The proteasome inhibitor lactacystin exerts its therapeutic effects on glioma via apoptosis: An in vitro and in vivo study

Abstract

Objective

To examine the effect and underlying mechanism of action of the proteasome inhibitor lactacystin on glioma, in vitro and in vivo.

Methods

Rat C6 glioma cells were cultured with or without lactacystin. Cell proliferation, apoptosis and mitochondrial membrane potential were determined. A glioma xenograft model was established in mice and animals were treated with 0, 1 or 5 µg/20 g body weight lactacystin for 7 days. Animals were sacrificed on day 17 after completion of treatment. Apoptosis in tumour tissue was examined by terminal deoxynucleotidyl transferase dUTP nick end labeling staining. Levels of B cell lymphoma 2 (Bcl-2), and Bcl2-associated X protein (Bax) protein and mRNA, were determined in C6 cells and tumour tissues.

Results

Lactacystin significantly inhibited the proliferation of C6 cells, increased apoptosis and reduced mitochondrial membrane potential in vitro, and suppressed tumour growth in vivo. Lactacystin increased the ratio of Bax to Bcl-2 at the mRNA and protein levels, both in vitro and in vivo.

Conclusions

The effects of lactacystin are associated with apoptosis induction. Proteasome inhibition may represent an effective treatment option for glioma.

Keywords

Apoptosis Bax Bcl-2 glioma lactacystin mitochondria

Introduction

Malignant gliomas account for ∼70% of the 22,500 malignant primary brain tumours diagnosed in adults in the US each year,¹ and are associated with high morbidity and mortality.² It is difficult to eliminate malignant gliomas because they are resistant to conventional therapies including surgery, radiotherapy and chemotherapy.¹ Research has therefore focused on finding new strategies for treating these tumours.

Inhibition of proteasome activity has been shown to reduce cell proliferation in melanoma, prostate cancer and glioma-derived tumour cell lines.^3–6 The proteasome is both a selective pathway for the disposal of short-lived proteins within eukaryotic cells,⁷ and is also essential for cellular processes including protein quality control, cell-cycle control, transcription, signalling, protein transport, DNA repair and stress responses.^8,9 Proteasome activity is higher in cancer tissue than in normal tissue,^10,11 suggesting that rapidly-growing malignant cells require higher proteasome activity. Inhibition of proteasome activity has become a new strategy for targeted chemotherapy, with several chemicals and natural compounds being shown to be effective in inducing tumour cell death.^12–15

The proteasome has three major enzymatic activities: chymotrypsin-like, trypsin-like and peptidylglutamylpeptide hydrolysing-like.¹⁶ Clinical trials of the chemically synthesized proteasome inhibitor bortizomib are underway for the treatment of leukaemia, but further studies are required in solid tumours. Lactacystin is a naturally occurring microbial product with potent proteasome inhibitory effects,¹⁷ binding to the catalytic core of the proteasome and inhibiting all three peptidase activities, in vitro and in vivo.¹⁸ The inhibition of chymotrypsin-like and trypsin-like activity by lactacystin is both rapid and irreversible.¹⁸

The antineoplastic activity of lactacystin has been shown to depend on inhibition of the ubiquitin proteasome pathway,⁶ and lactacystin is also able to enhance the activity of chemotherapeutic agents and radiosensitizers.^19,20 In vitro studies indicate that lactacystin induces cell death in rat and human glioma cell lines by triggering apoptosis, but these effects remain to be determined in vivo. The aim of the present study was to examine the effect (and underlying mechanism of action) of lactacystin on the C6 rat glioma cell line in vitro and C6 xenograft tumours in vivo.

Materials and methods

Cell Culture

The rat glioma cell line C6 was obtained from Shanghai Institute of Cell Biology, Chinese Academy of Sciences, Shanghai, China. Cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco, Rockville, MD, USA) supplemented with 10% fetal bovine serum, 2 mmol/l glutamine, 100 IU/ml penicillin and 100 µg/ml streptomycin, and maintained at 37°C in a humid environment with 5% carbon dioxide in air. Cells in the mid log growth phase were used in experiments.

Cell Viability Assay

Cells were seeded into 96-well plates (3 × 10⁴ cells/well) and cultured for 24 h, then incubated with 0, 2.5, 5 or 10 µmol/l lactacystin for a further 24 h. Cell viability was determined via 3 -(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (Beyotime, Jiangsu, China), according to the manufacturer’s instructions.

