Antitumorigenic Effect of Proteasome Inhibitors on Insulinoma Cells
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内分泌学杂志 2005年第4期
Laboratory for ?-Cell Biology (J.S., A.E.K., N.B., T.M.-P.), Steno Diabetes Center, Gentofte 2820, Denmark; Division of Medical Genetics (N.A.-P., C.B.), Centre Hospitalier Universitaire Vaudois, 1011 Lausanne, Switzerland; and Rolf Luft Center for Diabetes Research (T.M.-P.), Department of Molecular Medicine, Karolinska Institute, S-176 76 Stockholm, Sweden
Address all correspondence to: Thomas Mandrup-Poulsen, M.D., DMSc, Steno Diabetes Center, Niels Steensens Vej 2, DK-2820 Gentofte, Denmark. E-mail: tmpo@steno.dk. Address reprint requests to: Joachim St?rling, MSc, Steno Diabetes Center, Niels Steensens Vej 2, DK-2820 Gentofte, Denmark. E-mail: jstq@steno.dk.
Abstract
Malignant insulinoma is a critical cancer form with a poor prognosis. Because cure by surgery is infrequent, effective chemotherapy is in demand. Induction of cell death in tumor cells by proteasome inhibitors is emerging as a potential strategy in cancer therapy. Here we investigated whether inhibition of the proteasome has an antitumorigenic potential in insulinoma cells. Exposure of mouse ?TC3 insulinoma cells to the proteasome inhibitor N-Acetyl-Leu-Leu-Nle-CHO (ALLN) reduced cell viability, activated caspase-3, induced apoptosis, and suppressed insulin release. Treatment with ALLN also resulted in phosphorylation of c-jun N-terminal kinase (JNK) and an increase in in vitro phosphorylation of c-jun. In insulinoma cells with impaired JNK signaling, ALLN-induced apoptosis was significantly suppressed. Another proteasome inhibitor, lactacystin, also stimulated JNK activation, caused activation of caspase-3, suppressed cell viability, and induced apoptosis in ?TC3 and rat INS-1E cells. Both ALLN and lactacystin caused a marked decrease in the cellular amount of the JNK scaffold protein JNK-interacting protein 1/islet-brain-1. In primary pancreatic rat islet cells, proteasome inhibition reduced insulin secretion but had no impact on cell viability and even partially protected against the toxic effect of proinflammatory cytokines. Our findings demonstrate that proteasome inhibitors possess antitumorigenic and antiinsulinogenic effects on insulinoma cells.
Introduction
MALIGNANT INSULINOMA IS a rare but fatal cancer form with a poor prognosis. As opposed to patients with benign ?-cell tumors, which are normally cured by surgery, malignant insulinomas are often diagnosed after metastatic dissemination has occurred, thereby restraining the success of cure by surgery. Moreover, hyperinsulinemia and consequently hypoglycemia caused by the uncontrolled insulin release by the insulinoma is critical to these patients (1, 2). Effective chemotherapy of patients with malignant insulinomas is therefore in demand.
The 26S proteasome is a large cylindrical multiprotease complex possessing several distinct catalytic activities. The 26S proteasome complex consists of a 20S catalytic core attached to two 19S regulatory subunits. Conjugation of polyubiquitin to proteins via the action of an E1 ubiquitin-activating enzyme, an E2 ubiquitin-conjugating enzyme, and an E3 ubiquitin ligase targets them for ATP-dependent degradation by the proteasome. The degradation of most cellular proteins is regulated through this ubiquitin-proteasome system (3). It has become evident that the proteasome is an essential regulator of programmed cell death or apoptosis (4). Recent studies have revealed that inhibitors of the proteasome are effective inducers of apoptosis in different tumor cells in vitro (5, 6). Moreover, proteasome inhibitors have been shown to exert antitumor activity in animals models, and encouraging results have been obtained in preclinical and phase I and II studies (5, 7, 8, 9). Therefore, proteasome inhibition is emerging as a novel approach to the treatment of cancer. However, whether proteasome inhibitors have antitumorigenic effects on insulinoma cells remain to be clarified.
The c-jun N-terminal kinase (JNK) is a MAPK that is generally activated in response to various forms of cellular stresses including proinflammatory cytokines, UV light, and osmotic shock (10, 11). Activation of JNK is achieved in a three-module kinase cascade consisting of upstream MAPK2 and MAPK3 kinases. The scaffold protein JNK-interacting protein 1/islet-brain-1 (JIP-1/IB1) is known to regulate JNK activation by binding to JNK and the upstream kinases mixed lineage kinase-3 and MAPK kinase 7, which are thereby held in close proximity, thus facilitating signal propagation to JNK. Activated JNK can phosphorylate and thereby increase or inhibit the activity or function of a diverse set of proteins including transcription factors such as c-jun and activating transcription factor and members of the Bcl-2 family of apoptosis-regulating proteins (10, 12, 13). Recent studies have established that the JNK pathway plays a prominent role in apoptosis induced by the proinflammatory cytokine IL-1? in rodent insulinoma cells (14, 15).
In the present study, the putative antitumor effect of proteasome inhibition on insulinoma cells was examined. We show that proteasome inhibitors exert potent antitumorigenic effects on insulinoma cells, an effect that involves activation of the JNK pathway.
Materials and Methods
Materials
N-Acetyl-Leu-Leu-Nle-CHO (ALLN) and lactacystin (LC) were from Calbiochem (San Diego, CA). Reagents for SDS-PAGE were from Bio-Rad (Richmond, CA) and Invitrogen Life Technologies (Carlsbad, CA). The JNK substrate glutathione-S-transferase (GST)-c-jun [amino acids (aa) 1–79)] containing the two major phospho-acceptor sites for JNK (Ser63 and Ser73) was from Calbiochem and [32P]ATP (3000 Ci/mmol) was obtained from Amersham (Buckinghamshire, UK). Recombinant mouse IL-1? was from BD PharMingen (San Diego, CA) and recombinant rat interferon- was from R&D Systems (Minneapolis, MN). Antibodies against cleaved caspase-3, procaspase-3, JNK1/2, phospho-JNK1/2, p53 and p21 as well as horseradish peroxidase (HRP)-conjugated secondary antibodies were all from Cell Signaling (Beverly, MA). Antiactin and antitubulin antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA) and Sigma (St. Louis, MO), respectively. Antibody raised against JIP-1/IB1 have been described previously (16).
Cell and islet culture
Mouse ?TC3 and rat INS-1E/INS-1 insulinoma cells were cultured in RPMI 1640 medium with Glutamax-1 supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin (all from Invitrogen Life Technologies). In addition, the media for INS-1E/INS-1 cells contained 50 μM ?-mercaptoethanol (Sigma). Cells were trypsinized and passaged weekly. For experimentation, cells were seeded in either T25 tissue culture flasks or in 12-, 24-, or 96-well plates (Nunc, Roskilde, Denmark). Cells were precultured for 1–2 d before exposure to inhibitors. ?TC3 cells stably expressing the JNK-binding domain (JBD) (aa 1–280) of JIP-1/IB1 were generated through lentiviral delivery (16, 17). Expression of JBD and impaired JNK signaling in these cells were verified (Ref. 18 and data not shown). Pancreatic islets from neonatal rats were isolated by the collagenase method and fractionated on a Percoll gradient as described (19). Islets were cultured in RPMI 1640 medium supplemented with 0.5% human serum and 100 U/ml penicillin and 100 μg/ml streptomycin.
Apoptotic measurements
Hoechst/propidium iodide (PI).
The number of apoptotic ?TC3 cells was evaluated by PI and Hoechst 33342 nuclear staining. After treatment of cells with ALLN or LC for the specified time, cells were incubated with PI (12.5 μg/ml) and Hoechst 33342 (5 μg/ml) for 5 min at room temperature before visualization under inverted fluorescence microscope with UV excitation at 340–380 nm. A minimum of 1000 cells were counted for each experiment.
PhiPhiLux.
After treatment of INS-1E cells with LC for the specified time, cells were trypsinized, centrifuged (5 min at 500 x g) and washed in cold PBS. Cell pellets were resuspended in 20 μl of the caspase-3 substrate PhiPhiLux (OncoImmunin Inc., Gaithersburg, MD) and incubated for 1 h at 37 C. After washing in PBS, cells were visualized by fluorescence microscopy using a fluorescein filter.
