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Molecular Cancer Research 5, 783-792, August 1, 2007. doi: 10.1158/1541-7786.MCR-06-0433
© 2007 American Association for Cancer Research
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Cell Cycle, Cell Death, and Senescence

Ribotoxic Stress Sensitizes Glioblastoma Cells to Death Receptor–Induced Apoptosis: Requirements for c-Jun NH2-Terminal Kinase and Bim

Shuli Xia1,2, Yang Li1, Eliot M. Rosen5 and John Laterra1,2,3,4

1 The Kennedy-Krieger Institute and Departments of 2 Neurology, 3 Oncology, and 4 Neuroscience, School of Medicine, Johns Hopkins University, Baltimore, Maryland; and 5 Department of Oncology, Lombardi Cancer Center, Washington, District of Columbia

Requests for reprints: John Laterra, Room 400, Kennedy Krieger Research Institute, 707 N. Broadway, Baltimore, MD 21205. Phone: 443-923-2683; Fax: 443-923-2695. E-mail: laterra{at}kennedykrieger.org


   Abstract
Top
 Notes
 Abstract
Introduction
 Results
 Discussion
 Materials and Methods
 References
 
A prominent feature of glioblastoma is its resistance to death receptor–mediated apoptosis. In this study, we explored the possibility of modulating death receptor–induced cell death with the c-Jun-NH2-terminal kinase (JNK) activator anisomycin. Anisomycin activates JNK by inactivating the ribosome and inducing "ribotoxic stress." We found that anisomycin and death receptor ligand anti-Fas antibody CH-11 or tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) synergistically induce apoptosis in multiple human glioblastoma cell lines. For example, in U87 cells, anisomycin reduced the IC50 of CH-11 by more than 20-fold (from 500 to 25 ng/mL). Cell viability in response to anisomycin, CH-11, and their combination was 79%, 91%, and 28% (P < 0.001), respectively. Anisomycin and TRAIL were found to be similarly synergistic in glioblastoma cells maintained as tumor xenografts. The potentiation of death receptor–dependent cell death by anisomycin was specific because emetine, another ribosome inhibitor that does not induce ribotoxic stress or activate JNK, did not have a similar effect. Synergistic cell death was predominantly apoptotic involving both extrinsic and intrinsic pathways. Expression of Fas, FasL, FLIP, and Fas-associated death domain (FADD) was not changed following treatment with anisomycin + CH-11. JNK was activated 10- to 22-fold by anisomycin + CH-11 in U87 cells. Inhibiting JNK activation with pharmacologic inhibitors of JNKK and JNK or with dominant negative mitogen-activated protein kinase (MAPK) kinase kinase 2 (MEKK2) significantly prevented cell death induced by the combination of anisomycin + CH-11. We further found that anisomycin + CH-11 up-regulated the proapoptotic protein Bim by ~14-fold. Simultaneously inhibiting Bim expression and JNK activation additively desensitized U87 cells to anisomycin + CH-11. These findings show that anisomycin-induced ribotoxic stress sensitizes glioblastoma cells to death receptor–induced apoptosis via a specific mechanism requiring both JNK activation and Bim induction. (Mol Cancer Res 2007;5(8):783–92)


   Introduction
Top
 Notes
 Abstract
 Introduction
Results
 Discussion
 Materials and Methods
 References
 
Gliomas account for more than 50% of all primary brain tumors in adults. In spite of considerable progress in modern tumor therapy including aggressive surgery, radiotherapy, and chemotherapy, the outcome is very limited (1). Targeting apoptosis by employing the death receptor pathway constitutes a promising therapeutic strategy (2). Death receptor–induced apoptosis is initiated by ligand-induced multimerization of death receptors, i.e., Fas, tumor necrosis factor (TNF)–related apoptosis-inducing ligand (TRAIL), and TNF-{alpha} receptors, followed by recruitment of the cytoplasmic adapter protein Fas-associated death domain (FADD). Recruited FADD interacts with procaspase-8 or procaspase-10 through its death effector domain to form a death-inducing signaling complex (DISC; refs. 3-5). DISC stimulates caspase-8/10 activation that induces apoptotic cell death by activating caspase-3 either directly or indirectly via the mitochondrial apoptotic pathway (4, 5).

Targeting death receptors to trigger apoptosis in gliomas is an attractive concept for cancer therapy because the cytotoxic effect of death receptor activation is relatively selective to cancer cells compared with normal cells (6-8). However, various genetic and/or epigenetic alterations in glioblastoma lead to a dysfunctional cell death pathway and the impaired ability of cells to undergo apoptosis. Resistance of cancer cells to death receptor activation potentially occurs through the modulation of molecular targets involved in apoptosis pathways, such as differential expression of death receptors, constitutive activation of cell survival pathways, overexpression of antiapoptosis proteins, and defects in mitochondrial protein release (9-12). Therefore, the use of a death receptor activation approach alone may not be enough to trigger apoptosis. Harnessing the death-inducing pathway may ultimately require sensitization by other means. Studies have shown that in cancer cells from other origins, histone deacetylase inhibitors, retinoids, chemotherapeutic drugs, and irradiation and translation inhibitors can sensitize resistant cells to undergo apoptosis (13-16). These drugs influence cell sensitivity by changing the expression of cell surface death receptors and cellular proteins involved in DISC as well as proteins involved in mitochondrial apoptosis pathway and down-regulating cell survival pathways such as Akt and nuclear factor-{kappa}B (NF-{kappa}B; ref. 14-16). Thus, combining death receptor ligands with other approaches may prove to be useful in cancer therapy.