Flow Cytometry

The C6 glioma cells (3 × 10⁶ cells/flask) were cultured overnight, incubated with 0, 2.5, 5 or 10 µmol/l lactacystin for 24 h, and harvested by trypsinization and centrifugation (800 g for 5 min). For apoptosis analysis, cells were washed with PBS, adjusted to 1 × 10⁶ cells/ml, then fixed in 70% ethanol at 4°C overnight. The next day, cells were washed four times at 4°C with PBS, treated with 100 mg/l RNase at 37°C for 30 min, stained with 50 mg/l propidium iodide for 30 min and analysed by flow cytometry (FACScan™; Becton Dickinson, San Jose, CA, USA). The numbers of viable and apoptotic cells were determined for each culture condition, with a minimum of 20,000 cells analysed per sample. The rate of apoptosis was determined using CellQuest™ software (Becton Dickinson).

Mitochondrial membrane potential was determined in C6 cells grown and harvested as above. Cells were resuspended in culture medium, incubated with 10 µg/ml rhodamine 123 for 30 min at 37°C, harvested as before, washed four times with PBS and analysed by flow cytometry.

Glioma Xenograft Model

Athymic BALB/c nude mice (n = 36, aged 4 weeks, weight 20–22 g) were purchased from the Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences, Beijing, China. Mice were housed in a 12-h light/12-h dark cycle with free access to food and water, and were acclimatized to their surroundings for 2 days before the experiment began. The experimental protocol was approved by the Committee on the Use of Live Animals in Teaching and Research, Jilin University, and institutional guidelines were followed in handling the animals.

The C6 cells (5 × 10⁶ cells in 100 µl PBS) were implanted by subcutaneous injection into the right flank of each mouse. When the tumour reached ∼0.05 cm³ (after ∼7–10 days), mice were allocated to receive daily intraperitoneal injections of PBS (control group), 1.0 µg/20 g body weight lactacystin or 5.0 µg/20 g body weight lactacystin (all injections 100 µl) for 7 days (n ≥ 5/group). In each experimental group, approximately half of the mice were sacrificed by decapitation on day 9 after termination of treatment and tumours were removed for analysis. Part of each tumour was fixed in formalin and the remainder was frozen in liquid nitrogen. For the remaining mice, tumour volume (V) was determined daily from day 1 to day 17 after termination of treatment, by measuring the mid axis width (W) and length (L) of the tumour and using the equation V = (L × W²) × 0.52.

RT–PCR

Total RNA was extracted from frozen glioma xenograft tissue (0.5 g) or C6 cells (1 × 10⁶; cultured with 0, 2.5, 5 or 10 µmol/l lactacystin for 24 h) using TRIzol® (Invitrogen, Carlsbad, CA, USA), according to the manufacturer’s instructions. Total RNA (2 µg) was reverse transcribed using random hexamer primers and ThermoScript™ reverse transcriptase (Invitrogen) and treated with RNase H (Invitrogen). The resulting cDNA was used for polymerase chain reaction (PCR) for glyceraldehyde 3-phosphate dehydrogenase (GAPDH), Bcl-2 and Bcl2-associated X protein (Bax). The PCR reaction mix contained 2 µl cDNA, 0.2 µM primers, 0.2 mM dNTPs, 1.5 mM magnesium chloride, 1 × Taq reaction buffer and 5 U Taq DNA polymerase (Invitrogen). Primer sequences and primer-specific cycling conditions are given in Table 1. To ensure the exponential phase of amplification, the number of PCR cycles was optimized for each cDNA. The cycling programme involved preliminary denaturation at 94°C for 3 min, followed by the appropriate number of cycles of denaturation at 94°C for 40 s, annealing at the primer specific temperature for 30 s, and elongation at 72°C for 1 min, followed by a final elongation step at 72°C for 5 min. PCR products were separated in 2% agarose gels, visualized using ethidium bromide staining and UV light, and semiquantitated by reference to GAPDH band intensity.

Table 1.

Primer sequences and primer-specific conditions used in polymerase chain reaction for rat glyceraldehyde 3-phosphate dehydrogenase (GAPDH), B cell lymphoma 2 (Bcl-2) and Bcl2-associated X protein (Bax).

cDNA	Primer sequences	Annealing temperature °C	No. of cycles	Product size bp
GAPDH		57	27	617
Sense	5′-GGGTGATGCTGGTGCTGAGTATGT-3′
Antisense	5′-AAGAATGGGTGTTGCTGTTGAAGTC-3′
Bcl-2		64	30	723
Sense	5′-GAAGATCTAGGATGGCGCACGCTTG-3′
Antisense	5′-CGGAATTCTCACTTGTGGCCCAGATAGG-3′
Bax		58	28	468
Sense	5′-AGGGTTTCATCCAGGATCGAGC-3′
Antisense	5′-AGGCGGTGAGGACTCCAGCC-3′