Viability assay
Cell viability was assessed by the dimethylthiazol-diphenyltetrazolium bromide (MTT) assay (Promega, Madison, WI) measuring mitochondrial activity by the conversion of a tetrazolium salt to a colored formazan product by the enzyme succinate dehydrogenase. Briefly, insulinoma cells in triplicates (15,000 cells/well) or rat islets in duplicates (50 islets/well) in 96-well plates were treated with inhibitors for 2 d. Twenty-five (?TC3/INS-1E) or 10 (islets) μl of MTT Dye reagent was added to each well followed by incubation at 37 C for 1–3 h. Then 90 μl per well of MTT Stop solution were added followed by overnight incubation. Absorbance was measured at 578 nm.
Insulin release
Insulin release into the culture medium was measured by competitive enzyme-linked immunosorbent assay as described previously (20).
Cell lysis
Whole-cell extracts for in vitro kinase assay or Western blotting were prepared from cells after inhibitor treatment by lysing cells for 30 min on ice in lysis buffer containing 20 mM Tris-acetate (pH 7.0), 0.27 M sucrose, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 1% vol/vol Triton X-100, 5 mM sodium pyrophosphate, 10 mM sodium glycerophosphate, 1 mM benzamidine, 1 mM dithiothreitol (DTT), 1 mM Na3VO4, and 4 μg/ml leupeptin. Detergent-insoluble material was pelleted by centrifugation at 15,000x g for 5 min at 4 C. The supernatants constituting the whole-cell extracts were stored at –80 C until assayed. The protein concentration in extracts was determined using the Bio-Rad dye reagent concentrate (Bio-Rad Laboratories, Hercules, CA).
Western blotting
Whole-cell extracts were mixed with sodium dodecyl sulfate (SDS) sample buffer [125 mM Tris-HCl (pH 6.8), 4% SDS, 0.1 M DTT, 10% glycerol, and 0.02% bromophenol blue], boiled for 5 min, and subjected to 12% SDS-PAGE. Proteins were electrotransferred onto nitrocellulose membranes followed by blocking of nonspecific protein binding by incubating membranes in blocking buffer [1x TBS (pH 7.6), 0.1% Tween 20, 5% nonfat dry milk] for 1 h at room temperature. After washing in washing buffer [1x TBS (pH 7.6), 0.1% Tween 20], membranes were probed with antibodies overnight at 4 C. Membranes were then washed, incubated for 1 h at room temperature with HRP-conjugated secondary antibodies, and the immune complex visualized by chemiluminescence. Light emission was captured on x-ray film or detected using a Fuji Film luminescent image analyzer (LAS)-3000.
In vitro kinase assay
Phosphotransferase activity toward GST-c-jun and heat shock protein-25 was measured by mixing approximately 7 μl of whole-cell lysate (volumes adjusted to contain the same amount of protein) with 17 μl reaction buffer [2 μg GST-c-jun, 2 μg heat shock protein-2, 25 mM Tris-HCl (pH 7.4), 0.1 mM EGTA, 0.1 mM Na3VO4, 1 μM cAMP-dependent protein kinase inhibitor and 10 mM Mg-acetate] and 3 μl ATP mixture (1 mM ATP and 3 μCi [-32P]ATP). Phosphotransferase reactions were carried out at 30 C for 30 min. Reactions were terminated by the addition of 20 μl SDS sample buffer [125 mM Tris-HCl (pH 6.8), 4% SDS, 0.1 M DTT, 10% glycerol, and 0.02% bromophenol blue] and boiling for 5 min. Samples were then subjected to 12% SDS-PAGE and after eletrophoresis, gels were washed in fixation buffer (40% methanol and 10% acetate) for 15 min. The gels were dried and proteins visualized by autoradiography. Phosphorylations were quantitated by PhosphorImager analysis (Molecular Dynamics, Sunnyvale, CA).
Transient transfection and luciferase reporter assay
INS-1E cells were transiently transfected with the indicated vectors (Stratagene, La Jolla, CA) using Superfect (QIAGEN, Valencia, CA) as described by the manufacturer. The amounts of plasmids for each transfection were as follows: p53-driven luciferase reporter plasmid (0.2 μg) and constitutive Renilla luciferase expression vector (0.2 μg). Four hours post transfection, media was replaced with fresh media. The next day, cells were left untreated or treated with LC for 6 h. For preparation of cell lysate, cells were washed in PBS and lysed in passive lysis buffer (Promega). Luciferase measurements were carried out using the dual luciferase reporter system kit according to the specifications of the manufacturer (Promega). The p53-induced luciferase activity was normalized to the coexpressed Renilla luciferase.
Statistical analysis
In bar graphs, data are means ± SE. Statistical significance of differences between groups was calculated by paired or unpaired t test as appropriate. P < 0.05 was chosen as the level of significance.
Results
ALLN induces apoptosis in mouse ?TC3 insulinoma cells
We first examined by MTT assay the effect of the proteasome inhibitor ALLN on mouse insulinoma ?TC3 cell viability. ALLN dose-dependently suppressed (P < 0.0005, single-factor ANOVA) cell viability during a 2-d exposure (Fig. 1A). At 10, 20, 40, and 80 μM, ALLN suppressed cell viability by 36, 69, 83, and 86%, respectively. Apoptosis in ?TC3 cells after exposure to 20 μM ALLN was evaluated by PI and Hoechst 33342 nuclear staining. ALLN increased apoptosis from 0.90 ± 0.10 to 7.87 ± 0.30% (equivalent to a 8.7-fold increase) during a 24-h exposure (Fig. 1B). To further demonstrate an apoptotic process in cells treated with ALLN, caspase-3 activation was investigated. As demonstrated by Western blot analysis of caspase-3, ALLN induced cleavage of procaspase-3 into the active form (Fig. 1C). Finally, ALLN inhibited ?TC3 cell insulin release by 47% during 24 h (Fig. 1D). It was also observed that apoptosis induced by a combination of proinflammatory cytokines was significantly augmented by ALLN (data not shown). Together, these results demonstrate that the proteasome inhibitor ALLN is cytotoxic to ?TC3 insulinoma cells by inducing apoptosis.
FIG. 1. Effects of ALLN on ?TC3 cell viability and apoptosis. A, ?TC3 cells were left untreated [control (CTRL)], treated with 0.2% dimethylsulfoxide (DMSO), or exposed to the indicated concentrations (μM) of ALLN for 2 d. Viability was determined by MTT assay. Results are means ± SE of three independent experiments each performed in triplicate. B, ?TC3 cells were left untreated or exposed to 20 μM ALLN for 24 h. The number of apoptotic cells was determined by Hoechst and PI nuclear staining. Results are means ± SE of five independent experiments. C, Caspase-3 Western blot analysis of extracts from ?TC3 cells exposed to 20 μM ALLN for 24 h. Blots shown are representative of three independent experiments. D, Accumulated insulin release to culture medium after exposure to 20 μM ALLN for 24 h. Results are means ± SE of three independent experiments. *, P = 0.03 vs. CTRL; **, P < 0.001 vs. CTRL.
ALLN activates JNK
Previously, proteasome inhibition has been shown to activate JNK in noninsulinoma tumor cells (21, 22). To examine whether ALLN affects the JNK signaling pathway in insulinoma cells, ?TC3 cells were treated with 20 μM ALLN for 7 h. Western blot analysis of whole-cell extracts with antibodies against the phosphorylated, activated form of JNK1/2 revealed that ALLN induced phosphorylation of JNK1/2 (Fig. 2A). In accordance with this, extracts from cells treated with ALLN contained increased kinase activity toward GST-c-jun [aa 1–79], a JNK substrate that contains the two major phospho-acceptor sites for JNK (Ser63 and Ser73), as determined by in vitro kinase assay (Fig. 2, A and B). These results indicate that ALLN induces activation of JNK in insulinoma cells.
FIG. 2. Effect of ALLN on JNK activity. A, ?TC3 cells were treated with 20 μM ALLN or left untreated [control (CTRL)] for 7 h. Whole-cell extracts were prepared and subjected to Western blot analysis of phospho-JNK1/2 (P-JNK1/2) and JNK1/2 and to in vitro kinase assay using GST-c-jun as substrate in the presence of 32P-ATP. Blots and autoradiogram shown are representative of at least three independent experiments. B, Phosphorylation of GST-c-jun was quantitated by PhosphorImager analysis. Results are means ± SE. C, Control ?TC3 cells or JBD expressing ?TC3 cells (?TC3/JBD) were incubated with or without 20 μM ALLN for 24 h. The number of apoptotic cells was determined by Hoechst and PI nuclear staining. Results are means ± SE of four to six independent experiments. **, P < 0.008 vs. CTRL; #, P < 0.003.