We previously found that camptothecin sensitizes glioma cells to Fas-mediated apoptosis via a mechanism involving oxidative stress (15). Although c-Jun-NH2-terminal kinase/stress-activated protein kinase (JNK) was concurrently activated by camptothecin, the role of JNK activation in sensitizing glioma cells to Fas-induced cell death has remained unclear. Anisomycin interacts directly with and inhibits 28S rRNA and thereby indues "ribotoxic stress" and potently activates JNK (17-19). We used anisomycin to explore the role of JNK in the pathways that sensitize glioblastoma cells to apoptosis induced by death receptor activation. We found that death receptor activation, together with anisomycin, induced synergistic cell death in glioblastoma cells. JNK activation was required for cell death. We further found that the up-regulation of the proapoptotic protein Bim was also required for maximal apoptosis in response to anisomycin + death receptor activation.


   Results
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 Notes
 Abstract
 Introduction
 Results
Discussion
 Materials and Methods
 References
 
Anisomycin and Death Receptor Ligands Synergistically Induce Apoptosis in Glioblastoma Cultures
U87 human glioblastoma cells display modest levels of Fas receptor expression, but they are not sensitive to Fas activation–induced apoptosis (15, 20). We examined the response of U87 glioma cells to agonistic anti-Fas antibody (CH-11) alone or in conjunction with the strong JNK activator anisomycin. At the concentration of 50 ng/mL, CH-11 did not induce cell death by itself (Fig. 1A, bottom left ). There was no obvious cell death when U87 cells were treated with anisomycin at 0.1 µg/mL (Fig. 1A, top right). However, the majority of U87 cells died following the coapplication of anisomycin + CH-11 for 24 h (Fig. 1A, bottom right). Flow cytometry analysis with annexin V–FITC staining was used to quantify apoptotic cell death. After 24 h of treatment, annexin V–FITC–positive cells were 6.53% in control, 8.3% with anisomycin alone, 13.7% with CH-11 alone, and 62.7% with the combination of anisomycin + CH-11 (Fig. 1B). The flow cytometry data indicated that anisomycin was not death inducing on its own but sensitized cell death induced by CH-11.


Figure 1
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  		FIGURE 1. Anisomycin sensitizes glioblastoma cells to death receptor–mediated apoptosis. A. Phase contrast photomicrographs showing synergistic cell death with anisomycin + CH-11 after 24 h incubation (bottom right). Anisomycin (0.1 µg/mL; top right) and CH-11 alone (50 ng/mL; bottom left) does not cause obvious cell death. Bar, 10 µm. B. U87 cells were treated with drugs and stained with annexin V–FITC/propidium iodide. Percentage of annexin V–positive cells is plotted. Anisomycin alone (Ani) and CH-11 alone induce only 8.3% and 13.7% apoptosis, respectively. Anisomycin + CH-11 cause 62.7% of cells to undergo apoptosis. *, P < 0.001 versus each drug alone. C, D. Anisomycin and CH-11 synergistically induce U87 cell death as determined by MTT assay. C. Anisomycin alone (x) decreases the number of viable cells by 20% due to cell growth arrest. CH-11 alone ({diamondsuit}) at 500 ng/mL only induces about 20% cell loss. Both together ({blacksquare}) synergistically induce cell death. D. In U87 cells, CH-11 alone (50 ng/mL, x) causes only 6% cell loss. Anisomycin alone ({diamondsuit}) induces cell growth arrest. CH-11 together with anisomycin ({blacksquare}) at ≥0.03 µg/mL synergistically induces cell loss. E. Anisomycin sensitizes U373 cells to CH-11–induced apoptosis. CH-11 (50 ng/mL) induces about 7% cell death. F. Anisomycin from 0.3 to 10 µg/mL potentiates CH-11–induced cell death in U373 cells. U87 cells were treated with anisomycin or emetine alone (open columns) or in combination with CH-11 (50 ng/mL; filled columns) for 24 h followed by MTT assay. Anisomycin (0.1 µg/mL) but not emetine (10 µmol/L) sensitizes U87 cells to CH-11–induced cell death. *, P < 0.001 versus anisomycin alone. G. Anisomycin sensitizes TRAIL-induced cell death in U87 cells. Anisomycin (0.1 µg/mL) induces ~20% cell loss. TRAIL at 30 ng/mL induces ~22% cell death. Both drugs together cause ~68% cell loss. *, P < 0.001 versus each drug alone. H. Human glioblastoma cultures Mayo 22 (solid line) and Mayo 16 (dotted line) were treated with anisomycin, TRAIL, or their combination. The cells are not sensitive to TRAIL alone (50 ng/mL). Together with anisomycin (1 µg/mL), there is a 70% to 80% cell loss. Experiments were repeated at least thrice (n = 12). Values represent mean ± SE.

 
The response of U87 cells to anisomycin alone was further analyzed by cell counting and flow cytometry assay. U87 cells were treated with different concentrations of anisomycin for 48 h, and live cells were counted daily. At concentrations from 0.01 to 0.3 µg/mL, anisomycin induced cell growth arrest because the cell number remained the same as that before drug addition (data not shown). Cell cycle analysis with flow cytometry showed that, compared with control, anisomycin at the concentration of 0.1 µg/mL increased the fraction of cells in S phase and G2-M phase by more than 3- and 2-fold, respectively. Thus, there was a G2-M phase blockage with anisomycin. The apoptosis fraction (sub–G1-G0) remained very small following anisomycin treatment (data not shown).

