Neuro-Oncology Advance Access published February 24, 2014

Neuro-Oncology

Neuro-Oncology 2014; 0, 1 – 14, doi:10.1093/neuonc/nou012

The deadly connection between endoplasmic reticulum, Ca21, protein synthesis, and the endoplasmic reticulum stress response in malignant glioma cells Guyla G. Johnson, Misti C. White, Jian-He Wu, Matthew Vallejo, and Maurizio Grimaldi Laboratory of Neuropharmacology, Department of Biochemistry and Molecular Biology, Southern Research Institute, Birmingham, Alabama (G.G.J., M.C.W., J-H.W., M.G.); Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, Alabama (M.V.)

Background. The endoplasmic reticulum (ER) is involved in Ca2+ signaling and protein processing. Accumulation of unfolded proteins following ER Ca2+ depletion triggers the ER stress response (ERSR), which facilitates protein folding and removal of damaged proteins and can induce cell death. Unfolded proteins bind to chaperones, such as the glucose-regulated protein (GRP)78 and cause the release of GRP78-repressed proteins executing ERSR. Methods. Several glioma cell lines and primary astrocytes were used to analyze ERSR using standard western blots, reverse transcription –PCR, viability assays, and single cell Ca2+ imaging. Results. ERSR induction with thapsigargin results in a more intense ERSR associated with a larger loss of ER Ca2+, activation of ERassociated caspases (4/12) and caspase 3, and a higher rate of malignant glioma cell death than in normal glial cells. Malignant glioma cells have higher levels of protein synthesis and expression of the translocon (a component of the ribosomal complex, guiding protein entry in the ER), the activity of which is associated with the loss of ER Ca2+. Our experiments confirm increased expression of the translocon in malignant glioma cells. In addition, blockade of the ribosome-translocon complex with agents differently affecting translocon Ca2+ permeability causes opposite effects on ERSR deployment and death of malignant glioma cells. Conclusions. Excessive ER Ca2+ loss due to translocon activity appears to be responsible for the enhancement of ERSR, leading to the death of glioma cells. The results reveal a characteristic of malignant glioma cells that could be exploited to develop new therapeutic strategies to treat incurable glial malignancies. Keywords: caspase 4, Ca2+, ER stress response, glioma, astrocytes, thapsigargin, translocon.

The endoplasmic reticulum (ER) controls Ca2+ signaling1 and protein synthesis and folding. A high Ca2+ concentration ([Ca2+]) in the ER is essential to the function of chaperones, such as glucose-regulated protein (GRP)78.2 Depletion of ER Ca2+ is accompanied by protein unfolding3 and activation of the biochemical cascade known as the ER stress response (ERSR). Upregulation of GRP78, a Ca2+-dependent chaperone involved in ER protein folding, is the main indicator of ERSR activation.4 The primary role of ERSR is the repair and/or removal of damaged proteins. However, ERSR can trigger cell death.5,6 Lack of ER Ca2+ impedes chaperone activity7 and affects protein folding. Moreover, ER-associated caspases (4/12), in resting conditions, are bound to and inhibited by GRP78. But, in the presence of unfolded proteins, caspases are displaced by GRP78 and

activated by calpains, which are in turn activated by high local concentrations of cytoplasmic Ca2+ ([Ca2+]c).8 A widely used model to study ERSR, thapsigargin (THAP)induced ER Ca2+ depletion, is based on the irreversible loss of ER Ca2+ that takes place when the sarco/ER Ca2+ ATPase (SERCA), which is responsible for sequestering Ca2+ into the ER, is irreversibly inhibited. Several possible mechanisms can be invoked for ER Ca2+ loss following SERCA inhibition. These include chemical leakage and spontaneous inositol trisphosphate receptor activity. The contribution of the translocon complex to Ca2+ loss from the ER during protein synthesis has been largely underestimated and seldom investigated. The translocon, a heterotrimer of Sec61a, ß, g that spans the ER membrane,9 functions in translocating the nascent ribosomal proteins into the ER lumen

Received 25 April 2013; accepted 16 January 2014 # The Author(s) 2014. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: [email protected].

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Corresponding Author: Dr. Maurizio Grimaldi, MD, PhD, Laboratory of Neuropharmacology, Department of Biochemistry and Molecular Biology, Drug Discovery Division, Southern Research Institute 2000 9th Avenue South, Birmingham, AL 35205 ([email protected]).

Johnson et al.: ERSR, Ca2+, translocon, and glioma cell death

Materials and Methods All materials were purchased from Sigma unless otherwise specified. Puromycin was diluted in water; all other substances were diluted in dimethyl sulfoxide (DMSO) unless otherwise specified.

Preparation of Primary Cultures of Rat Cortical Astrocytes Cultures of astrocytes were obtained from E-17 fetuses of male and female origin according to a previously published protocol.15 Briefly, fetuses were obtained by C-section from a 17-day pregnant Wistar rat and quickly decapitated. The heads were placed in phosphate buffered saline (Gibco) containing 4.5 g/L glucose at room temperature. Cerebral cortices were dissected, minced, and digested with papain (Worthington). The tissue fragments were then mechanically dissociated. The cells were counted and plated in 75-cm2 flasks (4 × 107 cells/flask). The cultures were shaken, and the medium was changed after 6 –8 h to remove nonadherent cells (neurons). Thereafter, the medium was changed every 2 days. This yielded cultures consisting of 95% astrocytes, as characterized by immunoreactivity for glial fibrillary acidic protein, as previously published.16

Cell Lines For experiments, human U87MG (American Type Culture Collection [ATCC]), human LN22917,18 (a gift of Dr Bredel at the University of Alabama Birmingham [UAB]), human U11819,20 (a gift of Dr Bredel at UAB), and rat C6 cells (ATCC) were used up to passage 20 and then discarded. Rat cortical astrocytes were used directly from the primary vessel. All cells were cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 mg/mL streptomycin. All cells were maintained at 378C in a 5% CO2/95% humidified air atmosphere.

Single Cell [Ca2+]c Measurements Nearly confluent rat cortical astrocytes and C6 and U87MG derived from rat and human malignant glioma cells, respectively, were seeded onto 1.5-cm round #1 glass coverslips (Assistant).