JBD reduces ALLN-induced apoptosis
To examine the functional role of JNK in ALLN-induced apoptosis, we compared the ability of ALLN to induce apoptosis in control ?TC3 cells and ?TC3 cells stably transfected with the JBD of the JNK scaffold JIP-1/IB1 leading to inhibition of JNK signaling (14, 18, 23). Compared with control cells, expression of JBD yielded an approximately 50% (a 10.9-fold vs. a 5.5-fold induction) protection against ALLN-induced apoptosis (Fig. 2C). These findings suggest that JNK contributes to the apoptotic process induced by ALLN in insulinoma cells.
LC induces JNK activation and apoptosis
Because ALLN is not exclusively an inhibitor of the proteasome, but also inhibits e.g. calpain proteases, the effect of the more specific proteasome inhibitor LC (5, 24) on apoptosis and JNK was determined. An increase in the number of apoptotic cells from 2.7 ± 0.9 to 20.3 ± 0.5% (corresponding to a 7.5-fold increase) was observed after a 24-h treatment with 10 μM LC (Fig. 3A). Consistent with this, LC reduced ?TC3 cell viability (Fig. 3B) and induced cleavage of procaspase-3 (Fig. 3C). Also, incubation of cells with LC caused phosphorylation of JNK1/2 and an increase in in vitro kinase activity toward c-jun (Fig. 3, D and E). In contrast to these findings, a selective inhibitor of calpains, calpastatin peptide (25), neither activated JNK nor affected apoptosis in ?TC3 cells (data not shown).
FIG. 3. Effects of LC on ?TC3 cell viability, apoptosis and JNK activity. A, ?TC3 cells were incubated left untreated [control (CTRL)] or exposed to 10 μM LC for 24 h. The number of apoptotic cells was determined by Hoechst and PI nuclear staining. Results are means ± SE of three independent experiments. B, ?TC3 cells were incubated with or without 10 μM LC for 2 d. Viability was determined by MTT assay. Results are means ± SE of three independent experiments each performed in triplicate. C, Caspase-3 Western blot analysis of extracts from ?TC3 cells exposed to 10 μM LC for 24 h. Blots shown are representative of three independent experiments. D, ?TC3 cells were incubated with or without 10 μM LC for 7 h. Whole-cell extracts were prepared and subjected to Western blot analysis of phospho-JNK1/2 (P-JNK1/2) and JNK1/2 and to in vitro kinase assay using GST-c-jun as substrate in the presence of 32P-ATP. Blots and autoradiogram shown are representative of three to four independent experiments. E, Phosphorylation of GST-c-jun was quantitated by PhosphorImager analysis. Results are means ± SE. *, P < 0.02 vs. CTRL; **, P < 0.002 vs. CTRL.
Proteasome inhibitors cause down-regulation of JIP-1/IB1 protein
The JIP-1/IB1 JNK scaffold protein is highly expressed in pancreatic ?-cells (16). Because JIP-1/IB1 plays an important role in the regulation of the JNK pathway and because various stress stimuli including cytokines and UV light lead to a decrease in the cellular amount of JIP-1/IB1 in ?-cells (15, 26), thereby possibly affecting activation and routing of JNK signaling, we investigated whether proteasome inhibitors affect the JIP-1/IB1 protein level. Western blot analysis of JIP-1/IB1 in whole-cell lysates from ?TC3 cells exposed to ALLN or LC for 3 h showed a markedly reduced amount of JIP-1/IB1 as compared with untreated control cells (Fig. 4). JIP-1/IB1 down-regulation may thus be an important factor in proteasome inhibition-induced signaling via the JNK pathway and apoptosis.
FIG. 4. Effect of ALLN and LC on JIP-1/IB1 protein level. ?TC3 cells were left untreated [control (CTRL)] or treated with 20 μM ALLN or 10 μM LC for 3 h. Whole-cell extracts were prepared and subjected to Western blot analysis of JIP-1/IB1. Blots shown are representative of four independent experiments. The upper band detected corresponds to the full-length product of JIP-1/IB1, and the lower band is likely to be a 5' splicing product. Analysis of tubulin was used as control for equal protein loading.
Proteasome inhibitors induce JNK activation and apoptosis in rat INS-1E insulinoma cells
The effects of proteasome inhibition on INS-1E rat insulinoma cells were also investigated. As seen in Fig. 5A, both ALLN and LC caused phosphorylation of JNK1/2 as revealed by Western blotting. Viability analysis after a 2-d exposure to ALLN or LC showed a dose-dependent reduction in cell viability (Fig. 5B). In accordance with this, ALLN and LC stimulated cleavage of procaspase-3 (Fig. 5C). Apoptosis of INS-1E cells after LC exposure was in addition assessed by fluorescence microscopy using the PhiPhiLux system, which allows the detection of caspase-3 activity in living cells. In brief, cells are incubated with a profluorogenic protease substrate containing the specific peptide sequence recognized and cleaved by activated caspase-3. After cleavage by caspase-3, the PhiPhiLux substrate becomes fluorescent. Figure 5D shows that, after 48 h of treatment with LC, most cells were positive for caspase-3 activity. In line with these findings, ALLN and LC suppressed INS-1E cell insulin release to the culture medium during a 24-h exposure (Fig. 5E). Hypothetically, the inhibitory effect of proteasome inhibitors on insulin release could be related simply to the fact that fewer cells are secreting insulin due to the reduction in cell viability, and not because proteasome inhibitors per se impair insulin secretion in living cells. To address this, we also measured the cellular insulin content after treatment with ALLN or LC for 24 h to determine insulin release as percent of total insulin (insulin released + cellular insulin). The results showed that both proteasome inhibitors still markedly blocked insulin release (data not shown). This suggests that the inhibitory effect on insulin secretion by proteasome inhibitors is not simply due to a reduction in the number of cells that secrete insulin.
FIG. 5. Effects of ALLN and LC on JNK phosphorylation, viability, and apoptosis in INS-1E cells. A, INS-1E cells were left untreated [control (CTRL)] or exposed to 20 μM ALLN or 10 μM LC for 3 h. Whole-cell extracts were prepared and subjected to Western blot analysis of phospho-JNK1/2 (P-JNK1/2) and JNK1/2. Blots shown are representative of three independent experiments. B, INS-1E cells were incubated with or without the indicated concentrations (micromolar) of ALLN or LC for 2 d. Viability was determined by MTT assay. Results are means ± SE of three independent experiments each performed in triplicate. C, Caspase-3 Western blot analysis of extracts from INS-1E cells exposed to 20 μM ALLN or 10 μM LC for 24 h. Blots shown are representative of two independent experiments. D, INS-1E cells were left untreated or exposed to 10 μM LC for 48 h. Cells were trypsinized and incubated for 1 h with PhiPhiLux caspase-3 substrate. Cells positive for caspase-3 activity were visualized by fluorescence microscopy. Images shown are representative of four independent experiments. E, Accumulated insulin release to culture medium after exposure to 20 μM ALLN or 10 μM LC for 24 h. Results are means ± SE of four independent experiments. *, P < 0.05 vs. CTRL.
Proteasome inhibitors do not induce p53 accumulation in insulinoma cells
Because inhibition of proteasomal activity in other tumor cells previously was shown to lead to accumulation of the tumor suppressor protein p53 (27, 28), which is otherwise held at a low level due to degradation by the proteasome, the effect of proteasome inhibition on p53 protein level was investigated. Exposure of either INS-1 or ?TC3 cells to ALLN or LC for 2 or 4 h failed to induce accumulation of p53 as determined by Western blotting (Fig. 6, A and B). Furthermore, neither in control nor in proteasome inhibitor-treated cells expression of the cyclin-dependent kinase inhibitor p21, which is induced by p53, was detected (Fig. 6, A and B). Both p53 and p21 were detected in lysates from human embryonic kidney cells that were used as positive control. As an alternative method for investigating the effect of proteasome inhibition on p53, we transiently transfected cells with a p53-driven Luciferase reporter gene construct. It was observed that a 6-h exposure to LC only weakly (by 30%) stimulated an increase in p53-mediated reporter gene activity (Fig. 6C). These findings suggest that p53 does not accumulate and is not activated to any significant degree after proteasome inhibition in insulinoma cells.
FIG. 6. Effects of ALLN and LC on p53. INS-1 (A) or ?TC3 (B) cells were left untreated [control (CTRL)] or exposed to 20 μM ALLN or 10 μM LC for 2 or 4 h. Whole-cell extracts were prepared and subjected to Western blot analysis of p53, p21, and actin. Whole-cell extract from human embryonic kidney (HEK) 293 cells was used as positive control. Blots shown are representative of three independent experiments. C, At 1 d after transfection of INS-1E cells with a p53-driven luciferase reporter gene construct, cells were left untreated or exposed to 10 μM LC for 6 h. After cell lysis, luciferase activities were measured. Results are normalized to coexpressed Renilla luciferase. Results are means ± SE of three independent experiments. *, P < 0.05 vs. CTRL.
