Proteasome inhibitor MG-132 induces C6 glioma cell apoptosis via oxidative stress

Abstract

Aim: Proteasome inhibitors have been found to suppress glioma cell proliferation and induce apoptosis, but the mechanisms are not fully elucidated. In this study we investigated the mechanisms underlying the apoptosis induced by the proteasome inhibitor MG-132 in glioma cells.

Methods: C6 glioma cells were used. MTT assay was used to analyze cell proliferation. Proteasome activity was assayed using Succinyl-LLVY-AMC, and intracellular ROS level was evaluated with the redox-sensitive dye DCFH-DA. Apoptosis was detected using fluorescence and transmission electron microscopy as well as flow cytometry. The expression of apoptosis-related proteins was investigated using Western blot analysis.

Results: MG-132 inhibited C6 glioma cell proliferation in a time- and dose-dependent manner (the IC(50) value at 24 h was 18.5 μmol/L). MG-132 (18.5 μmol/L) suppressed the proteasome activity by about 70% at 3 h. It induced apoptosis via down-regulation of antiapoptotic proteins Bcl-2 and XIAP, up-regulation of pro-apoptotic protein Bax and caspase-3, and production of cleaved C-terminal 85 kDa PARP). It also caused a more than 5-fold increase of reactive oxygen species. Tiron (1 mmol/L) effectively blocked oxidative stress induced by MG-132 (18.5 μmol/L), attenuated proliferation inhibition and apoptosis in C6 glioma cells, and reversed the expression pattern of apoptosis-related proteins.

Conclusion: MG-132 induced apoptosis of C6 glioma cells via the oxidative stress.

Figure 1

Cellular viability assay and proteasome activity assay. (A) MTT assay of cell viability. The proliferation of C6 glioma cells was inhibited after 3 h incubation with MG-132; at 24 h, the maximal inhibitory effect was reached. (B) Proteasome activity assay. Proteasome activity in C6 glioma cells was significantly inhibited at 3 h by 18.5 μmol/L MG-132. (^cP<0.01 vs control group).

Figure 2

MG-132-induced oxidative stress. (A) ROS was generated in the MG-132-treated (18.5 μmol/L) C6 glioma cells at 3 h, increasing continuously to a peak at 24 h. When tiron (1 mmol/L) was used, ROS generation was blocked (B), and the viability of C6 glioma cells also increased significantly (C). Mean±SD. n=4. ^cP<0.01 vs control group, ^fP<0.01 vs MG-132 group.

Figure 3

C6 glioma cells displayed apoptotic morphological features after incubation with MG-132. Fluorescence microscopy (A: control group and B: MG-132 group) and transmission electron microscopy (C: control group and D: MG-132 group) showed chromatin condensation, nuclear condensation and nuclear fragmentation in the nucleus of C6 glioma cells treated with MG-132.

Figure 4

Tiron suppressed apoptosis and cell cycle arrest induced by MG-132. Flow cytometry analysis of apoptotic rate and cell cycle. In the control group (A), the apoptotic rate was 0.89%, and the percentage of the cells in G₂/M phase was 9.43%. Tiron effectively suppressed the increase of apoptosis rate (from 30.46% to 20.79%) and cell cycle arrest in G₂/M phase (from 17.31% to 12.77%) induced by MG-132 (B and C).

Figure 5

Tiron altered the expression of apoptosis-related proteins. Western blot analysis revealed that MG-132 induced down-regulation of anti-apoptotic proteins Bcl-2 and XIAP, up-regulated expression of pro-apoptotic protein Bax and caspase-3, and produced cleaved C-terminal 85 kDa PARP. Additionally, tiron reversed these alterations. ^cP<0.01 vs control; ^fP<0.01 vs MG-132 group.