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Before experiments, cells were washed in Krebs-Ringer buffer (KRB; 125 mM NaCl, 5 mM KCl; 1 mM Na2 HPO4, 1 mM MgSO4, 1 mM CaCl2, 5.5 mM glucose, 20 mM 4-(2-hydroxyethyl)-1piperazine ethanesulfonic acid, pH 7.3). Thereafter, they were loaded with 4 mM fura-2-acetoxymethyl ester (Fura-2AM, Invitrogen) for 22 min at room temperature under continuous gentle agitation. After loading, cells were washed once with KRB to allow unprocessed probe to be removed, then were incubated for an additional 22 min in KRB without Fura-2AM.16 The coverslips were mounted on a low-volume (150-mL), self-built perfusion chamber and set up on the motorized stage of an inverted microscope equipped with a 340 optimized 40× lens and a high-speed/highefficiency high-resolution charge-coupled camera connected to a self-built computer. Metafluor software (Molecular Devices) was used to control the system devices. Preparations were perfused with KRB through a peristaltic pump at a speed of 800 mL/min. Ca2+-free KRB was prepared by omitting Ca2+ and including 100 mM EGTA. Ratio measurements were obtained by collecting image pairs exciting the preparations at 340 and 380 nm every 2 s. The excitation wavelengths were changed through a highspeed mechanical filter changer connected via a short liquid guide to a stripped microscope epifluorescence path. The emission wavelength was set at 510 nm. The captured images were digitized with an acquisition board and analyzed by commercially available software (Metafluor, Molecular Devices). Ratio values were derived by averaging fluorescence intensity from the entire cytosolic area, obtained by delimiting the profile of the cells.

Western Blots The following antibodies were used: GRP78 (catalog #sc-13968), CCAAT/enhancer binding protein homologous protein (CHOP; #sc-7351), caspase 12 (#sc-21747) (all Santa Cruz Biotechnology); CHOP (#L63F7, Cell Signaling Technology); caspase 4 (#C 3392), caspase 3 (#9661); SEC61g (#11147-2-AP, Proteintech); and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; #2118, Cell Signaling). Briefly, cells were lysed in a commercial buffer (#9803; Cell Signaling), and protein content in cellular lysates was measured by bicinchoninic acid assay (Fisher Scientific). Samples were boiled for 5 min to denature the proteins. Aliquots (40 – 100 mg protein per lane) of total protein were separated by 8% – 16% gradient or 10% sodium dodecyl sulfate – polyacrylamide gel electrophoresis and transferred onto nitrocellulose or polyvinylidene difluoride membranes (Fisher Scientific) (Fig. 3 and Supplemental Figs. 1 and 2). Each membrane was blocked with 5% nonfat dry milk in TBS-T (20 mM Tris-HCl, pH 7.5, 137 mM NaCl, and 0.01% Tween-20) for 1 h at room temperature, followed by incubation with the appropriate primary antibodies. After washing with TBS-T, each membrane was incubated with horseradish peroxidase – conjugated anti-rabbit (#PI-31462, Fisher Scientific), anti-mouse (#PI-31430, Fisher Scientific), anti-goat (#sc-2020, Santa Cruz), or anti-rat (#sc-2032, Santa Cruz) secondary antibodies in TBS-T containing 5% nonfat dry milk. Detection was assessed by the use of an enhanced chemiluminescence reagent (Fisher Scientific), according to the manufacturer’s protocol. Densitometric analysis was performed using ImageJ Software (National Institutes of Health [NIH] freeware). Normalization of loading conditions was accomplished by calculating the ratio of the target protein band to the GAPDH band.

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when a leader peptide sequence is detected.10 Additionally, the translocon binds to GRP78,11 thus participating in protein folding during protein synthesis. During its activity, the translocon allows Ca2+ to exit the ER.12 Since malignant glioma cells have both high levels of translocon expression and protein synthesis,13,14 the enhancement of protein synthesis leading to persistent translocon activity could affect maintenance of high ER [Ca2+], rendering malignant glioma cells more susceptible to ERSR augmentation. The influence of translocon-mediated ER Ca2+ loss has not been determined in the dynamics of Ca2+ signaling and induction of ERSR in malignant glioma cells. Therefore, we sought to compare ERSR induction and showed differences in normal and malignant glial cells in various components of the ERSR pathway. Our results provide evidence that enhancement of translocon expression and activity resulting from the enhancement of ER protein synthesis plays an important part in destabilizing ER Ca2+ homeostasis, exacerbating both ERSR and ER caspase-dependent ERSR death pathways.

Johnson et al.: ERSR, Ca2+, translocon, and glioma cell death

RNA Isolation and Reverse Transcription PCR Analysis Total RNA from U87MG human glioma cells was isolated using TRI-Reagent (#TR-118, Molecular Research Center) according to the manufacturer’s guidelines. The mRNA levels of grp78 and ß-actin were analyzed by 1-step reverse transcription (RT) PCR using the Promega Access RT-PCR System (#A1250) for 23 cycles. Previously published primers were used for the RT-PCR analysis.4 Resulting cDNA was separated by electrophoresis on 1.5% NuSieve (#50091, Lonza)/1% agarose gel (#161-3101, BioRad Laboratories). ImageJ was used to quantitate cDNA intensities between samples. Normalization of loading conditions was performed calculating the ratio of the grp78 band to the loading control ß-actin band.

Cells were plated in half-area 96-well plates in DMEM supplemented with 10% fetal bovine serum, 100 units/mL of penicillin, and 100 mg/mL streptomycin. Each treatment point was set up in quadruplicate or more. Cells were allowed to attach overnight. At the start of the experiment, the plating medium was replaced with 50 mL medium containing the indicated treatment. An equal volume of Cell Titer Glo reagent (Promega) was added to terminate the reaction. Following 5 min of incubation in the dark, total luminescence was measured on a Wallac 1420 VICTOR2 multilabel reader (PerkinElmer).

Use of Laboratory Animals Adequate measures were taken to minimize unnecessary pain and discomfort to the animals and to minimize animal use, according to Southern Research Institute regulations, which meet or exceed NIH guidelines on animal handling and care (Guide for the Care and Use of Laboratory Animals).