LC is not cytotoxic to primary rat islet cells
To determine whether proteasome inhibition has functional effects on nontransformed primary ?-cells, isolated intact rat islets were exposed to LC for 2 d followed by MTT viability analysis. A 10-μM concentration of LC, which was observed to suppress ?TC3 and INS-1E insulinoma cell viability by 75% and 80%, respectively (Figs. 3B and Fig. 5B), had no effect on islet cell viability (Fig. 7A). In fact, the suppressive effect on islet viability induced by cytokines (a cocktail of IL-1? and interferon- was used because this is normally required to significantly reduce islet cell viability) was reversed from 71.5 ± 6.7% of control to 93.2 ± 2.6% by LC, suggesting an antiapoptotic effect of proteasome inhibition in primary ?-cells. Measurement of media insulin content revealed that LC inhibited accumulated islet insulin release by 47% during 2 d (Fig. 7B). Cytokines caused a 73% reduction in insulin release, which was not further affected by coincubation with LC. Similar trends of ALLN (20 μM) on islet viability and insulin secretion were found (data not shown). Finally, in vitro kinase activity toward GST-c-jun in lysates from islets exposed to LC was measured. A 3-h exposure of islets to LC failed to increase kinase activity toward GST-c-jun (Fig. 7, C and D). Interestingly, coincubation of islets with LC plus IL-1? (IL-1? was used alone because this cytokine is sufficient to cause significant activation of MAPKs) resulted in higher in vitro phosphorylations of GST-c-jun as compared with islets incubated with IL-1? alone (Fig. 7, C and D). Together, these results indicate that proteasome inhibition alone does not activate JNK and is not cytotoxic to primary islet cells, though accumulated insulin release is reduced.
FIG. 7. Effects of LC on primary rat islet cell viability, insulin release and JNK activity. A, Whole rat islets were left untreated [control (CTRL)] or exposed to 10 μM LC in the presence or absence of a combination of proinflammatory cytokines [cytokines (CTK): 150 pg/ml IL-1? + 5 ng/ml interferon-] for 2 d. Islet cell viability was determined by MTT assay. Results are means ± SE of three independent experiments each performed in duplicate. B, Accumulated insulin release to the culture medium after treatment of rat islets as in panel A. Results are means ± SE of three independent experiments. C, Rat islets were left untreated or treated with 10 μM LC in the presence or absence of IL-1? for 3 h. Whole-cell extracts were prepared and subjected to in vitro kinase assay using GST-c-jun as substrate in the presence of 32P-ATP. Autoradiogram shown is representative of four individual experiments. D, Phosphorylation of GST-c-jun was quantitated by PhosphorImager analysis. Results are means ± SE. *, P < 0.05 vs. CTRL; #, P < 0.05 vs. CTK or IL-1.
Discussion
The metastatic dissemination of malignant insulinomas and the ensuing hypoglycemia caused by hyperinsulinemia warrant effective chemotherapy of patients with this type of cancer. Proteasome inhibitors have recently gained attention as a novel strategy for the treatment of neoplastic diseases. Thus, in various tumor cells proteasome inhibitors such as ALLN and LC have been shown to induce apoptosis (5). However, until now there have been no studies describing the potential antitumorigenic effect of proteasome inhibitors on transformed ?-cells. In this study, the in vitro effects of proteasome inhibitors on viability, apoptosis and insulin secretion in insulinoma cells were investigated. Our data demonstrate a strong antitumorigenic effect of proteasome inhibitors on rodent insulinoma cells, but not on primary islet cells.
Proteasome inhibition by either ALLN or LC markedly reduced the viability of ?TC3 or INS-1E insulinoma cells after a 2-d exposure, at least in part due to an increase in apoptotic cell death. Our finding that apoptosis is induced by proteasome inhibition is consistent with observations in other noninsulinoma tumor cells (21, 27, 29, 30, 31, 32). Proteasome inhibitor-induced insulinoma cell apoptosis was associated with activation of caspase-3 as demonstrated by Western blotting showing proteolytic processing of the inactive 32-kDa procaspase-3 form into the active 17-kDa form and by fluorescence microscopy using the profluorescent caspase-3 substrate PhiPhiLux. These findings suggest that the classical apoptotic pathway involving the caspase cascade is activated in insulinoma cells by proteasome inhibitors.
The effect of proteasome inhibition on apoptosis was associated with activation of the JNK signaling pathway. Blocking JNK signaling by means of overexpressing JBD in ?TC3 cells reduced proteasome inhibitor-induced apoptosis by 50%. This finding is in agreement with previous findings showing that JNK contributes to proteasome inhibitor-induced apoptosis in 293 human kidney tumor cells and human Jurkat T tumor cells (21, 22, 33, 34).
Because JBD only partially protected against proteasome inhibitor-induced apoptosis, additional mechanisms to JNK is likely to be involved. One candidate is the tumor suppressor protein p53, which accumulates in Rat-1 and PC12 tumor cells treated with proteasome inhibitors and contributes to apoptosis (27). However, by immunoblotting and reporter gene assay, we found that p53 was not to any significant degree induced in insulinoma cells upon proteasome inhibition for up to 4 h. After treatment of other tumor cells with proteasome inhibitors, increased p53 and p21 expression is observed within 2–4 h (28). Based on these findings, p53 probably does not contribute to the apoptotic response in insulinoma cells upon treatment with proteasome inhibitors. However, because p53 is only one member among a family of tumor suppressor proteins, it cannot be excluded that other members might play a role in insulinoma cells.
The molecular mechanism behind proteasome inhibitor-induced JNK activation is unclear. Proteasome inhibition may lead to accumulation of upstream activators of JNK that are otherwise normally degraded by the proteasome. Alternatively, accretion of nondegraded proteins perturbs cell function and homeostasis and is sensed as cellular stress leading to activation of stress pathways, including JNK, and apoptosis. Tumor cells may be more sensitive to such signals because proteasome inhibition in primary islet cells failed to activate JNK and to suppress viability.
Our finding that ALLN and LC led to a marked reduction in the protein level of the JNK scaffold protein JIP-1/IB1 suggests that this event is involved in controlling the JNK pathway in proteasome inhibitor-exposed cells. JIP-1/IB1 normally facilitates the propagation of phosphorylations within the three-kinase module, leading to efficient activation of JNK. Very high expression of JIP-1/IB1 has been demonstrated in insulin-secreting ?-cells and brain (16) and may therefore render these cells extraordinary sensitive to various JNK-activating stimuli. However, a high concentration of JIP-1/IB1 (with JNK bound via the JBD) also physically prevents JNK from interacting with certain substrates e.g. c-jun and activating transcription factor 2 (23). Various stressful stimuli, however, including IL-1? (15) and proteasome inhibitors, lead to down-regulation of JIP-1/IB1, thereby affecting signal efficacy and routing of the JNK signaling pathway, because more JNK will be dissociated from JIP-1/IB1 and thus free to interact with (proapoptotic) downstream substrates.
In primary islet cells, LC affected neither JNK activity, as determined by in vitro c-jun phosphorylation, nor islet viability. This suggests that the effects of proteasome inhibitors on stress signaling and viability are specific for tumor ?-cells. In fact, proteasome inhibition partially reversed the suppressive effect on islet viability induced by cytokines. This observation may be relevant for the destruction of ?-cells in diabetes which is believed to be mediated by cytokines (35, 36). Interestingly, we observed that LC augmented IL-1?-induced islet kinase activity toward c-jun. This finding indicates that components in IL-1? signal transduction leading to JNK activation are subjected to degradation via the ubiquitin-proteasome pathway. Indeed, it has been shown that IL-1 receptor-associated kinase after IL-1 stimulation becomes phosphorylated and subsequently degraded by the proteasome (37). If proteasome inhibition increases IL-1? signaling in islets, then this would hypothetically result in aggravation of cytokine-induced cell death. However, this was not the case because LC afforded protection against the toxic effect of cytokines. One explanation for this might be that proteasomal activity is needed for distal signaling events (downstream JNK) that mediate cell death. Proteasome inhibition suppressed islet insulin release by 47%. This effect was comparable to that seen on insulinoma cells. Thus, proteasome inhibitors have similar antiinsulinogenic effects in both tumorigenic and nontumorigenic ?-cells. The mechanism underlying proteasome inhibition-mediated insulin release suppression remains to be elucidated.
In conclusion, this study shows for the first time that proteasome inhibitors exert antitumor effects on insulinoma cells in part via activation and possibly altered routing of the JNK signaling pathway. This, together with the observation that proteasome inhibitors do not affect primary islet cell viability, provide the basis for testing the efficiency of proteasome inhibitors in vivo in animal models of ?-cell tumorigenesis.