Cell death after CH-11 + anisomycin was further analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. U87 cell growth was inhibited when treated with anisomycin alone (Fig. 1D). Even very high concentrations of CH-11 (>500 ng/mL) induced only a small degree of cell death in U87 cells (Fig. 1C). This was changed by coapplication with a sublethal concentration of anisomycin. When anisomycin was coapplied with CH-11, U87 cells became very sensitive to CH-11. After 24 h incubation, in response to CH-11 (50 ng/mL) alone, anisomycin (0.1 µg/mL) alone, or their combination, cell viability was found to be 91%, 79%, and 28%, respectively (Fig. 1C). Anisomycin reduced the median inhibitory concentration (IC50) of CH-11 more than 20-fold in U87 cells, from >500 to 25 ng/mL (Fig. 1C). On the other hand, adding CH-11 together with various concentrations of anisomycin also changed cell response to anisomycin. CH-11 (50 ng/mL) with anisomycin at the range of 0.03 to 0.3 µg/mL induced rapid cell death (Fig. 1D). We used the isobologram equation as described in Materials and Methods to determine whether there was synergism between anisomycin and CH-11. In these cases, Ix was calculated to 0.68, indicating that CH-11 and anisomycin act synergistically to induce cell death in U87 cells. Similarly, anisomycin from 0.3 to 10 µg/mL also potentiated CH-11–induced cell death in U373 cells. Cell loss in the presence of CH-11 (50 ng/mL) alone, anisomycin (1 µg/mL) alone, or the combination of both was 7%, 23%, and 48% (P < 0.001 versus either drug alone), respectively (Fig. 1E). In contrast, another translation inhibitor, emetine, that inactivates the 28S ribosome, but does not induce ribotoxic stress or JNK activation (17), did not potentiate CH-11–induced cell death in U87 cells (Fig. 1F). CH-11–induced cell death was also not potentiated by the transcription inhibitor actinomycin D (data not shown). Thus, the death-potentiating effect of anisomycin was not simply due to nonspecific effects of protein synthesis inhibition.

We examined whether anisomycin sensitized glioblastoma cells to cell death induced by another death receptor ligand TRAIL. In response to TRAIL alone (30 ng/mL), fewer than 20% of U87 cells died. By adding anisomycin (0.03 µg/mL) together with TRAIL, more than 70% of cells died (P < 0.001 versus either drug alone, Fig. 1G). Thus, anisomycin potentiates death induced by multiple distinct death receptors. We extended these observations to human glioblastoma cell lines (Mayo 16 and Mayo 22) that more accurately model certain molecular and biological characteristics of human tumors. Because these cells were found to express DR4 but not Fas receptor (flow cytometry data not shown), their sensitivity to TRAIL was examined in the presence or absence of anisomycin. These cultures were not sensitive to TRAIL alone. Anisomycin (0.1-1 µg/mL) sensitized both Mayo 16 and Mayo 22 cells to TRAIL. For Mayo 16, cell viability in the presence of TRAIL (50 ng/mL), anisomycin (1 µg/mL), and their combination was 109%, 56%, and 28% (P < 0.001 versus either drug alone), respectively. For Mayo 22, cell viability in the presence of TRAIL (50 ng/mL), anisomycin (1 µg/mL), and their combination was 97%, 50%, and 12% (P < 0.001 versus either drug alone), respectively (Fig. 1H). An isobologram analysis of these cell responses to TRAIL (1-100 ng/mL) and anisomycin (0.03-1 µg/mL) alone or in combination was done. The Ix values for Mayo 22 and Mayo 16 cells calculated to 0.94 and 0.91, respectively. Thus, TRAIL and anisomycin synergistically induced cell death in primary glioblastoma cells as well.

Cell Death Pathways Involved in Apoptosis Induced by CH-11 + Anisomycin
Using the synergistic cell death in U87 cells induced by CH-11 + anisomycin as a model, we studied the mechanisms involved in the cell death induced by death receptor activation and anisomycin. Preincubation with the pan-caspase inhibitor z-VAD-FMK (50 µmol/L) for 30 min rescued more than 70% of U87 cells from death induced by anisomycin + CH-11 (Fig. 2A ). Thus, cell death induced by anisomycin + CH-11 was predominantly through caspase-dependent apoptosis. This was further confirmed based on the cleavage of caspase-3, caspase-8, and poly(ADP-ribose) polymerase (PARP) during the time course of drug treatment as detected by Western blot analysis (Fig. 2B). Caspase-8 was cleaved after 6 h of the combined treatment; whereas with either CH-11 alone or anisomycin alone, only weak bands of cleaved caspase-8 could be detected after 24 h of treatment. Caspase-3 and PARP were also cleaved at 12 h of combined treatment (Fig. 2B). The apoptosis response involved both extrinsic and intrinsic apoptosis pathways because the proapoptosis protein Bid, the linker between extrinsic and intrinsic apoptosis pathways, was also cleaved as early as 6 h after the combined treatment. There was a dramatic decrease in total Bid protein level consistent with Bid cleavage within 6 to 12 h of treating cells with both anisomycin + CH-11 (Fig. 2C).


Figure 2
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  		FIGURE 2. Synergistic cell death induced by anisomycin + CH-11 involves both intrinsic and extrinsic apoptotic pathways. A. U87 cells were preincubation with pan-caspase inhibitor Z-VAD-FMK (Z-Vad, 50 µmol/L) for 30 min before drug treatment. Z-VAD-FMK rescues ~70% of cells from death induced by anisomycin (0.1 µg/mL) + CH-11 (50 ng/mL). *, P < 0.001 versus control; +, P < 0.001 versus CH-11 + Ani; n = 9. B, C. U87 cells were treated with anisomycin (Ani, 0.1 µg/mL) and/or CH-11 (50 ng/mL) for the indicated times, and cell lysates were subjected to Western blot analysis. The cleavage products of caspase-8/3 and PARP are shown in (B). Caspase-8 is cleaved as early as 6 h after the combined treatment. Caspase-3 and PARP are cleaved after 12 h of the combined treatment. Proapoptotic protein Bid is cleaved in the presence of anisomycin + CH-11 as early as 6 h based on the loss of total Bid protein (C). Experiments were repeated thrice, and representative data are shown.

 
Extrinsic Apoptotic Pathway Modulation by Anisomycin
We examined the mechanisms by which anisomycin potentiates Fas-induced apoptosis. As a ribotoxin, anisomycin binds active 28S ribosomes and blocks protein synthesis. As a protein synthesis inhibitor, anisomycin might potentiate cell death by causing generalized cellular stress or by inhibiting the synthesis of certain antiapoptosis proteins (21-23). Therefore, we examined the expression level of proteins involved in the extrinsic and intrinsic apoptosis pathways. We first investigated modulation of the death receptor pathway, the critical components of which include Fas, FasL, FADD, and FLIP. Using flow cytometry analysis, we detected no substantive increase in the expression level of Fas after the combined treatment (Fig. 3A ). Cell-associated FasL was not detected in U87 cells, either with or without drugs (Fig. 3A). Using Western blot, we found no increase in FADD and or decrease in FLIP expression after the combined treatment (Fig. 3B). Thus, it appears that anisomycin does not potentiate Fas-induced cell death through changes in the expression of individual DISC components.


