resistant cell lines (LNZ308 and U373) exhibited the highest levels of basal NF-κB activity. For further investigation, we selected LN18, U87, LNZ308, and U373 and assessed NF-κB DNA-binding activity. Dose response analysis revealed that bortezomib markedly inhibited NF-κB DNA-binding activity in all four cell lines (Fig. 4B). Because IκB-α is a substrate of the ubiquitin-mediated proteasomal degradation pathway (31), bortezomib, a proteasome inhibitor, theoretically should stabilize IκB-α protein through inhibition of proteasome activity. When examining the effects of bortezomib on IκB-α degradation in three glioma cell lines, we surprisingly found that bortezomib decreased IκB-α levels while increasing phospho IκB-α levels in a dose-dependent manner which is consistent with the recent observation in multiple cancer cell lines (32). Furthermore, densitometric analysis showed that bortezomib effectively inhibited p65/ NF-κB levels (Fig. 4C). That prompted us to investigate the role of TRAIL and bortezomib on p65/NF-κB DNA-binding activity when the cells were treated with TRAIL or bortezomib or the combination of both. The EMSA data showed that (Fig. 4D), combination of TRAIL and bortezomib abolished p65/NF-κB DNA-binding activity and these observations support the idea that inhibition of NFκB pathway is critical for the enhancement of TRAIL sensitization in glioma cells.
Figure 4
Figure 4
Figure 4
Bortezomib inhibits NF-κB DNA-binding activity in glioma cell lines
p65/NF-κB gene silencing induces TRAIL cytotoxicity in glioma cells
To directly define the effects of p65/NF-κB inhibition on TRAIL-sensitivity, we compared the levels of TRAIL-induced apoptosis in cells transiently transfected with a p65/NF-κB-specific shRNA construct in LNZ308 and U373 cells. We also examined the effects of p65-NFκB shRNA knockdown on TRAIL sensitivity of LNZ308 and U373 cells. LNZ308 and U373 cells were transfected with p65/NF-κB shRNA, and 48 hours after transfection, Western blot analysis was done for p65/NF-κB expression and in parallel, cell viability was assessed by MTS assay. Immunoblotting confirmed that the p65/NF-κB-specific shRNA construct efficiently reduced p65/NF-κB protein levels (40-50% reduction compared to non-target shRNA) (Fig. 5A, top). As shown in Fig. 5A (bottom), p65/NF-κB knockdown significantly inhibited cell proliferation. To determine whether direct inhibition of p65/NF-κB enhanced TRAIL-sensitivity, LNZ308 and U373 cells were transiently transfected with non-target shRNA or shRNA-1 against p65/NF-κB. Forty-eight hours post-transfection, cells were treated with varying concentrations of TRAIL for 72 hours, and cell proliferation was assessed by MTS assay. As shown in Fig. 5B, TRAIL at the concentrations used had minimal or no effect on cell viability when added with non-target shRNA. In contrast, strong inhibition of cell proliferation was observed when p65/NF-κB shRNA interference and TRAIL were combined. Taken together, p65/NF-κB knockdown synergized with TRAIL to promote apoptosis in LNZ308 and U373 cells.
Figure 5
Figure 5
p65/NF-κB Knockdown induces TRAIL cytotoxicity in TRAIL-resistant glioma cells
Combined treatment with TRAIL and bortezomib does not induce cell death in non-neoplastic astrocytes and HUVECs
To characterize the potential interactions between bortezomib and TRAIL on normal cell viability, human astrocytes (HA), human cerebellar astrocytes (HAC) and human umbilical vein endothelial cells (HUVEC) were exposed to bortezomib or TRAIL or the combination of both and cell viability was assessed after 3 days by MTS assay. The combination of bortezomib and TRAIL had little or no effect on HA proliferation (Supplementary Figure 6, left). To investigate the molecular mechanisms for this differential sensitivity compared to glioma cells, HA, HAC, and HUVEC cells were exposed to bortezomib or TRAIL or the combination of both and a series of Western blot experiments were done to compare the expression of various components of the TRAIL signaling pathways. Unlike glioma cells (LNZ308 and U373), we found no expression of TRAIL receptors DR4 and DR5 or caspase activation. High expression of Bcl-xL was observed in HA, HAC, and HUVEC cells (Supplementary Figure 6, right). Taken together, these results may explain the preferential cytotoxicity of bortezomib and TRAIL co-treatment in glioma cells, sparing astrocytes and endothelial cells.
