Simvastatin is effective in killing the radioresistant breast carcinoma cells

Abstract

Background: Statins, small molecular 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors, are widely used to lower cholesterol levels in lipid-metabolism disorders. Recent preclinical and clinical studies have shown that statins exert beneficial effects in the management of breast cancer by increasing recurrence free survival. Unfortunately, the underlying mechanisms remain elusive.

Materials and methods: Simvastatin, one of the most widely prescribed lipophilic statins was utilized to investigate potential radiosensitizing effects and an impact on cell survival and migration in radioresistant breast cancer cell lines.

Results: Compared to parental cell counterparts, radioresistant MDA-MB-231-RR, T47D-RR andAu565-RR cells were characterized by upregulation of 3-hydroxy-3-methylglutharyl-coenzyme A reductase (HMGCR) expression accompanied by epithelial-to-mesenchymal transition (EMT) activation. Radioresistant breast cancer cells can be killed by simvastatin via mobilizing of a variety of pathways involved in apoptosis and autophagy. In the presence of simvastatin migratory abilities and vimentin expression is diminished while E-cadherin expression is increased.

Conclusions: The present study suggests that simvastatin may effectively eradicate radioresistant breast carcinoma cells and diminish their mesenchymal phenotypes.

Keywords: breast cancer cells; cancer stem cells; migration; radiotherapy; simvastatin.

Figure 1

Radiation-induced apoptosis in breast carcinoma cells. Radiation sensitivity of the investigated parental and radioresistant breast carcinoma cells was determined using apoptosis assay as described in the section Materials and methods. Grey bars represent parental cells and black bars the radioresistant cells. All experiments were performed at least three times in duplicates; * = p<0.05; ** = p<0.01; *** = p<0.001.

Figure 2

HMGCR expression in breast carcinoma cells. (A) Constitutive 3-hydroxy-3-methylglutharyl-coenzyme A reductase (HMGCR) expressions in parental and radioresistant breast carcinoma cells. Protein extracts from total cell lysates were subjected to Western blot analysis, and constitutive levels of HMGCR were determined in all investigated breast carcinoma cells; (B) Simvastatin-caused modulation of HMGCR expression in parental and radioresistant breast carcinoma cells was confirmed using Western blot analysis.

Figure 3

Simvastatin-regulated breast carcinoma cell migration. (A) Wound healing assay was used to determine how simvastatin affected breast carcinoma cell migration. Cell migration was assayed at a magnification of 4x (Lionheart Live Cell Microscope, BioTek, Bad Friedrichshall, Germany). The cell migration rates were determined as a ratio between the gap width at indicated time point and initial gap width at 0 hours; (B) Statistical evaluation of the gap width in breast carcinoma cells. Gap width was measured using the Gen5 (V. 3.08) software and the percentage of the gap width remained after 20 hours of cell incubation in presence of DMSO as a vehicle control or simvastatin (8 μM) was plotted. All experiments were performed at least three times in duplicates; * = p<0.05; ** = p<0.01; *** = p<0.001.

Figure 4

Mesenchymal and epithelial markers in breast carcinoma cells treated with simvastatin. (A) Simvastatin-dependent regulation of vimentin and E-cadherin expressions in parental and radioresistant breast carcinoma cells were evaluated using Western blot analysis as described in the section Materials and Methods. IDV was calculated for each protein band and normalized to the α-tubulin band density after background correction. IDV ratio means fold-change of vimentin or E-cadherin band densities in simvastatin-treated compared to the vehicle-treated breast carcinoma cells. (B) 3D holographic breast cancer cell microscopy. Parental and radioresistant MDA-MB-231, T47D, and Au565 cells were analyzed for their morphology using 3D Nanolive Explorer-FLUO as described in the Materials and Methods.

Figure 5

Cell death development in breast carcinoma cells treated with simvastatin or irradiation or their combination. At indicated time points, analysis of sub-G1 cell fraction was evaluated in the samples collected after treatment of parental and radioresistant breast carcinoma cells with simvastatin (8 μM) alone, irradiation (2 Gy) alone or combination of simvastatin (8 μM) and irradiation (2 Gy). All experiments were performed at least three times in duplicates; * = p<0.05; ** = p<0.01; *** = p<0.001.

Figure 6

Regulation of apoptosis- and autophagy-related proteins in breast carcinoma cells. Treatment-induced modulation of protein expression in parental and radioresistant breast carcinoma cells were investigated using Western blot analysis. Cells were either treated with simvastatin (8 μM), or irradiation alone at a dose of 2 Gy, or with combination treatment using simvastatin (8 μM) pretreatment (24 hours) followed by irradiation at a single dose of 2 Gy. Protein extractions were performed at the indicated time points, and then samples were analyzed using Western blotting as described in Materials and Methods.

Figure 7

3-hydroxy-3-methylglutharyl-coenzyme A reductase (HMGCR) expression and recurrence free survival in breast cancer patients. Prognostic value of the HMGCR expression at the mRNA level in breast cancer patients was evaluated using the KMplot database (http://kmplot.com/analysis/) and the results indicate that higher HMGCR expression was associated with worse overall survival in all cohorts of patients.