Simvastatin is effective in killing the radioresistant breast carcinoma cells

MDA-MB-231 (triple-negative type: estrogen, progesterone and HER2 receptor negative (ER-, PR-, HER2-), T47D (luminal A type: ER+, PR+, HER2-) and Au565 (Her2-positive type: ER-, PR-, HER2/neu+) cells were purchased from the American Type Culture Collection. All cell lines were grown in RPMI1640 medium supplemented with 2 mM L-glutamine, 50 U/mL penicillin, 50 μg/mL streptomycin (Thermo Fisher Scientific, Vienna, Austria), and 10% fetal bovine serum (FBS) (HyClone^Tm) (Thermo Fisher Scientific, Vienna, Austria). T47D cells were maintained in medium containing 10 μg/ml bovine insulin (Sigma Aldrich, Millipore Merck, Vienna, Austria). Cell cultures were incubated in a 5% CO₂ humidified atmosphere at 37°C.

Radiation-resistant cells, MDA-MB-231-RR, T47D-RR and Au565-RR (RR cells), were obtained from parental breast cancer cells after repetitive exposure to ionizing radiation (10 Gy) (16 MV x-rays) using an Elekta Precise Linear Accelerator (Elekta Oncology Systems, UK) at a dose rate of approximately 1.8 Gy/min. The cells were irradiated every 2 weeks when breast cancer cells recovered from their exposure to ionizing radiation. Cells which survived after irradiation (total dose of 100 Gy) were collected for further experiments. The newly received cell lines maintained resistance to ionizing radiation independently from a number of passages.

Simvastatin was purchased from Calbiochem (Merck Millipore, Vienna, Austria). Simvastatin was dissolved in dimethyl sulfoxide (DMSO, Sigma Aldrich, Merck Millipore, Vienna, Austria) and used at a final clinically relevant concentration of 8 μM.

Cells were irradiated at single doses of 2, 4, 6, and 8 Gy to determine radiation response of parental and RR breast carcinoma cells, and at a single clinically relevant dose of 2 Gy for all other experiments using an Elekta Precise Linear Accelerator (Elekta Oncology Systems, UK).

Parental and radioresistant MDA-MB-231, T47D, and Au565 cells were seeded into the glass-bottom dishes with a diameter of 35 mm (Ibidi, Switzerland), and cells were incubated for 24 hours at 37°C and 5% CO₂ humidified atmosphere. Next, cells were analysed for their morphology using 3D tomographic microscope with a 60x objective (3D Cell Exlplorer-FLUO, Nanolive SA, Switzerland), and 3D tomographic images (z-stacks) were collected.

Investigated breast carcinoma cells (parental and radioresistant MDA-MB-231, T47D and Au565) were seeded and cultured overnight in 6-well plates at 1,0 x 10^5 cells/well. Cells were exposed to ionizing radiation at single doses of 0, 2, 4, 6, and 8 Gy. After being cultured for 72 hours, irradiated cells were collected for apoptosis assay. Briefly, the cells were trypsinized and then pelleted by centrifugation at 300 g for 10 minutes at 4°C. The supernatant was discarded, and the pellet was washed once with cold PBS and further resuspended in Annexin-V binding buffer containing Annexin-V-APC and propidium iodide (PI) (AnxA100PI Kit, MabTag, Friesoythe, Germany). Cells were stained in darkness for 15 minutes, and the same volume (100 μL) of Annexin-V binding buffer was added to each sample and the prepared samples were analyzed by flow cytometry (BD FACSCantoTM II). The percentage of Annexin-V and PI positive cells was evaluated using the FlowJo_V10.6.2 software. Three independent experiments in duplicates were performed.

Breast carcinoma cells were seeded in 6-well plates and treated either with DMSO alone as a vehicle control, simvastatin (8 μM) alone, irradiation alone (2 Gy) or combination treatment using cell pretreatment with simvastatin (8 μM) for 24 hours followed by irradiation (2 Gy). Cell death development was studied during 72 hours after simvaststin treatment and different time points of 24 hours, 48 hours and 72 hours were selected for analysis.

To evaluate the induction of cell death, samples were analyzed as previously described.11 Briefly, all cells were harvested at the indicated time points followed by centrifugation for 10 minutes (300 g) at 4°C. The pellets were washed with cold PBS and resuspended in hypotonicfluorochrome solution (50 μg/mL propidium iodide (PI), 0.1% sodium citrate, 0.1% Triton X-100). The samples were stained in darkness for 30 minutes at 4°C followed by flow cytometry (BD FACSCantoTM II) analysis. To determine DNA fragmentation, PI fluorescence of individual nuclei was evaluated with an excitation wavelength of 488 nm and an emission wavelength of 670 nm. Gating was done on single nuclei to exclude doublets and debris from the analysis. Cell cycle analysis was performed using FlowJo_V10.6.2 software, and the Sub-G1 fraction was determined.

Western blot was performed as published previously12, 13 using E-cadherin, caspase-3, caspase-7, caspase-8, caspase-9, PARP-1, cytochrome C, XIAP, AIF, beclin-1, LC3 A/B rabbit monoclonal antibody (Cell Signaling Technology, Inc., Beverly, MA, USA), HMGCR rabbit monoclonal antibody (Abcam, UK), Vimentin, Smac/DIABLO mouse (Cell Signaling Technology, Inc., Beverly, MA, USA). Loading control was evaluated using α-tubulin rabbit monoclonal antibody (Cell Signaling Technology, Inc., Beverly, MA, USA). For evaluation of protein expression, X-ray films (GE Healthcare, Chicago, IL, USA) were scanned and analyzed by the Image StudioTM Lite 5.0 (LI-COR Biotechnology, Lincoln, NB, USA). The Integrated Density Value (IDV) was obtained as a ratio of normalized protein band densities in parental and radioresistant RR cells after background correction.