Acknowledgments
The technical assistance from Fie Hilles?, Anna Hlin Schram, and Hanne Foght is gratefully acknowledged.
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Address all correspondence to: Thomas Mandrup-Poulsen, M.D., DMSc, Steno Diabetes Center, Niels Steensens Vej 2, DK-2820 Gentofte, Denmark. E-mail: tmpo@steno.dk. Address reprint requests to: Joachim St?rling, MSc, Steno Diabetes Center, Niels Steensens Vej 2, DK-2820 Gentofte, Denmark. E-mail: jstq@steno.dk.
Abstract
Malignant insulinoma is a critical cancer form with a poor prognosis. Because cure by surgery is infrequent, effective chemotherapy is in demand. Induction of cell death in tumor cells by proteasome inhibitors is emerging as a potential strategy in cancer therapy. Here we investigated whether inhibition of the proteasome has an antitumorigenic potential in insulinoma cells. Exposure of mouse ?TC3 insulinoma cells to the proteasome inhibitor N-Acetyl-Leu-Leu-Nle-CHO (ALLN) reduced cell viability, activated caspase-3, induced apoptosis, and suppressed insulin release. Treatment with ALLN also resulted in phosphorylation of c-jun N-terminal kinase (JNK) and an increase in in vitro phosphorylation of c-jun. In insulinoma cells with impaired JNK signaling, ALLN-induced apoptosis was significantly suppressed. Another proteasome inhibitor, lactacystin, also stimulated JNK activation, caused activation of caspase-3, suppressed cell viability, and induced apoptosis in ?TC3 and rat INS-1E cells. Both ALLN and lactacystin caused a marked decrease in the cellular amount of the JNK scaffold protein JNK-interacting protein 1/islet-brain-1. In primary pancreatic rat islet cells, proteasome inhibition reduced insulin secretion but had no impact on cell viability and even partially protected against the toxic effect of proinflammatory cytokines. Our findings demonstrate that proteasome inhibitors possess antitumorigenic and antiinsulinogenic effects on insulinoma cells.
Introduction
MALIGNANT INSULINOMA IS a rare but fatal cancer form with a poor prognosis. As opposed to patients with benign ?-cell tumors, which are normally cured by surgery, malignant insulinomas are often diagnosed after metastatic dissemination has occurred, thereby restraining the success of cure by surgery. Moreover, hyperinsulinemia and consequently hypoglycemia caused by the uncontrolled insulin release by the insulinoma is critical to these patients (1, 2). Effective chemotherapy of patients with malignant insulinomas is therefore in demand.
The 26S proteasome is a large cylindrical multiprotease complex possessing several distinct catalytic activities. The 26S proteasome complex consists of a 20S catalytic core attached to two 19S regulatory subunits. Conjugation of polyubiquitin to proteins via the action of an E1 ubiquitin-activating enzyme, an E2 ubiquitin-conjugating enzyme, and an E3 ubiquitin ligase targets them for ATP-dependent degradation by the proteasome. The degradation of most cellular proteins is regulated through this ubiquitin-proteasome system (3). It has become evident that the proteasome is an essential regulator of programmed cell death or apoptosis (4). Recent studies have revealed that inhibitors of the proteasome are effective inducers of apoptosis in different tumor cells in vitro (5, 6). Moreover, proteasome inhibitors have been shown to exert antitumor activity in animals models, and encouraging results have been obtained in preclinical and phase I and II studies (5, 7, 8, 9). Therefore, proteasome inhibition is emerging as a novel approach to the treatment of cancer. However, whether proteasome inhibitors have antitumorigenic effects on insulinoma cells remain to be clarified.
The c-jun N-terminal kinase (JNK) is a MAPK that is generally activated in response to various forms of cellular stresses including proinflammatory cytokines, UV light, and osmotic shock (10, 11). Activation of JNK is achieved in a three-module kinase cascade consisting of upstream MAPK2 and MAPK3 kinases. The scaffold protein JNK-interacting protein 1/islet-brain-1 (JIP-1/IB1) is known to regulate JNK activation by binding to JNK and the upstream kinases mixed lineage kinase-3 and MAPK kinase 7, which are thereby held in close proximity, thus facilitating signal propagation to JNK. Activated JNK can phosphorylate and thereby increase or inhibit the activity or function of a diverse set of proteins including transcription factors such as c-jun and activating transcription factor and members of the Bcl-2 family of apoptosis-regulating proteins (10, 12, 13). Recent studies have established that the JNK pathway plays a prominent role in apoptosis induced by the proinflammatory cytokine IL-1? in rodent insulinoma cells (14, 15).
In the present study, the putative antitumor effect of proteasome inhibition on insulinoma cells was examined. We show that proteasome inhibitors exert potent antitumorigenic effects on insulinoma cells, an effect that involves activation of the JNK pathway.
Materials and Methods
Materials
N-Acetyl-Leu-Leu-Nle-CHO (ALLN) and lactacystin (LC) were from Calbiochem (San Diego, CA). Reagents for SDS-PAGE were from Bio-Rad (Richmond, CA) and Invitrogen Life Technologies (Carlsbad, CA). The JNK substrate glutathione-S-transferase (GST)-c-jun [amino acids (aa) 1–79)] containing the two major phospho-acceptor sites for JNK (Ser63 and Ser73) was from Calbiochem and [32P]ATP (3000 Ci/mmol) was obtained from Amersham (Buckinghamshire, UK). Recombinant mouse IL-1? was from BD PharMingen (San Diego, CA) and recombinant rat interferon- was from R&D Systems (Minneapolis, MN). Antibodies against cleaved caspase-3, procaspase-3, JNK1/2, phospho-JNK1/2, p53 and p21 as well as horseradish peroxidase (HRP)-conjugated secondary antibodies were all from Cell Signaling (Beverly, MA). Antiactin and antitubulin antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA) and Sigma (St. Louis, MO), respectively. Antibody raised against JIP-1/IB1 have been described previously (16).
Cell and islet culture
Mouse ?TC3 and rat INS-1E/INS-1 insulinoma cells were cultured in RPMI 1640 medium with Glutamax-1 supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin (all from Invitrogen Life Technologies). In addition, the media for INS-1E/INS-1 cells contained 50 μM ?-mercaptoethanol (Sigma). Cells were trypsinized and passaged weekly. For experimentation, cells were seeded in either T25 tissue culture flasks or in 12-, 24-, or 96-well plates (Nunc, Roskilde, Denmark). Cells were precultured for 1–2 d before exposure to inhibitors. ?TC3 cells stably expressing the JNK-binding domain (JBD) (aa 1–280) of JIP-1/IB1 were generated through lentiviral delivery (16, 17). Expression of JBD and impaired JNK signaling in these cells were verified (Ref. 18 and data not shown). Pancreatic islets from neonatal rats were isolated by the collagenase method and fractionated on a Percoll gradient as described (19). Islets were cultured in RPMI 1640 medium supplemented with 0.5% human serum and 100 U/ml penicillin and 100 μg/ml streptomycin.
Apoptotic measurements
Hoechst/propidium iodide (PI).
The number of apoptotic ?TC3 cells was evaluated by PI and Hoechst 33342 nuclear staining. After treatment of cells with ALLN or LC for the specified time, cells were incubated with PI (12.5 μg/ml) and Hoechst 33342 (5 μg/ml) for 5 min at room temperature before visualization under inverted fluorescence microscope with UV excitation at 340–380 nm. A minimum of 1000 cells were counted for each experiment.
PhiPhiLux.
After treatment of INS-1E cells with LC for the specified time, cells were trypsinized, centrifuged (5 min at 500 x g) and washed in cold PBS. Cell pellets were resuspended in 20 μl of the caspase-3 substrate PhiPhiLux (OncoImmunin Inc., Gaithersburg, MD) and incubated for 1 h at 37 C. After washing in PBS, cells were visualized by fluorescence microscopy using a fluorescein filter.
Viability assay
Cell viability was assessed by the dimethylthiazol-diphenyltetrazolium bromide (MTT) assay (Promega, Madison, WI) measuring mitochondrial activity by the conversion of a tetrazolium salt to a colored formazan product by the enzyme succinate dehydrogenase. Briefly, insulinoma cells in triplicates (15,000 cells/well) or rat islets in duplicates (50 islets/well) in 96-well plates were treated with inhibitors for 2 d. Twenty-five (?TC3/INS-1E) or 10 (islets) μl of MTT Dye reagent was added to each well followed by incubation at 37 C for 1–3 h. Then 90 μl per well of MTT Stop solution were added followed by overnight incubation. Absorbance was measured at 578 nm.