Figure 3
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  		FIGURE 3. Expression of extrinsic apoptosis signaling pathway constituents. A. Flow cytometry analysis of cell surface Fas and FasL in U87 cells. Mouse IgG was used as negative control. There is a slight decrease in cell surface Fas (open columns) in response to CH-11 or anisomycin + CH-11. Cell surface–bound FasL (filled columns) is not detected. B. U87 cells were treated with anisomycin (Ani, 0.1 µg/mL) and/or CH-11 (50 ng/mL) for the indicated times, and cell lysates were subjected to Western blot analysis. FLIP and FADD expression was not affected by anisomycin, either alone or combined with CH-11. Experiments were repeated thrice, and representative data are shown.

 
JNK Activation Is Necessary but not Sufficient in Potentiating Cell Death Induced by Anisomycin + CH-11
As mentioned above, anisomycin inhibits the 28S ribosome, which induces stress responses and JNK activation. We asked if the JNK pathway plays a direct role in CH-11/anisomycin synergistic cell killing. In U87 cells, anisomycin alone or in conjunction with CH-11 induced sustained and strong JNK activation as evidenced by Western blot analysis (Fig. 4A ). Compared with control, JNK1 and JNK2 were activated 5-fold and 12-fold with anisomycin alone at 12 h. JNK activation was sustained for at least 24 h without apparent decrease. CH-11 alone induced very weak JNK activation or had a borderline effect on JNK activation at up to 24 h. The combination of anisomycin + CH-11 caused stronger activation of JNK1 (10-fold, P < 0.001, n = 4) and JNK2 (22-fold, P < 0.001, n = 4) at 12 h than with anisomycin alone (Fig. 4A). We studied the contribution of JNK activation to synergistic cell death using the specific JNK pathway inhibitors CEP-11004 (inhibits the JNK upstream kinase JNKK1/2), the direct JNK inhibitor L-JNKI1 and a mitogen-activated protein kinase (MAPK) member p38 inhibitor SB203580 as a control. Western blot analysis indicated that CEP-11004 inhibited anisomycin-induced JNK1/2 phosphorylation by 78% (Fig. 4B). Furthermore, preincubation with CEP-11004 substantially (>80%) reduced JNK phosphorylation induced by anisomycin + CH-11 (Fig. 4C). Preincubation with CEP11004 or L-JNKI1 for 24 h significantly reversed cell death induced by anisomycin + CH-11. In cells treated with anisomycin + CH-11, ~31% of the cells remained viable. CEP-11004 or L-JNKI1 pretreatment increased the cell viability to 56% and 49%, respectively. In cells pretreated with SB203580, viability remained low at 27% (Fig. 4D). Compared with control (100%), CEP-11004, L-JNKI1, or SB203580 alone generated 115%, 109%, or 114% cell viability, respectively. Thus, they did not affect baseline cell viability. Annexin V–FITC/flow cytometry analysis revealed that CEP-11004 reduced apoptosis induced by anisomycin + CH-11 from 75% to 43% (Fig. 4E, P < 0.001). In contrast, the P38 inhibitor SB203580 did not elicit any protection against death or apoptosis (Fig. 4D and E). SB203580 and CEP-11004 by themselves had no effect on cell viability (Fig. 4E). We also used the dominant negative of MAPK kinase kinase 2 (MEKK2) or MEKK2KD, the upstream kinase of JNKK1/2, to study its effect on cell death. Cells were transiently transfected for 48 h and then exposed to anisomycin + CH-11. Western blot analysis indicated that transfection with MEKK2KD inhibited by 70% JNK activation induced by anisomycin + CH-11 (Fig. 4C). Cell viability was increased from 22% to 39% when U87 cells were transient transfected with MEKK2KD (Fig. 4F, P < 0.05). MEKK2KD alone had no effect on cell viability (104% versus control). This further confirms that JNK activation by anisomycin functions in the synergistic cell death induced by anisomycin + CH-11.


Figure 4
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  		FIGURE 4. JNK activation is required to sensitize U87 cells to CH-11. A. Time course of JNK1 and JNK2 activation (i.e., phosphorylation) in U87 cells treated with anisomycin (Ani, 0.1 µg/mL), CH-11 (50 ng/mL), or both. Anisomycin alone or anisomycin + CH-11 induce a sustained increase in JNK1 (p46) and JNK2 (p54) phosphorylation beginning as early as 3 h. B. U87 cells were preincubated with the specific JNK pathway inhibitor CEP-11004 (CEP, 4 µmol/L) for 24 h followed by anisomycin (0.1 µg/mL) treatment for 3 h. JNK activation was analyzed by Western blot. CEP-11004 inhibits about 78% JNK activation induced by anisomycin. C. U87 cells were pretreated with CEP for 24 h or transiently transfected with MEKK2KD for 48 h followed by anisomycin + CH-11 (50 ng/mL) for 6 h. JNK activation was analyzed by Western blot. CEP-11004 or MEKK2KD transfection inhibited more than 70% JNK activation induced by the combined treatment. D. U87 cells were incubated with CEP-11004, L-JNKI1, or the P38 inhibitor SB203580 (100 µmol/L) for 24 h before treatment with anisomycin + CH-11 for another 24 h. Cell death is partially reversed by CEP-11004 or L-JNKI1, but not by SB203580. *, P < 0.001; n = 9. E. Flow-cytometric assay showing the percentage of annexin V–positive cells. CEP-11004 but not SB203580 attenuates cell death. *, P < 0.001 versus anisomycin + CH-11; n = 9. F. U87 cells were transiently transfected with dominant negative of MEKK2 (MEKK2KD) followed by anisomycin + CH-11 for 24 h. MEKK2KD transfection increased cell viability from 22% to 39%. *, P < 0.05; n = 9. Buffer alone or MEKK2KD alone had no effect on cell viability.