Proteasome inhibitors have attracted recent attention as potential antitumor agents but the mechanism by which proteasome inhibitors induce apoptosis is poorly understood. The ubiquitin/proteasome pathway represents the primary mechanism by which the bulk of cellular proteins in proliferating cells are degraded. By regulating proteolysis, and thus intracellular protein levels, the proteasome plays an important role in cell growth and apoptosis (33). A TRAIL-sensitizing effect of bortezomib has been noted in various types of cancer (19, 22, 34), whereas the molecular mechanisms responsible for this synergy remain less clear-cut (35). Molecular targets responsible for the sensitizing effect of bortezomib on TRAIL-induced cell death include DR4 (18), DR5 (18, 20, 21), c-FLIP (20, 21), NF-κB (34, 36), p21 (19), and p27 (37). In addition, Bcl-2 family members also play a role in the combinational effect of bortezomib and TRAIL, including Bcl-2 (38), Bax (21), Bak (35), Bcl-xL (38), Bik (39), and Bim (40).
Although potential up-regulation of TRAIL receptors has been suggested as one explanation for a TRAIL-sensitizing effect of bortezomib in various cancer cell lines (18, 20, 21), including some gliomas (13) we did not observe this effect in any of the eight glioma cell lines we tested, suggesting that this mechanism may not be the primary contributor in these tumors. Because we observed a strong inverse association between NF-κB activation status and TRAIL response, we examined whether TRAIL sensitization by bortezomib reflected effects on this target. We not only observed a strong association between NF-κB inhibition by bortezomib and TRAIL sensitization, but also observed a similar potentiating effect of NF-κB-directed shRNA. This is consistent with observations in several other cell lines that the apoptosis potentiating effect of bortezomib reflects its effects on inhibiting the NF-κB signaling pathway (28, 41). Although inactivation of NFkB has been suggested to play a major role in the antitumorigenic effect of bortezomib in multiple myeloma (42) and melanoma cells (43), inhibition of NF-κB is not required to sensitize hepatocellular carcinoma cells and lymphoma cells to apoptosis (44, 45), emphasizing the cancer type-specific effects of this agent.
Given the involvement of NF-κB activation in glioma cell resistance to TRAIL-induced apoptosis, we examined the effects of bortezomib on the levels of IkB-α, a key protein that regulates NF-κB activation. In the tested cell lines, we found that bortezomib decreased IkB-α level. The observation that bortezomib reduced IkB-α levels in human glioma cells is novel and, these findings are consistent with previous studies demonstrating that PS-341 as well other proteasome inhibitors induce IkB-α degradation in endometrial carcinoma cell lines (46), HepG2 liver cancer cell line (47), multiple myeloma cells (48) and other tumor types (32). Recently, it has been shown that activated IκB kinase (IKK) complex phosphorylates IκB-α on serines 32 and 36, leading to subsequent ubiquitination and proteasome-mediated degradation of IκB-α (32). Our results show that bortezomib induces IκB-α phosphorylation on serines 32 and 36 accompanied by reduction of IκB-α levels. This finding is in agreement with previous work demonstrating that PS-341 and other proteasome inhibitors induce IKK-dependent IκB-α phosphorylation and degradation in cancer cell lines (48, 49). Our data suggest that bortezomib treatment not only induces IκB-α degradation, NF-κB inhibition and independent apoptosis induction, but also strongly enhances TRAIL-mediated cytotoxicity in malignant human glioma cell lines.
Our data also indicate that various targets of NF-κB may contribute to the apoptosis-modulating effects observed. For example, XIAP is an important anti-apoptotic protein that confers resistance to TRAIL-cytotoxicity (50) and our results demonstrated striking down-regulation of XIAP expression by bortezomib treatment in a dose-dependent manner. Likewise, Mcl-1 and p21 expression were significantly up-regulated in response to bortezomib, and it is conceivable that a combination of NF-κB-mediated effects may be involved in the TRAIL sensitization seen in the resistant U373 and LNZ308 cell lines.
In summary, we report that inhibition of p65-NFκB is an efficient strategy to broadly sensitize glioblastoma cells to TRAIL therapy, either via the death receptor (extrinsic) apoptosis pathway or via the mitochondrial (intrinsic) apoptosis pathway. The combination treatment is superior to single agent therapy in suppressing glioma cell viability. In addition, genetic silencing of p65/NF-κB shRNA significantly enhances TRAIL-induced apoptosis. From a therapeutic standpoint, our findings suggest that the levels of p65/NF-κB in glioma could have prognostic value for predicting the efficiency of TRAIL-based therapy. Moreover, our findings provide strong evidence that inhibition of p65/NF-κB is a promising approach to lower the threshold for apoptosis. In particular, bortezomib sensitizes glioma cells to TRAIL by inhibiting IκB-α and NF-κB DNA binding activity. Taken together, our results provide a strong rationale for combining TRAIL with proteasomal inhibition as a novel therapeutic combination for gliomas.
Supplementary Material
Acknowledgments
We thank Robert Lacomy, Peter Kang and Naomi Agostino for assistance.
This work was supported by National Institutes of Health Grant P01NS40923 (I.F.P) and by the Walter L. Copeland of The Pittsburgh Foundation (D.R.P).
Footnotes
Conflict of Interest: None declared
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