Thapsigargin Exposure Induces Higher Levels of GRP78 Expression and Larger ERSR in Malignant Glioma Cells Than in Astrocytes

Fig. 1. THAP affects GRP78 expression in normal glial cells and malignant glioma cells. (A) Primary rat cortical normal glial cells and C6 rat glioma cells were exposed to graded concentrations of THAP for 24 h. GRP78 expression was increased by THAP in a concentration-dependent manner. GRP78 upregulation in response to THAP, however, was more prominent in C6 cells than in normal glial cells. CTRL, control; VEH, vehicle. (B) Primary rat cortical normal glial cells and C6 rat and U87MG human malignant glioma cells were cultured in the presence of VEH (0.1% DMSO) or THAP (200 nM) for 24 h. THAP induction of ERSR caused greater expression of GRP78 in malignant glioma cells compared with normal glial cells. Panels A and B display representative western blots probed with GRP78 and GAPDH (loading control) antibodies. Digitized values from 3 or more independent experiments were averaged, and the SD was calculated and plotted in the line graphs. Normalization of loading conditions was performed by calculating the ratio of the GRP78 band to the GAPDH band. One-way ANOVA followed by Bonferroni’ post hoc test was used for statistical validation. *, **, *** indicate P ≤ .05, P ≤ .01, and P ≤ .001, respectively, vs corresponding astrocyte values. B B, B B B indicate P ≤ .01 and P ≤ .001, respectively, vs VEH treated corresponding cell types.

We analyzed GRP78 expression during ERSR induced by 24 h exposure to THAP (Fig. 1A). Astrocytes and C6 malignant glioma cells were exposed to graded concentrations (2.5 to 200 nM) of THAP, and GRP78 expression was measured by western blots. For both cell types, THAP exposure increased GRP78 expression in a concentration-dependent manner. The levels of induction, however, were higher in malignant glioma cells relative to astrocytes. Untreated astrocytes and C6 malignant glioma cells showed similar levels of GRP78. In astrocytes exposed to 200 nM of THAP, GRP78 expression reached 9-folds of induction,

while in C6 rat malignant glioma cells, we observed 20-folds of induction above baseline levels. Next, we determined whether the difference in the enhancement of GRP78 expression in C6 rat malignant glioma cells was a phenomenon peculiar to C6 cells or common to other malignant glioma cells. Therefore, we compared THAP-induced GRP78 expression in astrocytes, rat C6, and human U87MG malignant glioma cells. Baseline levels of GRP78 expression were not greatly different between these cells. GRP78 was upregulated

Statistical Analysis Experiments were performed at least 3 times on different cell preparations. When statistical validation was required, the values of the specified data points were analyzed by 1-way ANOVA followed by a Bonferroni post hoc test. Differences were considered statistically significant at P ≤ .05.

Results

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Cell Viability Determination

Johnson et al.: ERSR, Ca2+, translocon, and glioma cell death

following exposure to THAP (200 nM) for 24 h in all cell types. However, GRP78 expression levels achieved in C6 and U87MG malignant glioma cells were significantly higher than those achieved in astrocytes (Fig. 1B), confirming that the apparent hyperactivation of the chaperone system (ie, protein unfolding) is greater in malignant glioma cells than in astrocytes. Finally, we confirmed the finding in two additional human malignant glioma cell lines with different dedifferentiation marker expression,21 LN229 and U118, which showed enhanced ERSR, as evidenced by higher levels of GRP78 expression in response to THAP (Fig. 3A).

CHOP Induction Is Lower in Malignant Glioma Cells Than in Astrocytes

Fig. 2. CHOP expression, in response to ERSR induction, is differentially regulated in normal glial cells and in malignant glioma cells. Primary rat cortical astrocytes, rat C6, and human U87MG cells were cultured in media (CTRL) or in the presence of VEH (0.1% DMSO) or THAP (200 nM) for 24 h. Immunoblots using anti-CHOP antibodies showed a much higher induction of CHOP in normal glial cells than in malignant glioma cells. In U87MG cells, CHOP induction was almost absent when assessed in the same samples with an antibody obtained from Santa Cruz (main figure body) and much smaller than astrocytes when assessed with an antibody obtained from Cell Signaling (Fig. 2 inset and Supplemental Fig. 1). Normalization of loading conditions was performed calculating the ratio of the CHOP band to the GAPDH band. ***, **** indicate P ≤ .001 and P ≤ .0001, respectively, vs same cell control. B B, B B B B indicate P ≤ .01 and P ≤ .0001, respectively, vs THAP values in astrocytes.

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Fig. 3. Comparison of THAP effect on GRP78 and CHOP expression in different human glioma cell lines with different genetic pedigree. Cells were treated with THAP at 200 nM for 24 h. (A) THAP exposure caused significantly higher GRP78 upregulation in all human glioma cell lines tested compared with normal astrocytes (ie, increase protein unfolding and ERSR). (B) Following similar THAP exposure, CHOP was highly upregulated in normal astrocytes, while expression of this factor was negligible in all glioma cell lines tested. (C) Representative blots are shown. Experiments were repeated at least 3 times. Statistical analysis was performed by 1-way ANOVA followed by Bonferroni’ post hoc test. *, ***, **** indicate P ≤ .05, P ≤ .01, and P ≤ .001, respectively, vs values in THAP-treated astrocyte.

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During ERSR, the transcription factor CHOP is usually upregulated.6 CHOP regulates the expression of genes involved in mitochondrial apoptosis activation and is considered to be the main actuator of the ERSR-regulated death pathway.6 Based on this evidence, we analyzed THAP-induced CHOP expression in normal and malignant glial cells. Following 24 h exposure to THAP (200 nM), an increase in CHOP induction was observed in astrocytes, although to a much lower extent in C6 glioma cells and U87MG cells (Fig. 2, and inset). CHOP response to ERSR induction, however, followed a different pattern than GRP78. There was a higher level of CHOP induction in astrocytes compared with several human and rat malignant glioma cells (Fig. 2 and Fig. 3B and

C). In particular, U87MG, LN229, and U118 cells showed a lower induction of CHOP, even though the expression of GRP78 (protein unfolding) in these cells was larger than in the normal astrocytic counterpart. Because there were many reports indicating that CHOP is upregulated in glioma cells during ERSR, and because an array of commercial sources for anti-CHOP antibodies22 – 24 were employed in these studies, we confirmed our data with a second anti-CHOP antibody obtained from Cell Signaling as described by Ciechomska et al.22 Experiments conducted using previously used samples from the experiments shown in Figs. 2 and 3 and newly prepared samples probed with the anti-CHOP antibody obtained from Cell Signaling produced similar results to the ones shown in Figs. 2 and 3, with minimal differences possibly ascribed to the differences in the sensitivity of the two antibodies versus human and rat CHOP (see Fig. 2 inset and Supplemental Fig. 1). With the anti-CHOP antibody obtained from Cell Signaling, we were able to identify a small induction of CHOP in response to THAP in glioma cells. However, the induction was

Johnson et al.: ERSR, Ca2+, translocon, and glioma cell death

minimal compared with the large induction observed in astrocytes (see Fig. 2 inset and Supplemental Fig. 1). We also confirmed previous reports showing cyclosporine A induction of GRP78 and CHOP (data not shown).