Western Blotting

Total protein was extracted from frozen glioma xenograft tissue or C6 cells (cultured with 0, 2.5, 5 or 10 µmol/l lactacystin for 24 h) by homogenization in ice-cold Tris buffered saline (TBS; 15 mmol/l, pH 7.6) containing 250 mmol/l sucrose, 1 mmol/l magnesium chloride, 2.5 mmol/l ethylenediaminetetra-acetic acid, 1 mmol/l glycoletherdiaminetetra-acetic acid, 1 mmol/l dithiothreitol, 1.25 mg/ml pepstatin A, 10 mg/ml leupeptin, 2.5 mg/ml aprotinin, 1.0 mmol/l phenylmethylsulphonyl fluoride, 0.1 mmol/l sodium orthovanadate, 50 mmol/l sodium fluoride, and 2.0 mmol/l tetrasodium pyrophosphate. Homogenates were centrifuged at 10,000 g for 10 min at 4°C and the supernatant retained. Protein was quantified by Bio-Rad protein assay (Bio-Rad, Hercules, CA, USA), and equal quantities were electrophoresed on 10% sodium dodecyl sulphate–polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Membranes were blocked with 3% bovine serum albumin in TBS for 30 min at room temperature, then incubated overnight at 4°C with rabbit antirat Bax (1 : 500 dilution; Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit antirat Bcl-2 (1 : 1000 dilution; Santa Cruz Biotechnology) or rabbit antirat β-actin (1 : 1000 dilution, Santa Cruz Biotechnology). Membranes were then washed three times with PBS (pH 7.4) at room temperature, incubated with horseradish peroxidase-conjugated antirabbit Imuunoglobulin G (1 : 1500 dilution, Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature, and washed three times with PBS at room temperature. Immunoreactive proteins were visualized by enhanced chemiluminescence (Amersham Biosciences, Piscataway NJ, USA) and exposure to Kodak® X-omat LS film (Eastman Kodak Company, New Haven, CT, USA). Densitometry was performed with Kodak® 1D image analysis software (Eastman Kodak Company).

Tumour Pathology

Immediately after sacrifice, glioma tissue was excised and fixed in 4% paraformaldehyde in PBS for 24 h at 4°C. Tumours were then embedded in paraffin wax, cut into 4 -µm sections and deparaffinized. Similar sections were used for haematoxylin and eosin staining and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) staining. Sections were examined by light microscopy (×40 magnification).

TUNEL staining

Deparaffinized tissue sections were stained using an in situ cell death detection kit, POD (Roche Applied Science, Mannheim, Germany), according to the manufacturer’s instructions.

Statistical Analyses

Data were presented as mean ± SD of at least four independent experiments and compared using Student's t-test. Statistical analyses were performed using SPSS® software, version 17.0 (SPSS Inc., Chicago, IL, USA) for Windows®. A P-value < 0.05 was considered statistically significant.

Results

Preliminary studies identified the 50% growth-inhibitory concentration of lactacystin in C6 cells to be ∼10 µmol/l (data not shown). The current study therefore investigated the effects of 2.5, 5 or 10 µmol/l lactacystin on C6 cells. Lactacystin (2.5, 5 and 10 µmol/l) significantly inhibited the proliferation of C6 cells in vitro compared with no treatment (P < 0.01 for each comparison; Table 2). Lactacystin treatment (all concentrations) also significantly increased the number of apoptotic cells and decreased mitochondrial membrane potential compared with controls (P < 0.05 for all comparisons; Table 2).

Table 2.

Effect of incubation with lactacystin for 24 h on viability, apoptosis and mitochondrial membrane potential in rat glioma C6 cells in vitro.

Parameter	Control	Lactacystin, µmol/l
Parameter	Control	2.5	5.0	10.0
Viability	1.0 ± 0.062	0.754 ± 0.053**	0.703 ± 0.032**	0.521 ± 0.039**
Apoptosis	1.0 ± 0.023	1.089 ± 0.032*	1.352 ± 0.078**	1.47 ± 0.044**
Mitochondrial membrane potential	1.0 ± 0.046	0.925 ± 0.051*	0.843 ± 0.073**	0.728 ± 0.066**

Data presented as mean ± SD of at least four independent experiments, expressed relative to control values.

P <0.05, **P <0.01 vs control; Student’s t-test.