 
We asked if the magnitude of JNK activation in response to anisomycin alone was sufficient to sensitize cells to CH-11. We transfected U87 cells with wild-type JNKK1, JNKK2, and MEKK2 or their combination. All of the three constructs increased JNK1 and JNK2 activation. There was an ~4- to 12-fold increase in JNK1 activation with wild-type JNKK1, JNKK2, and MEKK2 and an ~2- to 3-fold increase in JNK2 activation with JNKK2 and MEKK2 (Fig. 5A ). However, when cells were transfected with JNKK1, JNKK2, or MEKK2 alone or their combination and then treated with CH-11 (500 ng/mL), there was no significant potentiation of cell death compared with CH-11 alone (Fig. 5B).


Figure 5
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  		FIGURE 5. JNK activation is not sufficient to sensitize U87 cells to CH-11–induced apoptosis. A. U87 cells were transiently transfected with wild-type JNKK1, JNKK2, and MEKK2. All three constructs increase JNK1 phosphorylation (p46) by 4- to 12-fold and JNK2 phosphorylation (p54) by 2- to 3-fold. B. U87 cells were transfected with wild-type JNKK1, JNKK2, and MEKK2 either alone or combined for 48 h followed by incubation with CH-11 (500 ng/mL) for 24 h. MTT assay was used to determine cell viability. Compared to control, none of the conditions sensitized cells to CH-11–induced cell death (n = 9).

 
Bim Up-Regulation and Cytochrome c Release Following Anisomycin + CH-11
Because the JNK pathway affects apoptosis mainly through the mitochondrial apoptotic pathway, we examined whether anisomycin + CH-11 affected critical components in the intrinsic apoptotic pathway, such as Bcl-2, Bcl-xL, Bad, Bax, Bak, Bim, survivin, and XIAP. Cells were simultaneously treated with anisomycin or CH-11 or their combination. The time course of the expression level of those pro- and antiapoptosis proteins was analyzed by Western blot. Among them, Bcl-xL and Bax were slightly down-regulated at 12 h following the combined treatment. There was no change in Bcl-2 and Bak protein levels, and there was no detectable expression of Bad and survivin in U87 cells (data not shown). XIAP protein was moderately down-regulated (3-fold) after 12 h exposure to either anisomycin alone or in combination with CH-11. However, CH-11 alone had little effect on XIAP levels (Fig. 6A ).


Figure 6
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  		FIGURE 6. Bim is potently induced by anisomycin + CH-11 and required for maximal cell death from anisomycin + CH-11. A, B. Western blot analysis of specific intrinsic apoptosis pathway proteins during cell treatment with anisomycin (Ani, 0.1 µg/mL), CH-11 (50 ng/mL), or both. Bcl-2, Bcl-xL, and Bax are slightly down-regulated following 12 h incubation with anisomycin + CH-11. BimL is strongly induced by anisomycin and CH-11 from 3 to 12 h. There is also a weak induction of BimS most evident in response to anisomycin + CH-11. XIAP was decreased ~70% at 12 h following anisomycin + CH-11 treatment. Cytochrome c is released 12 h after treatment with anisomycin + CH-11 (B). C. U87 cells were transfected with siRNA targeting both BimL and BimS (Bim siRNA) or with negative control siRNA (Neg-Con). After 48 h, BimL protein is specifically inhibited by ~70%. D. U87 cells were transfected with Bim siRNA or with negative control siRNA (Neg-Con) followed by anisomycin (0.1 µg/mL) + CH-11 (50 ng/mL) for 24 h. Cell death induced by anisomycin + CH-11 is partially reversed by Bim siRNA. Negative control siRNA had no effect on cell viability. *, P < 0.05; n = 9. E, F. U87 cells were transfected with Bim siRNA for 24 h followed by incubation with CEP-11004 (4 µmol/L) for another 24 h. Cells were then treated with anisomycin + CH-11 for 24 h followed by MTT assay (E) or flow cytometry analysis (F). Blocking JNK activation with CEP-11004 and inhibiting Bim induction with Bim siRNA additively protected U87 cells from cell death induced by CH-11 + anisomycin. *, P < 0.001 versus anisomycin + CH-11; n = 8.

 
Anisomycin alone or in combination with CH-11 induced strong and sustained up-regulation of BimL protein levels (Fig. 6A). In cells treated with CH-11, there was only a slight increase in BimL protein levels. The response to anisomycin ± CH-11 was time dependent, with an increase as early as 3 h and a maximum at 12 h. BimL protein induction was more pronounced in response to anisomycin + CH-11. There was a 2-fold increase in BimL protein levels at 12 h of treating cells with anisomycin + CH-11 compared with anisomycin alone. Western blot analysis showed that cytosolic cytochrome c levels were also increased after treatment with anisomycin + CH-11 at 12 h (Fig. 6B), which was consistent with the time course of BimL up-regulation. BimS was also increased at 3 and 6 h of anisomycin + CH-11 treatment. To further assess the role of Bim protein in the cell death response to anisomycin + CH-11, Bim expression was inhibited using small interfering RNA (siRNA). Bim siRNA decreased BimL protein levels by >70% (Fig. 6C). When U87 cells were pretreated with Bim siRNA for 48 h and then treated with anisomycin + CH-11, cell viability was increased by ~20% (P < 0.05; Fig. 6D). Thus, Bim directly contributes to the cell death response to anisomycin + CH-11. To examine whether JNK phosphorylates or up-regulates Bim in our system, we pretreated the cells with the JNK pathway inhibitor CEP-11004 and found that it did not inhibit Bim induction in response to either anisomycin or anisomycin + CH-11 (data not shown). Furthermore, when U87 cells were simultaneously treated with Bim siRNA for 48 h and CEP-11004 for 24 h followed by anisomycin + CH-11, cell viability was 40%, 51%, and 62% with Bim siRNA, CEP-11004, and Bim siRNA + CEP-11004, respectively (Fig. 6E). We also did flow cytometry with annexin V staining in cells treated with Bim siRNA, CEP-11004, or their combination. Anisomycin + CH-11 induced about 55% apoptosis. In the presence of Bim siRNA, CEP-11004, or their combination, the percentages of apoptosis was 38%, 26%, and 16%, respectively (Fig. 6F). Thus, blocking JNK activation and inhibiting Bim induction additively protected U87 cells from cell death induced by anisomycin + CH-11. This is consistent with cooperative actions of JNK activation and Bim up-regulation in the U87 cell death response to anisomycin and Fas activation.