ERSR Activation Results in Marked Cytotoxicity in Malignant Glioma Cells but Not in Astrocytes

Fig. 4. ERSR activation induces cytotoxicity in malignant glioma cells. (A) Astrocytes and the 4 glioma cell lines were treated with 200 nM THAP for 24, 48, and 72 h. Viability was then assessed using the commercially available kit Cell Titer Glo. Astrocytes did not show significant toxicity from the treatment at any of the times tested. Conversely, all glioma cell lines showed a significant and time-dependent toxicity that appeared to culminate at 72 h with almost complete extinction of the cell line. Even in the case of U118, which showed less toxicity, the death was higher than 60%. (B) Primary rat cortical normal glial cells, rat C6, and human U87MG, LN229, and U118 glioma cells were treated with THAP (200 nM) for 72 h. Astrocytes showed a modest death following treatment with 200 nM THAP for 72 h (,17% cell death). Conversely, malignant glioma cells treated with 200 nM THAP showed a marked death exceeding 90% in C6 and 95% in U87MG and LN229. Experiments were repeated 3 times or more. Statistical analysis was performed by 1-way ANOVA followed by Bonferroni’ post hoc test. **** indicates P ≤ .0001 vs same cell control. B B B B indicates P ≤ .0001 vs astrocyte correspondent treatment.

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It could be expected that given the differences in CHOP induction, glioma cells should be particularly indifferent to ERSR, while astrocytes’ viability should be strongly affected. Therefore, we measured ERSR-induced cell death in the cell panel used in this study. Astrocytes, C6, U87MG, LN229, and U118 cells were treated with THAP (200 nM) for 24, 48, and 72 h, and viability was

measured using Cell Titer Glo and exosaminidase conversion as described in a previous work25 (cell viability was expressed as percent of the values for untreated cells of the corresponding type) (Fig. 4A). Astrocytes, unexpectedly, did not appear to be particularly sensitive to THAP toxicity, while all the glioma cells showed a marked and time-dependent viability reduction, which appeared to plateau at 72 h but was already significant at 48 h (Figs. 4A, 10, and 11). After 72 h of exposure to THAP, astrocytes showed very little inhibition of viability (213%), while at the same time point C6, U87MG, LN229, and U118 showed 92%, 98%, 88%, and 77%, respectively, viability reduction (Fig. 4B). Interestingly, there was a correlation between the magnitude of GRP78 induction and viability reduction. In fact, the least sensitive cells appeared to be astrocytes, followed by LN229 cells, which

Johnson et al.: ERSR, Ca2+, translocon, and glioma cell death

showed relatively lower levels of GRP78 compared with the other 3 glioma cell lines.

ERSR Led to the Activation of ER-associated Caspases 12/4 in Malignant Glioma Cells but Not in Normal Glial Cells In view of the differences among ERSR magnitude, CHOP induction, and death between normal and malignant glial cells, we hypothesized that other ERSR-associated death mechanisms could be responsible for the observed differences. Therefore, we measured the activation of ER-associated caspases in response to THAP. Baseline expression and the low levels of activation of

caspase 4 (humans)/12 (rat) were similar between these cell types. However, exposure of C6, and U87MG malignant glioma cells to THAP (200 nM) for 24 h caused activation of rat-specific caspase 12 in C6 cells (Fig. 5A) and human-specific caspase 4 in U87MG cells (Fig. 5B). In astrocytes, caspase 12 was not affected by THAP (Fig. 5A). We concluded that the activation of ER-associated caspases strongly correlated with GRP78enhanced induction and higher toxicity observed in malignant glioma cells.

ERSR Induction Caused Caspase 3 Activation in Malignant Glioma Cells but Not in Normal Glial Cells

Thapsigargin Exposure Causes Larger Ca2+ Changes in Malignant Glioma Cells Than in Normal Glial Cells The ERSR-inducing effect of THAP is associated with its capacity to cause complete and irreversible emptying of the ER Ca2+ content. We determined whether the effect of THAP was different in the

Fig. 5. ERSR induction activates ER-associated caspases 12/4 in malignant glioma cells but not in normal glial cells. (A) Primary rat cortical astrocyte normal glial cells and C6 malignant glioma cells were treated with VEH (0.1% DMSO) or THAP (200 nM) for 24 h. A cleaved caspase 12 band was evident in THAP-treated C6 cells but was absent in normal glial cells. (B) U87MG glioma cells were treated with VEH (0.1% DMSO) or THAP (200 nM) for 24 h. The conversion of procaspase 4 to active caspase 4 was evident in THAP-treated U87MG cells. Normalization of loading conditions was performed calculating the ratio of the active caspase band to the GAPDH band. Experiments were repeated 3 times or more. Statistical analysis was performed by 1-way ANOVA followed by Bonferroni’ post hoc test. ** indicates P ≤ .05 vs same cell control normal glial cells. B B indicates P ≤ .05 vs same treatment in astrocytes.

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Fig. 6. ERSR induction in malignant glioma cells is accompanied by activation of caspase 3. Primary rat cortical normal glial cells and C6 rat and U87MG human malignant glioma cells were treated with VEH (0.1% DMSO) or THAP (200 nM) for 24 h. Caspase 3 cleavage/activation was evident in both glioma cell types but was essentially absent in normal glial cells. Normalization of loading conditions was performed calculating the ratio of the active caspase band to the GAPDH band. **, **** indicate P ≤ .05 and P ≤ .0001, respectively, vs same cell control; B B B B B B , indicate P ≤ .05 and P ≤ .0001, respectively, vs same treatment in astrocytes.

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Since the activation of ER-associated caspases has the potential to directly cause caspase 3 activation and initiate the terminal branch of the apoptosis cascade,26 we studied the activation of caspase 3 in astrocytes and malignant glioma cells following exposure to THAP (200 nM) for 24 h by quantitative western blotting. THAP caused significant activation of caspase 3 in C6, U87MG, LN229, and U118 malignant glioma cells but not in astrocytes (Fig. 6). The pattern of caspase 3 activation in the cell panel correlated well with the activation of GRP78 and ER-associated caspases and cytotoxicity, but not with CHOP.