Lactacystin treatment (1.0 µg or 5.0 µg/20 g body weight for 7 days) resulted in significantly smaller tumours than control in the in vivo glioma xenograft model, beginning from day 1 after completion of treatment and continuing until sacrifice at day 17 (P < 0.01 for each comparison; Table 3). There were no significant differences in tumour volume between the two treatment groups at any time point. Tumour volume increased relative to baseline in all experimental groups throughout the 17-day period.

Table 3.

Effect of lactacystin on tumour volume in a mouse xenograft glioma model generated using the rat glioma cell line, C6. Animals were treated with 0 (control), 1 or 5 µg/20 g body weight lactacystin for 7 days (n = 5 per group).

Time point	Tumour volume, cm³
Time point	Control	Lactacystin 1.0 µg/20 g	Lactacystin 5.0 µg/20 g
Before treatment	0.051 ± 0.016	0.049 ± 0.018	0.052 ± 0.015
Days after completion of treatment
1	0.108 ± 0.036	0.038 ± 0.020**	0.040 ± 0.017**
5	0.182 ± 0.052	0.054 ± 0.052**	0.059 ± 0.022**
9	0.365 ± 0.061	0.101 ± 0.012**	0.111 ± 0.032**
13	1.025 ± 0.121	0.495 ± 0.067**	0.487 ± 0.049**
17	3.867 ± 0.318	1.231 ± 0.225**	1.149 ± 0.134**

Data presented as mean ± SD.

P < 0.01 vs control group; Student’s t-test.

Haematoxylin and eosin and TUNEL staining of tumour tissue on day 9 after termination of lactacystin treatment revealed round nuclei with discernible chromatin and an absence of brown TUNEL staining in tumours from control mice (Figure 1A). In contrast, tumours from animals treated with lactacystin showed polygonal condensed nuclei (Figures 1B, C) with brown TUNEL staining indicating apoptosis.

Figure 1.

Representative light photomicrographs of haematoxylin and eosin (HE; upper panels) and terminal deoxynucleotidyl transferase dUTP nick end labelling staining (TUNEL; lower panels) of tumour tissue from a mouse xenograft glioma model generated using the rat glioma cell line, C6. Animals were treated with 0 (control), 1.0 or 5.0 µg/20 g body weight lactacystin for 7 days and sacrificed at 9 days after completion of treatment. (A) Nuclei of tumours from control animals were round, with discernible chromatin and an absence of brown TUNEL staining. (B, C) Tumours from animals treated with lactacystin showed polygonal condensed nuclei with brown TUNEL staining indicating apoptosis.

Treatment with lactacystin resulted in significant increases at the mRNA and protein level in the ratio of Bax to Bcl-2 compared with controls on day 9 after termination of treatment, at all concentrations studied, both in vitro and in vivo (P < 0.05 for all comparisons; Table 4).

Table 4.

Effect of lactacystin on the relative levels of B cell lymphoma 2 (Bcl-2) and Bcl2-associated X protein (Bax) mRNA (assessed via reverse transcription–polymerase chain reaction) and protein (assessed via Western blotting) in rat glioma C6 cells in vitro (24-h treatment) and an in vivo mouse xenograft glioma model (7 days treatment; assessed 9 days after treatment completion).

Lactacystin treatment	Bax/Bcl-2 ratio
Lactacystin treatment	mRNA	Protein
In vitro, µmol/l
Control	0.93 ± 0.032	0.83 ± 0.095
2.5	0.96 ± 0.027*	0.74 ± 0.061
5.0	1.03 ± 0.051**	1.12 ± 0.054*
10.0	1.08 ± 0.046**	1.18 ± 0.056**
In vivo, µg/20 g
Control	0.62 ± 0.036	0.58 ± 0.069
1.0	0.85 ± 0.064**	0.76 ± 0.12**
5.0	0.93 ± 0.059**	1.28 ± 0.21**

Data presented as mean ± SD of at least four independent experiments.

P < 0.05, **P < 0.01 vs control; Student’s t-test.

Discussion

The present study used rat C6 glioma cells to examine the therapeutic effects of lactacystin in vitro and in vivo. C6 cells are derived from malignant cerebral neuroblastoma and have similar biological features to human glioma cells, including their rapid growth rate, aggressive nature and high angiogenic activity,¹³ making them useful in screening for potential glioma therapies.

Although several in vitro reports have demonstrated the inhibition of tumour cell proliferation by lactacystin,^21–23 there are few in vivo studies concerning the therapeutic effects on malignant tumours. Intracranially delivered lactacystin suppressed tumour growth and prolonged survival in a rat model of cerebral glioma, but the underlying mechanism of action remained unclear.⁶ Other in vitro studies have shown that lactacystin exerted its antineoplastic effects via triggering apoptosis,²² which is in accordance with the present findings that lactacystin induced apoptosis both in vitro and in vivo.