   Discussion
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
Materials and Methods
 References
 
Despite the fact that many glioblastoma cells express death receptors, they are not sensitive to apoptosis induced by death receptor agonists (15, 24). Resistance to apoptosis induced by death receptor activation can occur at different points in the apoptotic cascade. These include defects in death receptors (10), FADD or caspase-8 (11), overexpression of cFLIP, Bcl-2, Bcl-xL, and IAPs (9, 25), loss of Bax or Bak function (26), reduced release of mitochondrial proteins (27), and constitutively active NF-{kappa}B in resistant cells (28). Therefore, developing strategies to overcome resistance is important for the successful application of death receptor agonists for clinical therapy. Previous work establishes that a variety of chemoprevention drugs can be used to enhance Apo2L/TRAIL sensitivity in glioma cells (14, 16). We reported that oxidative stress induced by camptothecin and Adriamycin sensitizes glioma cells to apoptosis induced by Fas activation. JNK was activated by camptothecin, but the role of JNK activation in sensitizing glioma cells to Fas-induced cell death remained to be clarified (15). Here, we examined the cooperative proapoptotic interactions of anisomycin, a strong JNK activator, and the agonistic anti-Fas antibody (CH-11). Anisomycin was found to potentiate Fas-mediated glioma cell death via caspase-dependent mechanisms involving both the JNK and the intrinsic pathway proapoptotic protein Bim.

Anisomycin has been shown to bind and alter the structure of 28S RNA of the eukaryotic ribosome (17). This results in so-called ribotoxic stress with at least two consequences: JNK activation and protein synthesis inhibition. JNK, also known as stress-activated protein kinase, is activated when cells are exposed to environment stress. It phosphorylates a variety of cytoplasmic and nuclear proteins such as the transcription factor c-Jun, activating transcription factor-2, Elk-1, etc. (for a review, see ref. 29). Whereas the function of JNK under physiologic conditions is largely unknown, the role of JNK in various stress-induced models of apoptosis has been well documented. JNK plays an important role in apoptosis induced by the withdrawal of nerve growth factor, excitotoxic stress, and UV radiation (for reviews, see refs. 29, 30). Recent studies show that sustained JNK activation signals caspase-dependent cell death in cancer cells (31). In glioma cells, JNK activation seems to play a role in apoptosis induced by a variety of stimuli, including nitric oxide and radiation (32). In this study, we found that the JNK activator anisomycin potently sensitizes glioma cells to death receptor ligand–mediated apoptosis. A direct role for JNK activation in this synergistic death response was illustrated by the inhibition of apoptosis by three functionally distinct inhibitors of JNK activation: CEP-11004, L-JNKI1, and dominant negative MEKK2. Both CEP-11004 and MEKK2KD transfection substantially inhibit JNK1 activation induced by anisomycin + CH-11. CEP-11004 and L-JNKI1 partially enhanced cell survival in the setting of anisomycin + CH-11. The percentage of cells protected by MEKK2 transfection was ~17% and is consistent with the estimated 30% transfection efficiency under the conditions of these experiments. We also asked if the degree of JNK activation was sufficient to sensitize cells to apoptosis in response to CH-11. Overexpressing JNKK1, JNKK2, and MEKK2, either alone or combination, under conditions that activated JNK1 ~4- to 12-fold and JNK2 ~2- to 3-fold failed to sensitize cells to even 10 times higher concentrations of CH-11. The activation rate of JNK1 under these conditions was comparable to that observed with anisomycin alone. Together, these results suggest that JNK activation by anisomycin is necessary but not sufficient to sensitize glioblastoma cells to CH-11–induced apoptosis and further suggests the existence of a parallel JNK-independent death-inducing pathway. It remains possible that higher levels of JNK2 activation are required to sensitize cells to CH-11 because JNK2 was more potently activated by anisomycin than in response to JNK/MEKK2 transfection.

Anisomycin has been shown to inhibit protein synthesis in various cell lines (17, 18, 22, 23). Translation inhibitors have been shown to increase the sensitivity of many cell types to apoptosis induced by the TNF family members (21, 33, 34). This effect has been attributed to diminished translation of apoptosis inhibitory proteins such as FLIP and IAPs (33, 34). The variable effects of anisomycin on apoptosis-regulating proteins in our studies do not support a similar mechanism. Anisomycin alone only modestly down-regulated the antiapoptotic proteins XIAP and Bcl-2, but at the same time up-regulated the antiapoptotic protein Bcl-xL. Although more substantial changes in apoptosis regulators were seen in cells after 12 to 24 h of treatment with anisomycin + CH-11, these changes cannot be attributed solely to anisomycin, and their interpretation is complicated by the fact that cells were actively entering apoptosis at these times.