Johnson et al.: ERSR, Ca2+, translocon, and glioma cell death

Expression of the Sec61 Translocon Is Higher in Malignant Glioma Cells Than in Normal Glial Cells

Fig. 7. [Ca2+]c indicates difficult homeostasis in malignant glioma cells. (A) Differences in [Ca2+]c between cell types observed using THAP: THAP (2 mM), a SERCA inhibitor that indirectly reveals ER Ca2+ loss, results in an increase of [Ca2+]c in normal glial cells, C6 cells, and U87MG cells. The elevation of [Ca 2+]c , however, was larger in C6 and largest in U87MG compared with normal glial cells. (B) ATP effect in the presence of extracellular Ca2+: Exposure to ATP, a purinergic P2Y receptor agonist, results in the elevation of [Ca2+]c in normal glial cells and C6 cells. The inset bar graphs show the average ratio values obtained for each cell type 20 s after the peak value and the statistical evaluation of the differences. (C) ATP effect in the absence of extracellular Ca2+: Exposure of normal glial cells and C6 cells to ATP in Ca2+-free bathing solution resulted in responses with identical profiles. Of note is the similarity in the refilling phase (ie, capacitative Ca2+ entry) triggered by addition of Ca2+-containing bathing solution at the end of the experiment. These

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Since the function of the translocon during ER-associated protein synthesis is coupled with Ca2+ leakage from the ER, we analyzed baseline expression of its Sec61 subunit g (Sec61g) in astrocytes and in malignant glioma cells by use of quantitative western blots, with the aim to determine translocon levels in these cells. Malignant glioma cells expressed higher levels of Sec61g relative to astrocytes (Fig. 8). These data confirm previous reports suggesting that translocon expression is elevated in cancer cells, including malignant glioma cells.13,14 data indicate that the ER Ca2+ content and capacitative Ca2+ entry are not different between the 2 cell types. Statistical differences are displayed in the inset bar graphs. Experiments were repeated 3 times or more. Statistical analysis was performed by 1-way ANOVA followed by Bonferroni’ post hoc test. ** indicates P ≤ .01; N.S. indicates differences not statistically significant.

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cell panel proposed in this study and whether ER Ca2+ homeostasis was involved in the enhanced ERSR and death of malignant glioma cells. THAP treatment evoked a significantly higher elevation of [Ca2+]c in malignant glioma cells relative to astrocytes (Fig. 7A). Analysis of the data, presented in the Fig. 7A inset, showed a significant difference in THAP-induced ER Ca2+ loss between malignant glioma cells and astrocytes (statistical analysis provided in Fig. 7A inset). We sought to determine why the effect of THAP was greater in malignant glioma cells. The inositol 1,4,5-trisphosphate receptor (InsP3-R) is a potential contributor to ER Ca2+ loss.27,28 Exposure to ATP, acting via the purinergic 2Y-type receptor, causes phospholipase C activation, InsP3 production, and mobilization of ER Ca2+ in most cells and, in particular, in C6 and astrocytes.16,29,30 Astrocytes and C6 cells were challenged with ATP in the presence and absence of extracellular Ca2+ to assess differences in phospholipase C/InsP3-evoked Ca2+ transient responses between the 2 cell types (Fig. 7B and C). The peak phases of the responses to ATP challenge were similar for C6 cells and astrocytes (Fig. 7B). When the ATP challenge was conducted in the absence of extracellular Ca2+, the peak phase was identical for astrocytes and C6 cells, indicating that agonist-induced ER Ca2+ release was similar (comparable peak responses) and implying that the resting Ca2+ store contents were also similar (Fig. 7C). However, we observed a difference in the plateau phase of the ATP response between the two cell types (Fig. 7B, analysis provided in the inset to Fig. 7B). This result could have several mechanistic bases. The identical profiles of Ca2+ entry in the two cell types once Ca2+ was reintroduced into the extracellular environment at the end of the experiments (Fig. 7C) strongly support the conclusion that enhancement of capacitative Ca2+ entry (the Ca2+ influx triggered by emptying of ER stores) was not responsible for the observed differences. In addition, our previous experiments indicate that extrusion mechanisms have a limited, if any, role in shaping Ca2+ transient responses in glial cells (unpublished results). Ruling out changes in store content, capacitative Ca2+ entry enhancement, and abnormalities in extrusion mechanisms, it appears that the increased response to THAP is due to an enhanced ER Ca2+ loss ascribable to other mechanisms.

Johnson et al.: ERSR, Ca2+, translocon, and glioma cell death

Translocon Blocking Agents Have Opposite Effects on Ca2+ Homeostasis in U87MG Malignant Glioma Cells Anisomycin, a peptidyl transferase inhibitor that locks the translocon in a Ca2+ impermeable configuration, did not affect resting Ca2+ levels. However, anisomycin exposure prevented THAPinduced ER Ca2+ release in U87MG cells (Fig. 9A), which is in agreement with the finding reported in other cell types.31 Likewise, THAP-induced ER Ca2+ release was inhibited following anisomycin treatment also in C6 cells (data not shown). Puromycin, another translocon inhibitor, is a tRNA mimetic that purges the translocon of nascent proteins, hence preventing ER-associated protein synthesis. In doing so, puromycin locks the translocon in a Ca2+ permeable state, allowing unrestricted ER Ca2+ efflux.12 U87MG cells exposed to puromycin (3 mM) released substantial amounts of Ca2+ into the cytoplasm (Fig. 9B). Consistent with the proposed mechanism, puromycin’s effect on Ca2+ has a significant latency compared with other direct Ca2+ mobilizing substances such as ATP and THAP.