A study using transmission electron microscopy reported that inhibition of proteasome activity was associated with dilated rough endoplasmic reticulum (ER), dense mitochondrial deposits and cytoplasmic vacuolization.²⁴ These nonapoptotic changes were present even when apoptosis was blocked by caspase inhibitors, suggesting that proteasome inhibition may cause cell death by a nonapoptotic mechanism. This is consistent with our earlier finding that proteasome inhibition activated autophagy in human SHG-44 glioma cells.¹³ Autophagy is an intracellular protein disposal system that collaborates with the proteasome pathway responsible for protein degradation. Although overactivation of autophagy may result in cell death by self digestion, the autophagy induced by proteasome inhibition renders glioma cells more able to survive environmental change.¹³

Following activation by apoptotic signals such as anticancer agents, apoptotic endonuclease mediates regulated DNA fragmentation and chromatin condensation.²⁵ This DNA fragmentation forms the basis of TUNEL staining for detecting apoptotic nucei. Lactacystin treatment of tumour-bearing mice resulted in visibly higher numbers of apoptotic cells compared with untreated animals, in the present study. In addition, lactacystin has been shown to have no effect on promoting tumour cell differentiation.²⁶

The significant in vitro increase in apoptotic cells and decrease in mitochondrial membrane potential induced by lactacystin in the present study suggest that the effect of lactacystin on apoptosis may be related to mitochondria. Mitochondria are the major source of energy in all eukaryotic cells, producing ATP through oxidative phosphorylation and the citric acid cycle.²⁷ Mitochondrial dysfunction has been shown to participate in the induction of apoptosis and may be central to the apoptotic pathway.²⁸ The loss of mitochondrial membrane potential would lead to the release of cytochrome C and the formation of a multimeric apoptosome complex that initiates caspase activation cascades.²⁹ Mitochondrial membrane potential disruption and cytochrome C release are prominent features of apoptosis triggered by proteasome inhibition in human glioma cells.³⁰ The proteasome inhibitor MG-132 was found to induce apoptosis in human glioma SHG-44 cells via enhanced oxidative stress.³¹ As mitochondria have a crucial role in the generation of oxidative stress, it is possible that proteasome inhibition may cause mitochondrial dysfunction.

The mitochondrial proteins Bcl-2 and Bax are essential for activating apoptosis. Following a death signal, the proapototic protein Bax translocates to the outer mitochondrial membrane, promoting permeabilization and the release of various apoptogenic factors.³² The antiapoptotic protein Bcl-2 is is located on the outer mitochondrial membrane and prevents apoptosis by inhibiting the activation of Bax.³³ The ratio of Bax to Bcl-2 is therefore used to represent the likelihood of apoptosis. The expressional and transcriptional Bax to Bcl-2 ratios were significantly elevated by lactacystin treatment in the present study, both in vitro and in vivo. These findings suggest that lactacystin-induced apoptosis in glioma is associated with quantitative alterations in mitochondrial proteins. It has been shown that Bax is normally degraded via the proteasome pathway,⁴ and the increase in Bax seen in the present study may therefore be partly due to decreased degradation. Taken together, these data suggest that lactacystin induces apoptosis via the mitochondrial pathway.

Others have shown that proteasome inhibitors induce mitochondria-independent apoptosis in human glioma cells,³⁴ possibly via an endoplasmic reticulum pathway.³⁵ In addition, activation of the c-Jun N-terminal kinase/c-Jun pathway is closely associated with growth inhibition in human glioblastoma cells.⁵ Proteasome inhibitors have been shown to induce damage to nuclear DNA in cancer cells,³⁶ in addition to nucleolar aggregation of proteasome target proteins and polyadenylated RNA.³⁷ It is likely that inhibition of glioma cell proliferation may be mediated via activation of multiple pathways.

In conclusion, the present study demonstrated that the therapeutic effects of lactacystin are associated with the induction of apoptosis. Such effects may be mediated via a mitochondrial pathway, involving Bcl-2 and Bax. Proteasome inhibition may represent an effective treatment option for glioma.

Footnotes

Declaration of Conflicting Interest

The authors declare that there are no conflicts of interest.

Funding

This work was supported by the National Nature Science Foundation of China (81072071, 30973110) and the Nature Science Foundation of Jilin Province (201115068, 20121809).

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