Insulin release
Insulin release into the culture medium was measured by competitive enzyme-linked immunosorbent assay as described previously (20).
Cell lysis
Whole-cell extracts for in vitro kinase assay or Western blotting were prepared from cells after inhibitor treatment by lysing cells for 30 min on ice in lysis buffer containing 20 mM Tris-acetate (pH 7.0), 0.27 M sucrose, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 1% vol/vol Triton X-100, 5 mM sodium pyrophosphate, 10 mM sodium glycerophosphate, 1 mM benzamidine, 1 mM dithiothreitol (DTT), 1 mM Na3VO4, and 4 μg/ml leupeptin. Detergent-insoluble material was pelleted by centrifugation at 15,000x g for 5 min at 4 C. The supernatants constituting the whole-cell extracts were stored at –80 C until assayed. The protein concentration in extracts was determined using the Bio-Rad dye reagent concentrate (Bio-Rad Laboratories, Hercules, CA).
Western blotting
Whole-cell extracts were mixed with sodium dodecyl sulfate (SDS) sample buffer [125 mM Tris-HCl (pH 6.8), 4% SDS, 0.1 M DTT, 10% glycerol, and 0.02% bromophenol blue], boiled for 5 min, and subjected to 12% SDS-PAGE. Proteins were electrotransferred onto nitrocellulose membranes followed by blocking of nonspecific protein binding by incubating membranes in blocking buffer [1x TBS (pH 7.6), 0.1% Tween 20, 5% nonfat dry milk] for 1 h at room temperature. After washing in washing buffer [1x TBS (pH 7.6), 0.1% Tween 20], membranes were probed with antibodies overnight at 4 C. Membranes were then washed, incubated for 1 h at room temperature with HRP-conjugated secondary antibodies, and the immune complex visualized by chemiluminescence. Light emission was captured on x-ray film or detected using a Fuji Film luminescent image analyzer (LAS)-3000.
In vitro kinase assay
Phosphotransferase activity toward GST-c-jun and heat shock protein-25 was measured by mixing approximately 7 μl of whole-cell lysate (volumes adjusted to contain the same amount of protein) with 17 μl reaction buffer [2 μg GST-c-jun, 2 μg heat shock protein-2, 25 mM Tris-HCl (pH 7.4), 0.1 mM EGTA, 0.1 mM Na3VO4, 1 μM cAMP-dependent protein kinase inhibitor and 10 mM Mg-acetate] and 3 μl ATP mixture (1 mM ATP and 3 μCi [-32P]ATP). Phosphotransferase reactions were carried out at 30 C for 30 min. Reactions were terminated by the addition of 20 μl SDS sample buffer [125 mM Tris-HCl (pH 6.8), 4% SDS, 0.1 M DTT, 10% glycerol, and 0.02% bromophenol blue] and boiling for 5 min. Samples were then subjected to 12% SDS-PAGE and after eletrophoresis, gels were washed in fixation buffer (40% methanol and 10% acetate) for 15 min. The gels were dried and proteins visualized by autoradiography. Phosphorylations were quantitated by PhosphorImager analysis (Molecular Dynamics, Sunnyvale, CA).
Transient transfection and luciferase reporter assay
INS-1E cells were transiently transfected with the indicated vectors (Stratagene, La Jolla, CA) using Superfect (QIAGEN, Valencia, CA) as described by the manufacturer. The amounts of plasmids for each transfection were as follows: p53-driven luciferase reporter plasmid (0.2 μg) and constitutive Renilla luciferase expression vector (0.2 μg). Four hours post transfection, media was replaced with fresh media. The next day, cells were left untreated or treated with LC for 6 h. For preparation of cell lysate, cells were washed in PBS and lysed in passive lysis buffer (Promega). Luciferase measurements were carried out using the dual luciferase reporter system kit according to the specifications of the manufacturer (Promega). The p53-induced luciferase activity was normalized to the coexpressed Renilla luciferase.
Statistical analysis
In bar graphs, data are means ± SE. Statistical significance of differences between groups was calculated by paired or unpaired t test as appropriate. P < 0.05 was chosen as the level of significance.
Results
ALLN induces apoptosis in mouse ?TC3 insulinoma cells
We first examined by MTT assay the effect of the proteasome inhibitor ALLN on mouse insulinoma ?TC3 cell viability. ALLN dose-dependently suppressed (P < 0.0005, single-factor ANOVA) cell viability during a 2-d exposure (Fig. 1A). At 10, 20, 40, and 80 μM, ALLN suppressed cell viability by 36, 69, 83, and 86%, respectively. Apoptosis in ?TC3 cells after exposure to 20 μM ALLN was evaluated by PI and Hoechst 33342 nuclear staining. ALLN increased apoptosis from 0.90 ± 0.10 to 7.87 ± 0.30% (equivalent to a 8.7-fold increase) during a 24-h exposure (Fig. 1B). To further demonstrate an apoptotic process in cells treated with ALLN, caspase-3 activation was investigated. As demonstrated by Western blot analysis of caspase-3, ALLN induced cleavage of procaspase-3 into the active form (Fig. 1C). Finally, ALLN inhibited ?TC3 cell insulin release by 47% during 24 h (Fig. 1D). It was also observed that apoptosis induced by a combination of proinflammatory cytokines was significantly augmented by ALLN (data not shown). Together, these results demonstrate that the proteasome inhibitor ALLN is cytotoxic to ?TC3 insulinoma cells by inducing apoptosis.
FIG. 1. Effects of ALLN on ?TC3 cell viability and apoptosis. A, ?TC3 cells were left untreated [control (CTRL)], treated with 0.2% dimethylsulfoxide (DMSO), or exposed to the indicated concentrations (μM) of ALLN for 2 d. Viability was determined by MTT assay. Results are means ± SE of three independent experiments each performed in triplicate. B, ?TC3 cells were left untreated or exposed to 20 μM ALLN for 24 h. The number of apoptotic cells was determined by Hoechst and PI nuclear staining. Results are means ± SE of five independent experiments. C, Caspase-3 Western blot analysis of extracts from ?TC3 cells exposed to 20 μM ALLN for 24 h. Blots shown are representative of three independent experiments. D, Accumulated insulin release to culture medium after exposure to 20 μM ALLN for 24 h. Results are means ± SE of three independent experiments. *, P = 0.03 vs. CTRL; **, P < 0.001 vs. CTRL.
ALLN activates JNK
Previously, proteasome inhibition has been shown to activate JNK in noninsulinoma tumor cells (21, 22). To examine whether ALLN affects the JNK signaling pathway in insulinoma cells, ?TC3 cells were treated with 20 μM ALLN for 7 h. Western blot analysis of whole-cell extracts with antibodies against the phosphorylated, activated form of JNK1/2 revealed that ALLN induced phosphorylation of JNK1/2 (Fig. 2A). In accordance with this, extracts from cells treated with ALLN contained increased kinase activity toward GST-c-jun [aa 1–79], a JNK substrate that contains the two major phospho-acceptor sites for JNK (Ser63 and Ser73), as determined by in vitro kinase assay (Fig. 2, A and B). These results indicate that ALLN induces activation of JNK in insulinoma cells.
FIG. 2. Effect of ALLN on JNK activity. A, ?TC3 cells were treated with 20 μM ALLN or left untreated [control (CTRL)] for 7 h. Whole-cell extracts were prepared and subjected to Western blot analysis of phospho-JNK1/2 (P-JNK1/2) and JNK1/2 and to in vitro kinase assay using GST-c-jun as substrate in the presence of 32P-ATP. Blots and autoradiogram shown are representative of at least three independent experiments. B, Phosphorylation of GST-c-jun was quantitated by PhosphorImager analysis. Results are means ± SE. C, Control ?TC3 cells or JBD expressing ?TC3 cells (?TC3/JBD) were incubated with or without 20 μM ALLN for 24 h. The number of apoptotic cells was determined by Hoechst and PI nuclear staining. Results are means ± SE of four to six independent experiments. **, P < 0.008 vs. CTRL; #, P < 0.003.
JBD reduces ALLN-induced apoptosis
To examine the functional role of JNK in ALLN-induced apoptosis, we compared the ability of ALLN to induce apoptosis in control ?TC3 cells and ?TC3 cells stably transfected with the JBD of the JNK scaffold JIP-1/IB1 leading to inhibition of JNK signaling (14, 18, 23). Compared with control cells, expression of JBD yielded an approximately 50% (a 10.9-fold vs. a 5.5-fold induction) protection against ALLN-induced apoptosis (Fig. 2C). These findings suggest that JNK contributes to the apoptotic process induced by ALLN in insulinoma cells.