Inhibition of Translocon Ca2+ Permeability Prevents THAP-induced ERSR Activation, ER-associated Caspase Activation, and Cytotoxicity To prove that translocon-associated Ca2+ loss is responsible for ERSR modulation, the effects of anisomycin on ERSR deployment was determined. U87MG cells were exposed to THAP (200 nM) in the presence of anisomycin (3 mM) for 24 h, and ERSR deployment (protein unfolding) was assessed. Anisomycin per se did not affect either GRP78 protein or mRNA expression (did not cause protein unfolding). Malignant glioma cells treated with anisomycin

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Fig. 9. Translocon blocking agents have opposite effects on Ca2+ and on ERSR in malignant glioma cells. (A) Effect of anisomycin (ANISO) on THAP-induced ER Ca 2+ loss: cells were treated with anisomycin as described31 and then challenged with THAP (2 mM). Anisomycin inhibited THAP-induced ER Ca2+ loss and the subsequent elevation of [Ca2+]c. The inset bar graph in panel A shows the highest average ratio values obtained from each treatment and the statistical evaluation of the differences. Obtained values were analyzed by t-tests. ** indicates P ≤ .01 vs THAP alone cells. (B) Effect of puromycin (PURO) on ER Ca2+ loss: U87MG human malignant glioma cells were plated on coverslips and pretreated for 20 min with puromycin (3 mM) as described.12 Cells were moved to a perfusion chamber, and a brief acquisition time was allowed to establish baseline [Ca2+]c. The cells were then challenged again with puromycin (3 mM). [Ca2+]c was measured by single-cell Ca2+ imaging. Puromycin caused a large increase in [Ca 2+]c . Experiments were repeated 3 times or more. Statistical analysis was performed by 1-way ANOVA followed by Bonferroni’ post hoc test. ** indicates P ≤ .01 vs corresponding normal glial cells.

and exposed to THAP, however, failed to upregulate GRP78 protein expression (Fig. 10A) and/or its mRNA (Fig. 10B). Comparable results were observed when these experiments were performed using C6 rat glioma cells (data not shown). The lack of GRP78 mRNA induction indicates that anisomycin blunts ERSR induction by preventing ER Ca2+ depletion rather than by preventing protein synthesis. In fact, if GRP78 induction were inhibited at the protein synthesis level, GRP78 mRNA should have been enhanced by transcriptional activity of activating transcriptional factor 6. Therefore, our data indicate that THAP-induced ERSR is prevented when the Ca2+ permeability of the translocon is pharmacologically prevented with anisomycin. Since ERSR was inhibited by preventing the ER Ca2+ loss that occurs during translocon activity, the activation of other components of the ERSR cascade was also assessed. Anisomycin also prevented THAP-induced activation

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Fig. 8. Malignant glioma cells express higher levels of the translocon Sec61g subunit than normal glial cells. Primary rat cortical normal glial cells and C6 rat and U87MG human malignant glioma cells were probed for expression of Sec61g -translocon protein. Malignant glioma cells had a larger basal expression of the Sec61g subunit relative to normal glial cells. Normalization of loading conditions was performed by calculating the ratio of the Sec61g band to the GAPDH band. Experiments were repeated 3 times or more. Statistical analysis was performed by 1-way ANOVA followed by Bonferroni’ post hoc test. *, ** indicate P ≤ .05 and P ≤ .01, respectively, vs corresponding normal glial cells.

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of caspase 4 in U87MG (Fig. 10C). Additionally, THAP-induced caspase 12 activation was inhibited following anisomycin treatment in C6 cells (data not shown). The effect of inhibiting translocon Ca2+ permeability on THAP-induced U87MG toxicity was also determined. U87MG cells were treated with anisomycin, THAP, or both anisomycin and THAP for 48 h, and their viability was determined. Although anisomycin is reportedly cytotoxic, the toxicity was limited to a 42% reduction in cell viability, which allowed us to use anisomycin in these experiments. THAP-treated U87MG cells showed a 79% reduction in viability. Anisomycin prevented the toxic effect of THAP, reducing death levels to those of anisomycin alone (Fig. 10D). These data indicate that the inhibition of the

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translocon-associated ER Ca2+ loss blocks ERSR activation and prevents ERSR-induced cytotoxicity in malignant glioma cells.

Translocon Blockade in a Ca2+ Permeable Configuration Mimics THAP-induced ERSR Activation, Caspase 4 Activation, and Cytotoxicity To further confirm that translocon-associated Ca2+ loss is responsible for ERSR modulation, the effects of puromycin on ERSR deployment were determined in U87MG glioma cells. U87MG cells treated with puromycin (3 mM) for 24 h, which caused inhibition of ER-associated protein synthesis in a fashion similar to

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Fig. 10. Effects of anisomycin (AN) translocon inhibition on ERSR induction in malignant glioma cells. (A) U87MG cells were treated for 24 h with anisomycin (3 mM), THAP (200 nM), or anisomycin + THAP. THAP-induced ERSR activation, as indicated by increases in GRP78 expression, was attenuated when ER Ca2+ loss was inhibited with anisomycin. GAPDH immunoreactivity is presented as a loading control. (B) U87MG cells were treated for 24 h with anisomycin (3 mM), THAP (200 nM), or anisomycin + THAP. THAP-induced ERSR activation, as measured by increases in grp78 mRNA expression, was attenuated when ER Ca2+ loss was inhibited with anisomycin. ß-Actin was used as a loading control, a reaction control, and for comparison of grp78 band intensity. (C) THAP-induced caspase 4 activation was reduced when ER Ca2+ loss was inhibited by anisomycin. GAPDH was used as a loading control. (D) U87MG cells were treated for 48 h with anisomycin (3 mM), THAP (200 nM), or anisomycin + THAP. Inhibition of ER Ca2+ loss resulted in complete inhibition of THAP-induced cytotoxicity. Experiments were repeated 3 times or more. Statistical analysis was performed by 1-way ANOVA followed by Bonferroni’ post hoc test. *, ***, **** indicate P ≤ .05, P ≤ .001, and P ≤ .0001, respectively, vs control; B B, B B B, B B B B indicate P ≤ .05, P , .001, and P ≤ 0.0001, respectively, vs THAP treatment. N.S. indicates lack of difference vs AN.

Johnson et al.: ERSR, Ca2+, translocon, and glioma cell death

anisomycin, showed an increase of GRP78 protein expression and mRNA levels (i.e. ERSR induction), an effect completely prevented by 3 mM anisomycin (Fig. 11A and B). Treatment of U87 MG with puromycin also caused activation of caspase 4, as indicated by the appearance of the activated caspase 4 band (Fig. 11C). This evidence shows that ER-associated caspases are activated by enhanced ER Ca2+ loss through open translocons. The effect of puromycin on caspase 4 activation was also completely inhibited by anisomycin (Fig. 11C). Lastly, U87MG cell viability was assessed following exposure to puromycin (3 mM) for 48 h. Following puromycin exposure, we observed a 93% reduction of viability (Fig. 10D). This effect of puromycin was completely eliminated by concomitant exposure to anisomycin (Fig. 11D).