One of the most dramatic responses to anisomycin + CH-11 was the activation of caspase-8, an extrinsic pathway effector of apoptosis that is upstream to the family of Bcl proteins and XIAPs. Both caspase-8 and Bid activations were seen as early as 6 h after the combined treatment. This was concurrent with the significant increase in JNK activation, which occurs after 6 h of the combined treatment. This is consistent with a role for JNK in the initiation of the apoptosis cascade. Western blot and flow cytometry analysis revealed that the expression levels of critical components in the extrinsic apoptotic pathway, including Fas, FADD, and FLIP, were not changed by anisomycin, either alone or combined with CH-11. It is possible that anisomycin + CH-11 increased caspase-8 activity by altering the dynamics of DISC formation independent of changes in expression of its constituent components. More extensive studies focusing on how anisomycin and death receptor agonists synergistically alter DISC function and caspase-8 activation may shed light on the mechanisms involved in the synergistic cell killing effect of anisomycin.

The BH3-only proapoptotic protein Bim mediates apoptosis by binding to antiapoptotic members of the Bcl-2 family (e.g., Bcl-2 and Bcl-xL) or by activating proapoptotic members Bax and Bak (35, 36). In normal cells, Bim is sequestered in motor complexes that interact with the cytoskeleton. Regulation of Bim protein to trigger apoptosis includes its transcriptional up-regulation, its phosphorylation, or its release from cytoplasmic sequestration sites (37). The mechanism that accounts for the release of Bim from motor complexes has not been established. One possible mechanism is phosphorylation (38). JNK has been found to phosphorylate or up-regulate Bim in sympathetic neurons and cerebellar granule neurons (38-40). In U87 cells, preincubating glioma cells with CEP-11004 did not inhibit Bim induction in response to anisomycin + CH-11 (data not shown). On the contrary, CEP-11004 and Bim siRNA cooperatively protected U87 cells from cell death induced by anisomycin + CH-11. Thus, JNK activation and Bim up-regulation seem to constitute two parallel and cooperative pathways required for glioma cell death in response to anisomycin and Fas activation.

Innovative approaches designed to harness the death receptor cascade for treating malignant glioma are currently under development. Our in vitro findings in multiple glioblastoma cell lines predict that inducing cellular stress with ribotoxic strategies might synergize with therapeutic death receptor pathway activation. Attempts to translate our findings to in vivo applications should proceed with caution due to potential toxicity. Anisomycin, the ribotoxin used in our in vitro experiments, has been given systemically to mice at doses ranging from 20 to 140 ng/mL to treat lung adenocarcinoma with minimal side effects reported (22). In addition, anisomycin (s.c., 20-200 mg/kg) has been used successfully in neurobehavioral studies (41, 42). Other compounds that induce ribotoxic stress through alternate mechanisms may be applicable as well (19). Although Fas receptor agonists are not applicable in vivo, TRAIL has been safely applied in animal models. Finally, direct intratumoral delivery could serve as a rational approach to reduce systemic toxicity of apoptosis-inducing therapeutics such as ribotoxins and/or death receptor agonists.

In conclusion, we show in this report that anisomycin sensitizes glioma cells to death receptor–mediated apoptosis via a mechanism requiring both JNK activation and Bim up-regulation. Understanding the cell death signaling pathways and mechanisms of this synergistic cell death response could shed light on inherent mechanisms of glioma cell resistance to death receptor–induced apoptosis and lead to novel approaches to primary brain tumor therapy.


   Materials and Methods
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
References
 
Reagents
Reagents were purchased from Sigma unless otherwise mentioned. Drugs were made in stock and diluted in cell culture medium. Anisomycin was dissolved in DMSO at 5 mg/mL. Anti-Fas antibody (clone CH-11) was purchased from Upstate. Human recombinant TRAIL was purchased from Chemicon. Pan-caspase inhibitor Z-VAD-FMK was dissolved in DMSO at 50 mmol/L. JNKK inhibitor CEP-11004 was kindly provided by Cephalon Inc. (West Chester, PA) and dissolved in DMSO at 4 mmol/L (43). JNK inhibitor L-JNKI1 was purchased from Alexis Biochemicals. All primary antibodies used for Western blot were obtained from Cell Signaling Technology unless otherwise stated, and the concentrations used for Western blotting were according to the manufacturer's recommendations. All the secondary antibodies conjugated to horseradish peroxidase were purchased from Jackson Immunoresearch Laboratories and were used in Western blotting at 1:1,000 dilution.

Cell Culture
U87 and U373 glioblastoma cell lines were originally purchased from American Type Culture Collection. U87 cells were grown in EMEM (Cellegro) supplemented with 10% fetal bovine serum (Cellegro), 0.15% sodium bicarbonate (Life Technologies), 1 mmol/L sodium pyruvate (Life Technologies), 0.1 mol/L nonessential amino acids (Life Technologies), and 500 µg/mL penicillin-streptomycin (Life Technologies). U373 cells were grown in DMEM (Life Technologies) supplemented with 10% fetal bovine serum, 20 mmol/L HEPES (Life Technologies), and penicillin-streptomycin. All cells were grown at 37°C in a humidified incubator with 5% CO2.

The human glioblastoma cells, designated Mayo 16 and Mayo 22 were kindly provided by Dr. C. David James (University of California, San Francisco, CA). These cells were maintained by serial passage as subcutaneous xenographs as previously described (44). Tumor cells were dissociated from resected xenographs and cultured briefly at 37°C in a humidified incubator with 5% CO2 for up to five passages in EMEM (4.5 g/L glucose), supplemented with 10% fetal bovine serum and 500 µg/mL penicillin-streptomycin to assess responses to anisomycin and/or TRAIL.

MTT Assay
MTT was used to assay cell viability. Cells were plated at 5,000 cells per well in 24-well tissue culture plates and cultured for 48 to 72 h before treating with drugs. After 24 h of treatment, MTT was added to each well at a final concentration of 150 µg/mL, and the cells were incubated for 1 to 2 h at 37°C. The medium was then removed, and the formation reaction product was dissolved with DMSO and quantified spectrophotometrically at 570 nm using a Spectra MAX 340pc plate reader (Molecular Devices). The results are expressed as a percentage of absorbance measured in control cultures after subtracting the background absorbance from all values. We used the isobologram equation (45), Ix = (a/A)+(b/B), to determine if CH-11 and anisomycin synergistically induced U87 cell death. In this equation, A is the IC50 concentration of CH-11; B is the IC50 concentration of anisomycin; a and b are the concentration of CH-11 (a) or anisomycin (b) required to produce the same effect in combination with the other agent. If Ix < 1, then the combination effect is synergistic; if Ix = 1, the effect is additive.