LC induces JNK activation and apoptosis
Because ALLN is not exclusively an inhibitor of the proteasome, but also inhibits e.g. calpain proteases, the effect of the more specific proteasome inhibitor LC (5, 24) on apoptosis and JNK was determined. An increase in the number of apoptotic cells from 2.7 ± 0.9 to 20.3 ± 0.5% (corresponding to a 7.5-fold increase) was observed after a 24-h treatment with 10 μM LC (Fig. 3A). Consistent with this, LC reduced ?TC3 cell viability (Fig. 3B) and induced cleavage of procaspase-3 (Fig. 3C). Also, incubation of cells with LC caused phosphorylation of JNK1/2 and an increase in in vitro kinase activity toward c-jun (Fig. 3, D and E). In contrast to these findings, a selective inhibitor of calpains, calpastatin peptide (25), neither activated JNK nor affected apoptosis in ?TC3 cells (data not shown).
FIG. 3. Effects of LC on ?TC3 cell viability, apoptosis and JNK activity. A, ?TC3 cells were incubated left untreated [control (CTRL)] or exposed to 10 μM LC for 24 h. The number of apoptotic cells was determined by Hoechst and PI nuclear staining. Results are means ± SE of three independent experiments. B, ?TC3 cells were incubated with or without 10 μM LC for 2 d. Viability was determined by MTT assay. Results are means ± SE of three independent experiments each performed in triplicate. C, Caspase-3 Western blot analysis of extracts from ?TC3 cells exposed to 10 μM LC for 24 h. Blots shown are representative of three independent experiments. D, ?TC3 cells were incubated with or without 10 μM LC for 7 h. Whole-cell extracts were prepared and subjected to Western blot analysis of phospho-JNK1/2 (P-JNK1/2) and JNK1/2 and to in vitro kinase assay using GST-c-jun as substrate in the presence of 32P-ATP. Blots and autoradiogram shown are representative of three to four independent experiments. E, Phosphorylation of GST-c-jun was quantitated by PhosphorImager analysis. Results are means ± SE. *, P < 0.02 vs. CTRL; **, P < 0.002 vs. CTRL.
Proteasome inhibitors cause down-regulation of JIP-1/IB1 protein
The JIP-1/IB1 JNK scaffold protein is highly expressed in pancreatic ?-cells (16). Because JIP-1/IB1 plays an important role in the regulation of the JNK pathway and because various stress stimuli including cytokines and UV light lead to a decrease in the cellular amount of JIP-1/IB1 in ?-cells (15, 26), thereby possibly affecting activation and routing of JNK signaling, we investigated whether proteasome inhibitors affect the JIP-1/IB1 protein level. Western blot analysis of JIP-1/IB1 in whole-cell lysates from ?TC3 cells exposed to ALLN or LC for 3 h showed a markedly reduced amount of JIP-1/IB1 as compared with untreated control cells (Fig. 4). JIP-1/IB1 down-regulation may thus be an important factor in proteasome inhibition-induced signaling via the JNK pathway and apoptosis.
FIG. 4. Effect of ALLN and LC on JIP-1/IB1 protein level. ?TC3 cells were left untreated [control (CTRL)] or treated with 20 μM ALLN or 10 μM LC for 3 h. Whole-cell extracts were prepared and subjected to Western blot analysis of JIP-1/IB1. Blots shown are representative of four independent experiments. The upper band detected corresponds to the full-length product of JIP-1/IB1, and the lower band is likely to be a 5' splicing product. Analysis of tubulin was used as control for equal protein loading.
Proteasome inhibitors induce JNK activation and apoptosis in rat INS-1E insulinoma cells
The effects of proteasome inhibition on INS-1E rat insulinoma cells were also investigated. As seen in Fig. 5A, both ALLN and LC caused phosphorylation of JNK1/2 as revealed by Western blotting. Viability analysis after a 2-d exposure to ALLN or LC showed a dose-dependent reduction in cell viability (Fig. 5B). In accordance with this, ALLN and LC stimulated cleavage of procaspase-3 (Fig. 5C). Apoptosis of INS-1E cells after LC exposure was in addition assessed by fluorescence microscopy using the PhiPhiLux system, which allows the detection of caspase-3 activity in living cells. In brief, cells are incubated with a profluorogenic protease substrate containing the specific peptide sequence recognized and cleaved by activated caspase-3. After cleavage by caspase-3, the PhiPhiLux substrate becomes fluorescent. Figure 5D shows that, after 48 h of treatment with LC, most cells were positive for caspase-3 activity. In line with these findings, ALLN and LC suppressed INS-1E cell insulin release to the culture medium during a 24-h exposure (Fig. 5E). Hypothetically, the inhibitory effect of proteasome inhibitors on insulin release could be related simply to the fact that fewer cells are secreting insulin due to the reduction in cell viability, and not because proteasome inhibitors per se impair insulin secretion in living cells. To address this, we also measured the cellular insulin content after treatment with ALLN or LC for 24 h to determine insulin release as percent of total insulin (insulin released + cellular insulin). The results showed that both proteasome inhibitors still markedly blocked insulin release (data not shown). This suggests that the inhibitory effect on insulin secretion by proteasome inhibitors is not simply due to a reduction in the number of cells that secrete insulin.
FIG. 5. Effects of ALLN and LC on JNK phosphorylation, viability, and apoptosis in INS-1E cells. A, INS-1E cells were left untreated [control (CTRL)] or exposed to 20 μM ALLN or 10 μM LC for 3 h. Whole-cell extracts were prepared and subjected to Western blot analysis of phospho-JNK1/2 (P-JNK1/2) and JNK1/2. Blots shown are representative of three independent experiments. B, INS-1E cells were incubated with or without the indicated concentrations (micromolar) of ALLN or LC for 2 d. Viability was determined by MTT assay. Results are means ± SE of three independent experiments each performed in triplicate. C, Caspase-3 Western blot analysis of extracts from INS-1E cells exposed to 20 μM ALLN or 10 μM LC for 24 h. Blots shown are representative of two independent experiments. D, INS-1E cells were left untreated or exposed to 10 μM LC for 48 h. Cells were trypsinized and incubated for 1 h with PhiPhiLux caspase-3 substrate. Cells positive for caspase-3 activity were visualized by fluorescence microscopy. Images shown are representative of four independent experiments. E, Accumulated insulin release to culture medium after exposure to 20 μM ALLN or 10 μM LC for 24 h. Results are means ± SE of four independent experiments. *, P < 0.05 vs. CTRL.
Proteasome inhibitors do not induce p53 accumulation in insulinoma cells
Because inhibition of proteasomal activity in other tumor cells previously was shown to lead to accumulation of the tumor suppressor protein p53 (27, 28), which is otherwise held at a low level due to degradation by the proteasome, the effect of proteasome inhibition on p53 protein level was investigated. Exposure of either INS-1 or ?TC3 cells to ALLN or LC for 2 or 4 h failed to induce accumulation of p53 as determined by Western blotting (Fig. 6, A and B). Furthermore, neither in control nor in proteasome inhibitor-treated cells expression of the cyclin-dependent kinase inhibitor p21, which is induced by p53, was detected (Fig. 6, A and B). Both p53 and p21 were detected in lysates from human embryonic kidney cells that were used as positive control. As an alternative method for investigating the effect of proteasome inhibition on p53, we transiently transfected cells with a p53-driven Luciferase reporter gene construct. It was observed that a 6-h exposure to LC only weakly (by 30%) stimulated an increase in p53-mediated reporter gene activity (Fig. 6C). These findings suggest that p53 does not accumulate and is not activated to any significant degree after proteasome inhibition in insulinoma cells.
FIG. 6. Effects of ALLN and LC on p53. INS-1 (A) or ?TC3 (B) cells were left untreated [control (CTRL)] or exposed to 20 μM ALLN or 10 μM LC for 2 or 4 h. Whole-cell extracts were prepared and subjected to Western blot analysis of p53, p21, and actin. Whole-cell extract from human embryonic kidney (HEK) 293 cells was used as positive control. Blots shown are representative of three independent experiments. C, At 1 d after transfection of INS-1E cells with a p53-driven luciferase reporter gene construct, cells were left untreated or exposed to 10 μM LC for 6 h. After cell lysis, luciferase activities were measured. Results are normalized to coexpressed Renilla luciferase. Results are means ± SE of three independent experiments. *, P < 0.05 vs. CTRL.