Discussion ERSR results when proteins unfold in the ER.4 Low levels of ERSR activation are protective, particularly in transformed cells.32

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Continued activation of ERSR, however, reduces the cells’ endurance to further stress. Under such conditions, pro-death mechanisms prevail and trigger programmed cell death.33 In the continued presence of ERSR-inducing agents, tumor cells have increased chemosensitivity and susceptibility to cell death, while normal cells have lower ERSR-mediated cell death.34 – 36 Our data clearly show that ERSR is hyperactivated in malignant glioma cells after exposure to THAP, a SERCA blocker that prevents ER Ca2+ uptake. Such hyperactivation triggers death mechanisms, an effect that is absent in normal glial cells. Excessive proliferation demands can be reflected in the upregulation of ERSR due to higher rates of protein synthesis needed in rapidly proliferating cells and, therefore, could participate in the development of the labile equilibrium that we demonstrated is at work in glioma cells. However, our data seem to suggest that even if higher proliferation played a role in the deployment of ERSR, it does not appear to reflect on all ERSR factors. For example, even though astrocytes grow at a slower rate than glioma cells, their levels of CHOP are significantly higher in response to ERSR activation with THAP. Therefore, the astrocytes’ CHOP

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Fig. 11. Puromycin (PURO) translocon blockade induces ERSR and causes caspase 4 activation and cytotoxicity in malignant glioma cells. (A) U87MG cells were treated for 24 h with anisomycin (ANISO; 3 mM), puromycin (3 mM), or anisomycin + puromycin. Puromycin-induced ER Ca2+ loss was associated with ERSR activation, as measured by an increase in GRP78 expression. Puromycin-induced ERSR activation was completely inhibited by anisomycin. (B) ERSR induction, as measured by an increase in GRP78 mRNA levels, was increased by puromycin. This effect of puromycin was prevented by treatment with anisomycin. (C) Caspase 4 was activated following treatment with puromycin for 24 h, and anisomycin prevented even caspase 4 activation by puromycin. (D) U87MG cells were treated for 48 h with anisomycin (3 mM), puromycin (3 mM), or anisomycin + puromycin. Puromycin caused cytotoxicity, which was reduced following treatment with anisomycin. Experiments were repeated 3 times or more. Statistical analysis was performed by 1-way ANOVA followed by Bonferroni’ post hoc test. **** indicates P ≤ .0001 vs control; B B B B indicates P ≤ .0001 vs PURO. N.S. indicates lack of difference vs ANISO.

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Ultimately, activation of these caspases can activate caspase 3 and trigger the final and irreversible steps of apoptosis.26 Our analyses indicated that caspases 4 and 12 are markedly activated following THAP-induced ERSR in malignant glioma cells but are unaffected in astrocytes. These results are consistent with other findings of our study, such as higher levels of GRP78 and cell death in malignant glioma cells. The view that ER-associated caspase activation is mainly responsible for the death of malignant glioma cells during ERSR is strengthened by our finding that caspase 3 is also activated by ERSR in malignant glioma cells. Our previous studies on the pharmacological modulation of ERSR in malignant glioma cells using nonsteroidal anti-inflammatory drugs has also shown that glioma cell death during ERSR, even in the presence of CHOP elevation, is predominantly due to ER-associated caspase activation. 25 Our previous work also pointed out that activation of CHOP and caspase 4 can cause an additive effect on the activation of caspase 3, but such an additive effect does not result in additional cell death in U87MG.25 It appears from our previous data that mitochondrial sufferance is necessary to increase CHOP expression during ERSR in U87MG.25 Ca2+ regulates cell signaling, proliferation, and apoptosis.44 These functions are all affected in cancer cells.45 Since Ca2+ is involved in ERSR deployment, we sought to determine whether Ca2+ homeostasis is also affected in malignant glioma cells and could be involved in the effects we report in this study. We found a difference in Ca2+ responses triggered by THAP and Ca2+mobilizing neurotransmitters. In resting conditions, high [Ca2+]ER is maintained in cells by the equilibrium of the ER Ca2+ reuptake mechanism (SERCA) and by active and passive ER Ca2+ losses.46,47 ER Ca2+ losses usually occur as a result of InsP3-R activity, chemical diffusion, and the activity of ER proteins.46 Our data demonstrate that ATP/InsP3 responses are characterized by higher Ca2+ levels during the plateau phase of the response and that THAP-induced [Ca2+]c elevation is greater in malignant glioma cells. Since we observed that in astrocytes and C6 the peak responses to ATP are similar, and there are no differences in the absence of extracellular Ca2+ in the peak values in response to ATP, we conclude that the Ca2+ content of the ER between normal glial and malignant glioma cells is similar. In addition, the finding that extracellular Ca2+ influx, measured after complete ER Ca2+ depletion triggered by exposure to maximal ATP concentrations in a Ca2+-free environment,30 follows similar patterns in these cells excludes substantial differences between normal glial cells and malignant glioma cells in the latter function. These results, by excluding other possible avenues, implicate Ca2+ loss from the ER as responsible for the enhancement of ER Ca2+ mobilization from the ER observed in malignant glioma cells. We thus investigated active, but not signaling related, ER Ca2+ loss. Transformed cells have high levels of protein synthesis.48 Furthermore, translocons, which are responsible for translocating nascent protein chains into the ER in the presence of the leader peptide sequence, are upregulated in malignant glioma cells.14 In particular, other authors have shown that the expression of the translocon Sec61g subunit is higher in malignant glioma cells than in normal glial cells,13,14 reflecting the overall higher levels of the translocon complex in these cells. The activity of the ribosome-translocon complex is associated with ER Ca2+ permeability.31,49,50 In rat liver microsomes, ER Ca2+