Flow Cytometry/Apoptosis Assay
Apoptosis was quantified using the Annexin V–FITC/propidium iodide apoptosis kit (BD Biosciences) according to the manufacturer's instructions. Briefly, U87 cells were trypsinized (Life Technologies), pelleted by centrifugation, resuspended in Annexin V binding buffer (150 mmol/L NaCl, 18 mmol/L CaCl2, 10 mmol/L HEPES, 5 mmol/L KCl, 1 mmol/L MgCl2). FITC-conjugated Annexin V (1 µg/mL) and propidium iodide (50 µg/mL) were added to cells and incubated for 30 min at room temperature in the dark. Analyses were done on a FACScan (Becton Dickinson). The data were analyzed with CellQuest software (Becton Dickinson).

Cell Cycle Analysis
Cell cycle analysis was done as previously described (46). Briefly, monolayer U87 cells were trypsinized, pelleted by centrifugation, resuspended in 1 mL of ice-cold PBS, and fixed by adding 4 mL of ice-cold ethanol under gentle vortexing. Fixed cells were collected by centrifugation, resuspended in 1 mL of PBS, and treated with 20 µg of DNase-free RNase (Roche) for 30 min at 37°C. Cells were labeled with propidium iodide (100 µg/mL) for 10 min at room temperature. Analyses were done on a FACScan. Raw data were gated to remove doublets and cellular debris. The resultant cell cycle histograms were analyzed with CellQuest software.

Fas and FasL Expression
Cell surface Fas and FasL were subjected to antibody staining and measured by flow cytometry. Briefly, untreated or treated U87 cell monolayers were harvested by trypsinization and centrifugation. The suspended cells were incubated with 5% normal goat serum in PBS and then with either anti-Fas (1:500) or anti-FasL (1:500) antibody (BD Biosciences) for 1 h at room temperature. Mouse immunoglobulin G (IgG) was used as the negative control. After washing twice in PBS, cells were incubated with the goat anti-mouse IgG conjugated with FITC (Jackson Immunoresearch Laboratories) at room temperature for 30 min. The cells were washed again in PBS and analyzed using a Becton Dickinson FACScan. The data were processed using CellQuest software.

Western Blot Analysis
Cells grown in 10-cm-diameter tissue culture dishes were lysed with radioimmunoprecipitation assay buffer [50 mmol/L Tris-HCl (pH, 7.4), 150 mmol/L NaCl, 1% NP40, 0.25% Na-deoxycholate] containing 1x protease and phosphatase inhibitor cocktail (Calbiochem). After sonication for 15 s, the suspensions were centrifuged at 3,000 x g for 10 min. Protein concentrations were determined using the Coomassie Protein Assay Reagent (Pierce). About 30 µg of protein were subjected to 4% to 20% SDS-PAGE electrophoresis and then transferred to nitrocellulose membrane for 1 h. The membrane was then blocked in 5% nonfat dry milk (Carnation, Nestle Food Co.) in TBS Tween 20 for 1 h and then incubated overnight at 4°C in 5% nonfat dry milk or 5% bovine serum albumin containing the primary antibody. Membranes were subsequently rinsed in TBS Tween 20 and then incubated for 1 h at room temperature with secondary antibody conjugated with horseradish peroxidase. After incubation, membranes were rinsed, and antibody binding was detected with the enhanced chemiluminescence system (Amersham). Membranes were stripped in buffer [2% SDS, 100 mmol/L ß-mercaptoethanol, 50 mmol/L Tris (pH 6.8)] at 55°C for 0.5 h and reprobed for actin (Santa Cruz Biotechnology). Signals were quantified by densitometer (Molecular Dynamics) and normalized to actin.

siRNA Transfection
siRNA Target Finder program (Ambion) was used to generate a siRNA construct (purchased from Ambion) targeting both BimL and BimS. The target sequence is GGUAGACAAUUGCAGCCUGUU. U87 cells were cultured at 50% to 70% confluency on a 10-cm dish containing 6 mL medium. For each transfection, 32 µL transfection reagent siPort (Ambion) was mixed with 1,100 µL optimum medium (Life Technologies) and incubated at room temperature for 20 min. About 3 µL of 20 µmol/L negative control siRNA (Silencer Negative Control 1 siRNA, Ambion) or Bim siRNA solution was added to the mixture and incubated at room temperature for another 20 min. The mixture was then added to the cell medium and incubated for 48 h before MTT assay or protein extraction. The silencing effect of the siRNA construct on Bim expression was analyzed by Western blot.

Plasmid Transfection
MEKK2, JNKK1, JNKK2, and MEKK2KD were generously provided by Dr. B. Su (The University of Texas M. D. Anderson Cancer Center, Houston, TX) and have been described previously (47, 48). Target cells were transfected using Fugene-6 transfection reagent (Roche) according to manufacturer's specifications, with a 5:1 ratio of transfection reagent volume (5 µL) to DNA mass (1 µg) into a 10-cm dish. Cells were incubated with transfection mixture for 48 h before drug treatment. Cell transfection efficiencies were ~30% as determined by green fluorescent protein transfection under identical conditions. After 24 h of drug treatment, cells were subjected to MTT assay or flow cytometry assay.

Statistical Analysis
Statistical analysis consisted of one-way ANOVA followed by the Tukey multiple comparison tests using Prizm (GraphPad). All experiments reported here represent at least three independent replications. Data are represented as mean value ± SE.


   Notes
Top
 Notes
Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Grant support: NIH grants NS 95704 (J. Laterra) and ES09169 (E.M. Rosen).

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 12/22/06; revised 4/20/07; accepted 5/ 7/07.


   References
Top
 Notes
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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