LC is not cytotoxic to primary rat islet cells
To determine whether proteasome inhibition has functional effects on nontransformed primary ?-cells, isolated intact rat islets were exposed to LC for 2 d followed by MTT viability analysis. A 10-μM concentration of LC, which was observed to suppress ?TC3 and INS-1E insulinoma cell viability by 75% and 80%, respectively (Figs. 3B and Fig. 5B), had no effect on islet cell viability (Fig. 7A). In fact, the suppressive effect on islet viability induced by cytokines (a cocktail of IL-1? and interferon- was used because this is normally required to significantly reduce islet cell viability) was reversed from 71.5 ± 6.7% of control to 93.2 ± 2.6% by LC, suggesting an antiapoptotic effect of proteasome inhibition in primary ?-cells. Measurement of media insulin content revealed that LC inhibited accumulated islet insulin release by 47% during 2 d (Fig. 7B). Cytokines caused a 73% reduction in insulin release, which was not further affected by coincubation with LC. Similar trends of ALLN (20 μM) on islet viability and insulin secretion were found (data not shown). Finally, in vitro kinase activity toward GST-c-jun in lysates from islets exposed to LC was measured. A 3-h exposure of islets to LC failed to increase kinase activity toward GST-c-jun (Fig. 7, C and D). Interestingly, coincubation of islets with LC plus IL-1? (IL-1? was used alone because this cytokine is sufficient to cause significant activation of MAPKs) resulted in higher in vitro phosphorylations of GST-c-jun as compared with islets incubated with IL-1? alone (Fig. 7, C and D). Together, these results indicate that proteasome inhibition alone does not activate JNK and is not cytotoxic to primary islet cells, though accumulated insulin release is reduced.
FIG. 7. Effects of LC on primary rat islet cell viability, insulin release and JNK activity. A, Whole rat islets were left untreated [control (CTRL)] or exposed to 10 μM LC in the presence or absence of a combination of proinflammatory cytokines [cytokines (CTK): 150 pg/ml IL-1? + 5 ng/ml interferon-] for 2 d. Islet cell viability was determined by MTT assay. Results are means ± SE of three independent experiments each performed in duplicate. B, Accumulated insulin release to the culture medium after treatment of rat islets as in panel A. Results are means ± SE of three independent experiments. C, Rat islets were left untreated or treated with 10 μM LC in the presence or absence of IL-1? for 3 h. Whole-cell extracts were prepared and subjected to in vitro kinase assay using GST-c-jun as substrate in the presence of 32P-ATP. Autoradiogram shown is representative of four individual experiments. D, Phosphorylation of GST-c-jun was quantitated by PhosphorImager analysis. Results are means ± SE. *, P < 0.05 vs. CTRL; #, P < 0.05 vs. CTK or IL-1.
Discussion
The metastatic dissemination of malignant insulinomas and the ensuing hypoglycemia caused by hyperinsulinemia warrant effective chemotherapy of patients with this type of cancer. Proteasome inhibitors have recently gained attention as a novel strategy for the treatment of neoplastic diseases. Thus, in various tumor cells proteasome inhibitors such as ALLN and LC have been shown to induce apoptosis (5). However, until now there have been no studies describing the potential antitumorigenic effect of proteasome inhibitors on transformed ?-cells. In this study, the in vitro effects of proteasome inhibitors on viability, apoptosis and insulin secretion in insulinoma cells were investigated. Our data demonstrate a strong antitumorigenic effect of proteasome inhibitors on rodent insulinoma cells, but not on primary islet cells.
Proteasome inhibition by either ALLN or LC markedly reduced the viability of ?TC3 or INS-1E insulinoma cells after a 2-d exposure, at least in part due to an increase in apoptotic cell death. Our finding that apoptosis is induced by proteasome inhibition is consistent with observations in other noninsulinoma tumor cells (21, 27, 29, 30, 31, 32). Proteasome inhibitor-induced insulinoma cell apoptosis was associated with activation of caspase-3 as demonstrated by Western blotting showing proteolytic processing of the inactive 32-kDa procaspase-3 form into the active 17-kDa form and by fluorescence microscopy using the profluorescent caspase-3 substrate PhiPhiLux. These findings suggest that the classical apoptotic pathway involving the caspase cascade is activated in insulinoma cells by proteasome inhibitors.
The effect of proteasome inhibition on apoptosis was associated with activation of the JNK signaling pathway. Blocking JNK signaling by means of overexpressing JBD in ?TC3 cells reduced proteasome inhibitor-induced apoptosis by 50%. This finding is in agreement with previous findings showing that JNK contributes to proteasome inhibitor-induced apoptosis in 293 human kidney tumor cells and human Jurkat T tumor cells (21, 22, 33, 34).
Because JBD only partially protected against proteasome inhibitor-induced apoptosis, additional mechanisms to JNK is likely to be involved. One candidate is the tumor suppressor protein p53, which accumulates in Rat-1 and PC12 tumor cells treated with proteasome inhibitors and contributes to apoptosis (27). However, by immunoblotting and reporter gene assay, we found that p53 was not to any significant degree induced in insulinoma cells upon proteasome inhibition for up to 4 h. After treatment of other tumor cells with proteasome inhibitors, increased p53 and p21 expression is observed within 2–4 h (28). Based on these findings, p53 probably does not contribute to the apoptotic response in insulinoma cells upon treatment with proteasome inhibitors. However, because p53 is only one member among a family of tumor suppressor proteins, it cannot be excluded that other members might play a role in insulinoma cells.
The molecular mechanism behind proteasome inhibitor-induced JNK activation is unclear. Proteasome inhibition may lead to accumulation of upstream activators of JNK that are otherwise normally degraded by the proteasome. Alternatively, accretion of nondegraded proteins perturbs cell function and homeostasis and is sensed as cellular stress leading to activation of stress pathways, including JNK, and apoptosis. Tumor cells may be more sensitive to such signals because proteasome inhibition in primary islet cells failed to activate JNK and to suppress viability.
Our finding that ALLN and LC led to a marked reduction in the protein level of the JNK scaffold protein JIP-1/IB1 suggests that this event is involved in controlling the JNK pathway in proteasome inhibitor-exposed cells. JIP-1/IB1 normally facilitates the propagation of phosphorylations within the three-kinase module, leading to efficient activation of JNK. Very high expression of JIP-1/IB1 has been demonstrated in insulin-secreting ?-cells and brain (16) and may therefore render these cells extraordinary sensitive to various JNK-activating stimuli. However, a high concentration of JIP-1/IB1 (with JNK bound via the JBD) also physically prevents JNK from interacting with certain substrates e.g. c-jun and activating transcription factor 2 (23). Various stressful stimuli, however, including IL-1? (15) and proteasome inhibitors, lead to down-regulation of JIP-1/IB1, thereby affecting signal efficacy and routing of the JNK signaling pathway, because more JNK will be dissociated from JIP-1/IB1 and thus free to interact with (proapoptotic) downstream substrates.
In primary islet cells, LC affected neither JNK activity, as determined by in vitro c-jun phosphorylation, nor islet viability. This suggests that the effects of proteasome inhibitors on stress signaling and viability are specific for tumor ?-cells. In fact, proteasome inhibition partially reversed the suppressive effect on islet viability induced by cytokines. This observation may be relevant for the destruction of ?-cells in diabetes which is believed to be mediated by cytokines (35, 36). Interestingly, we observed that LC augmented IL-1?-induced islet kinase activity toward c-jun. This finding indicates that components in IL-1? signal transduction leading to JNK activation are subjected to degradation via the ubiquitin-proteasome pathway. Indeed, it has been shown that IL-1 receptor-associated kinase after IL-1 stimulation becomes phosphorylated and subsequently degraded by the proteasome (37). If proteasome inhibition increases IL-1? signaling in islets, then this would hypothetically result in aggravation of cytokine-induced cell death. However, this was not the case because LC afforded protection against the toxic effect of cytokines. One explanation for this might be that proteasomal activity is needed for distal signaling events (downstream JNK) that mediate cell death. Proteasome inhibition suppressed islet insulin release by 47%. This effect was comparable to that seen on insulinoma cells. Thus, proteasome inhibitors have similar antiinsulinogenic effects in both tumorigenic and nontumorigenic ?-cells. The mechanism underlying proteasome inhibition-mediated insulin release suppression remains to be elucidated.
In conclusion, this study shows for the first time that proteasome inhibitors exert antitumor effects on insulinoma cells in part via activation and possibly altered routing of the JNK signaling pathway. This, together with the observation that proteasome inhibitors do not affect primary islet cell viability, provide the basis for testing the efficiency of proteasome inhibitors in vivo in animal models of ?-cell tumorigenesis.
Acknowledgments
The technical assistance from Fie Hilles?, Anna Hlin Schram, and Hanne Foght is gratefully acknowledged.
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