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response to ERSR induction seems to behave in the opposite manner to what one would predict based on the differences observed in the proliferation rates. Differently, GRP78 upregulation is greater in glioma cells than in astrocytes, effectively following predicted behavior based on proliferation rates. However, even in this case one would expect that C6 would be the more sensitive cells because their proliferation rates are higher. It appears that even if proliferation rates contribute to ERSR deployment differences, they do not directly correlate with the transduction factor levels during the ERSR cascade deployment, suggesting that other factors may be at play. Differences between normal and malignant glioma cells render the latter more susceptible to death by ERSR. Our data show that malignant glioma cells produce higher levels of unfolded proteins during ERSR, as signaled by higher levels of GRP78 expression. Although it is reported that ERSR results in comparable upregulation of GRP78 and CHOP,22,34 – 36 the studies did not compare normal with transformed cells.6 Our experiments carried out parallel analyses of the regulation of GRP78, CHOP, and other ERSR-involved death pathway factors in normal and transformed glial cells. During ERSR, CHOP is highly induced in normal glial cells, while its induction is much lower in malignant glioma cells. If CHOP were the main executor of ERSR-mediated cell death, normal glial cells would be more susceptible to ERSR-induced death than malignant glioma cells. Our data indicate that THAP-induced ERSR reduces viability of malignant glioma cells to a larger extent than normal glial cells. These data conflict with the hypothesis that CHOP is central in determining cell death during ERSR, at least in glioma cells and astrocytes. We do not know why high levels of CHOP expression in normal glial cells do not lead to activation of mitochondrial-dependent apoptosis, but such a failure of CHOP induction in causing cell death has been reported by others. CHOP is proapoptotic in Schwann cells unless counteracted by low-density lipoprotein receptor-related protein 1.37 In mouse models of Charcot-MarieTooth disease, Schwann cells exhibit CHOP activation but limited apoptosis.38 Additionally, in oligodendrocytes, CHOP expression exhibits antiapoptotic activity.39 It has been shown that CHOP upregulation in glioma cells could be dependent on mitochondrial functional impairment.25 However, even in the presence of high levels of CHOP induction in glioma cells, it appears that cell death correlated with other apoptotic determinants, rather than CHOP.25 Further studies are obviously needed to clarify the reasons for the failure of CHOP to cause cytotoxicity in astrocytes. There are other mechanisms through which ERSR can lead to cell death, besides CHOP proapoptotic effects. For example, GRP78 binds and prevents activation of ER-associated caspases, such as caspase 4 in humans and caspase 12 in rats.40 – 43 In most specimens derived from patients bearing malignant gliomas, caspase 4 is almost invariably upregulated (http://tcga. cancer.gov). Unfolded proteins generated during THAP-induced ERSR compete with the ER-associated caspase binding/immobilization by GRP78, causing the release of caspase 4/12. Once released, these caspases must be activated by the protease calpain, which in turn is activated by high local [Ca2+]c.8 In an environment of high [Ca 2+]c caused by exposure to THAP, ER-associated caspases are cleaved and thereby activated by Ca2+-enabled calpain.8 The simultaneous effect of THAP on the generation of unfolded proteins and elevation of [Ca2+]c activates ER-associated caspases.8

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ERSR cascade. These are reflected in the death of malignant glioma cells, triggered by protein unfolding mediated by ER Ca2+ depletion. Furthermore, increased activity of the Sec61 translocon is essential for ERSR deployment abnormalities. These data, along with abnormalities in the expression of caspase 4 reported in patient-derived specimens of malignant glioma cells, indicate that this system is a possible target for the development of new and effective antiglioma medications.

Supplementary Material Supplementary material is available online at Neuro-Oncology (http://neuro-oncology.oxfordjournals.org/).

Funding G.G.J. was supported by NIH-NIDA grant #1R03DA031669 to M.G.; M.C.W. and J.H.W. were supported by grants NIH-NIA #1R21AG038782 to M.G. and 1R03DA031669 to M.G.; M.G. was partially supported by NIH-NIA #1R21AG038782 and NIH-NIDA 1R03DA031669 both to M.G.

Conflict of interest statement. None declared.

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efflux has been associated with translocon activity.50 In LNCaP prostate cancer cells, THAP-induced ER Ca2+ release is attenuated following pretreatment with puromycin, which causes ER Ca2+ depletion.12 Additionally, anisomycin inhibition of the translocon in a Ca2+-impermeable configuration decreases ER Ca2+ release induced by THAP.31 In HeLa cells, the translocon interacts with calmodulin, a Ca2+-dependent signaling molecule, resulting in a reduction of ER Ca2+ efflux.49 Malignant glioma cells have enhanced ER Ca2+ loss, higher translocon expression levels, and lower levels of SERCA (unpublished results). These characteristics could affect ER Ca2+ homeostasis and cause these cells to respond to ER Ca2+ perturbations with high levels of protein unfolding, aggravated by more profound and longer-lasting ER Ca2+ depletion because of the relative SERCA deficit. Our results from experiments with the translocon inhibitors puromycin and anisomycin provide support for the hypothesis that translocon-mediated ER Ca2+ efflux is involved in ERSR deployment in malignant glioma cells by aggravating ER Ca2+ loss. Anisomycin and puromycin both inhibit ER resident protein synthesis by blocking translocon activity.51 – 54 Since their effect on translocon Ca2+ permeability is opposite, they provide a very useful tool to verify our hypothesis. Anisomycin, a peptidyl transferase inhibitor, prevents Ca2+ efflux from the translocon while blocking the activity of the translocon.31 Our experiments show that anisomycin prevents THAP-induced ER Ca2+ loss. As a consequence, in the presence of anisomycin, THAP induction of GRP78 is prevented. To exclude the possibility that the lack of GRP78 induction in cells treated with anisomycin could be due to inhibition of protein synthesis associated with translocon inhibition, we measured GRP78 mRNA expression, which is not affected by anisomycin.52,55,56 Our rationale for this experiment was that if the lack of GRP78 induction were due to inhibition of protein synthesis, levels of GRP78 mRNA would be elevated by THAP because of activating transcriptional factor 6 –dependent GRP78 mRNA induction, which is protein synthesis independent. The data clearly show that GRP78 mRNA is not upregulated following treatment with THAP in the presence of anisomycin. In addition, lack of caspase 4 activation and THAP-induced death of malignant glioma cells following concomitant treatment with THAP and anisomycin strengthen the hypothesis that translocon-dependent Ca2+ efflux is a determinant factor in THAP-induced ERSR aggravation. The results obtained following exposure to puromycin, a protein synthesis inhibitor/translocon blocker that, differently from anisomycin, locks the translocon complex in a Ca2+ permeable configuration,46 are also consistent with our hypothesis. Puromycin mimics the effect of THAP on ERSR and synergizes with it, although with a saturable ceiling corresponding to the maximal THAP effect (data not shown), which indicates a common mechanism. In fact, puromycin causes [Ca2+]c elevation, increases GRP78 protein (although it is an inhibitor of ER-associated protein synthesis) and mRNA expression, activates ER-associated caspase 4, and causes death of malignant glioma cells. All of the effects of puromycin are prevented by anisomycin, supporting the view that translocon activity and the associated ER Ca2+ leakage could be the basis for the differences in ER Ca2+ loss between normal glial cells and malignant glioma cells, rather than the inhibition of ER-associated protein synthesis. In conclusion, malignant glioma cells, in contrast to normal glial cells, have abnormalities affecting multiple levels of the

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