Evaluation of the Effect of Proteasome Inhibitor (MG132) in Regulating Cell Death, Apoptosis, Caspase Activity, the Amount of Reactive Oxygen Species and Mitochondrial Membrane Potential in Breast Cancer Cell Line (MCF-7)

Breast cancer imposes a great burden of cancer-related mortality and morbidity in women worldwide. Scientific efforts are in progress to improve the efficiency of current therapeutic strategies and reduce chemoresistance (1). Due to the fact that the homeostasis of cancer cell growth is dependent on the balance between cell proliferation and cell death, the emerging role of pro-apoptotic agents to promote apoptosis and attenuate cancer cell evasion from apoptosis has opened up promising cancer therapeutic approaches (2). On the other hand, the process of protein breakdown, which involves the loss of toxic, incorrectly folded, or accumulated proteins play a critical role in normal cell fate. The protein breakdown is mainly implemented by the proteasome system that breaks the proteins into short peptides and their constituent amino acids and transfer them to the cytoplasm to reuse in the synthesis of new proteins (3). If protein breakdown is disrupted, the accumulation of incorrectly folded proteins leads to errors and induction of apoptosis (4). Due to the great importance of proteosomes for cells, inhibition of their function has been proposed as a way to induce apoptosis in cancer cells (5). MG132 has been considered as a proteosome pathway inhibitor and postulated to regulate cancer cell growth and death, recently (6). It has been proposed that MG132 synergized with bevacizumab and/or cisplatin to inhibit cancer cell proliferation by triggering reactive oxygen species generation (6-8). However, the lack of sufficient evidence regarding the relevance of MG132 on breast cancer cell growth provoked us to unravel the possible effect of MG132 and its underlying mechanism in breast cancer. Thus, this study is designed to elucidate the effect of MG132 on growth regulation and induction of apoptosis by emphasizing the role of caspases, reactive oxygen species, and mitochondria in MCF-7 cancer cells.

In this study, the human breast cancer cell lines, MCF-7 which pathologically originated from invasive carcinoma of the ducts of the breast was obtained from Pasture Institute of Iran and cultured in RPMI 1640 medium supplemented with 10% (v/v) fetal bovine serum, 100 U/ml of penicillin and 100 µg/ml of streptomycin and maintained at 37 ºC, 5% CO2 and 100% humidity in the incubator. Cells were treated with different concentrations of MG132 (0.5, 1, 5, and 10 μmol) at incubation times of 12, 24, and 48 hours. The cytotoxic effect of MG132 on MCF-7 growth was investigated using MTT assay and the results were expressed in terms of the percentage of viable cells relative to the control. Annexin-V-FITC staining and PI staining were used to diagnose early and late apoptosis using flow cytometry. The level of mitochondrial membrane potential (Δψm) was investigated using JC-1 lipophilic dye and its accumulation in mitochondria, which is associated with fluorescence emission and change of emission from green (520nm) to red (590 nm). The formation of reactive oxygen species (ROS) after treatment with different concentrations of MG132 was performed using a fluorescence probe dichlorofluorescein diacetate (DCFH-DA). To evaluate the possible involvement of caspases, the activity of caspase 3 and caspase 8 was examined by the ELISA method. To determine the specificity and accuracy, all experiments were repeated at least three times. The non-parametric one-way analysis of variance (ANOVA) with Dennet’s post hoc test and Tukey´s post hoc test were applied for analysis of differences using Graph Pad Prism version 7 (Graph Pad Software, San Diego California)

Based on data, a significant reduction in the percentage of viable breast cancer cells (MCF-7) was detected following treatment by MG132 that was occurred in a dose and time-dependent manner. Treatment of MCF-7 cells with 5 μmol and 10 μmol of MG132 for 48 hours reduced cell viability by about 40% and 50%, respectively. Annexin-V and PI double staining method was applied to evaluate whether the cytotoxic effect of MG132 was related to the induction of apoptosis. According to the protocol, annexin-V positive, PI negative cells accounts as early apoptotic cells and annexin-V positive, PI-positive cells account for late apoptotic cells. Decreasing the percentage of viable cells after treatment with 5 and 10 μmol of MG132 after 48 hours has increased the percentage of early apoptotic cells. The percentage of early apoptotic cells was 15% after treatment with 5 μmol of MG132 and 30% after treatment with 10 μmol of MG132. Also, due to the considerable role of the caspase cascade as executors of apoptosis, the activity of caspase 3 and 8 was assessed. A significant increase in the caspase-3 activity was observed after treatment with 10 μmol of MG132 in MCF-7 cells.  Also, the level of caspase-8 activity in the mentioned time showed a significant increase in both 5 and 10 μmol of MG132 indicating that the MG132-induced apoptosis in MCF-7 cells occurred in a caspase-dependent manner. Based on the results of this study, a significant increase in the intracellular ROS level of MCF-7 cells was observed while cells were treated with 5 and 10 μmol of MG132 for 48 hours. Increases in intracellular ROS levels indicate MG132-induced apoptosis in MCF-7 cells is associated with the induction of oxidative stress. Also, reduced mitochondria membrane potential (ΔΨm) reflects mitochondrial impairment and accounts as a hallmark of apoptosis (9). Our data showed that treatment of MCF-7 cells with 1, 5, and 10 μmol of MG132 for 48 hours, reduced the mitochondria membrane potential significantly, indicating the fact that MG132 influences mitochondria to induce apoptosis in breast cancer.

The data presented in this study revealed that MG132 inhibits the growth of breast cancer cells and induces apoptosis by activating caspases 3 and 8, increasing intracellular ROS level, and decreasing mitochondrial membrane potential (Δψm). Also, our results showed a significant difference in the effect of MG132 on the mentioned assays in untreated (control) breast cancer cells compared to the treated cells and the observed effects on the treated cells depending on the concentration of MG132. These results emphasize the effective role of inhibiting the proteosome system through MG132 in stopping the proliferation of MCF-7 cells and inducing apoptosis and the potential of this combination to design more effective therapies in controlling the growth of breast cancer cells.