Scratch assay or wound healing assay was performed to evaluate two-dimensional cancer cell migration. Parental and radioresistant breast carcinoma cells were grown to confluence in 6-well plates. A scratch was made on the monolayer using a sterile 200 μL-pipette tip. The monolayer was rinsed three times with PBS and placed in the appropriate complete medium with either simvastatin dissolved in DMSO (8 μM) or DMSO as a vehicle control. Phase contrast images were made during 20 hours at a magnification of 4x (Lionheart Live Cell Microscope, BioTek, Bad Friedrichshall, Germany), and the gap width was measured using the Gen5 (V. 3.08) software. The percentage of the gap width which remained after 20 hours of cell incubation was plotted.

GraphPad Prism software was used to plot the graphs and for statistical evaluation. All the values are represented as the means ± standard error of the mean. Statistical comparisons were performed by one-way analysis of variance (ANOVA) followed by Bonferroni post-hoc comparisons to analyze the differences between each group. Statistical significance was defined as: * = P ≤ 0,05, ** = P ≤ 0,01, *** = P ≤ 0.001 and **** = P ≤ 0.0001.

To confirm that newly received breast carcinoma cells (RR cells) possess radiation resistance, parental and RR cells were exposed to different doses of ionizing radiation and their susceptibility to apoptosis was determined. As it is seen in Figure 1, all three MDA-MB-231-RR, T47D-RR, and Au565-RR cells were less sensitive to irradiation than their parental counterparts. Although there are no significant differences in apoptosis development in parental and RR cells irradiated at lower single doses (2 and 4 Gy), cell exposure to ionizing radiation at higher doses of 6 and 8 Gy was accompanied by more pronounced cell death in parental cells. Thus, irradiation at a dose of 8 Gy induced 30.93 ± 2.47% AnnexinV-PI-positive cells in MDA-MB-231-RR, 30.46 ± 2.71% in T47D-RR, and 30.75 ± 3.08% in Au565-RR cells versus 46.15 ± 7.67%, 61.40 ± 1.60%, and 38.69 ± 1.22% in parental MDA-MB-231, T47D, and Au565 breast carcinoma cells, respectively.

Figure 1

Since HMGCR is a target for simvastatin, its expression was evaluated in parental and RR breast carcinoma cells (Figure 2A). It was found that triple-negative MDA-MB-231-RR and hormone receptor positive T47D-RR cells were characterized by up-regulation of HMGCR in comparison with their parental counterparts. It is necessary to note that parental T47D breast carcinoma cells did not express HMGCR whereas T47D-RR cells showed HMGCR overexpression. Surprisingly, Her2-positive Au565-RR demonstrated slight downregulation of HMGCR compared to the parental Au565 cells.

Figure 2

Administration of simvastatin alone at a clinically relevant doses of 8 μM resulted in significant downregulation of HMGCR in all investigated breast carcinoma cells (Figure 2B). Cell exposure to the clinically relevant single dose of irradiation of 2 Gy led to the substantial increase of HMGCR expression especially at 24 hours after the treatment. Simvastatin-pretreated parental and RR breast carcinoma cells were protected from radiation-induced HMGCR upregulation, which did not significantly differ from those in cells treated with simvastatin alone.

Next, we have investigated whether RR breast carcinoma cells are altered in their migratory capacities (Figure 3A, 3B). Triple-negative MDA-MB-231-RR and hormone receptor-positive T47D-RR breast carcinoma cells showed increased migratory properties compared to their parental counterparts. Using scratch assay, it was observed that MDA-MB-231-RR cells were able to close the gap within 20 hours, whereas parental MDA-MB-231 cells demonstrated 51.62 ± 2.55% of the original gap width left at this time point. Very similar data were received when migratory capacities of T47D-RR breast carcinoma cells were compared with those in parental T47D cells. Thus, parental T47D cells had 83.08 ± 1.71% of the gap open 20 hours after scratching, whereas radioresistant T47D-RR cells demonstrated only 22.97 ± 2.05% of the original gap width open. In contrast, parental Her2-neu-positive Au565 cells were more migratory than their radioresistant Au565-RR counterparts with the gap closure of more than ~95% and ~ 60%, respectively.

Figure 3

Simvastatin significantly reduced migratory abilities of parental and radioresistant MDA-MB-231-RR, radioresistant T47D-RR, and parental Au565 breast carcinoma cells. The most prominent inhibition of cell migration was observed in radioresistant MDA-MB-231-RR and T47D-RR cells (~ 5-fold simvastatin-caused reduction of migration in MDA-MB-231-RR cells versus ~ 1.6-fold in parental MDA-MB-231 cells, and ~ 3-fold simvastatin-induced reduction of migration in T47D-RR cells versus no effect observed in simvastatin-treated parental T47D cells). Surprisingly, radioresistant Au565-RR cells were not affected in their migratory capacities by simvastatin, and the gap was equally closed by the untreated and treated Au565-RR cells.

Considering that radioresistant breast carcinoma cells were affected in their sensitivity to ionizing radiation and altered in their migratory capacities, we next analyzed the levels of expression of mes-enchymal (vimentin) and epithelial (E-cadherin) markers in the investigated carcinoma cells (Figure 4A). It was found that all radioresistant breast carcinoma cells acquired a more mesenchymal phenotype compared to parental cells. Interesting to note that radioresistant MDA-MB-231-RR cells lost E-cadherin expression accompanied by up-regulation of vimentin. Parental T47D cells had a pure epithelial phenotype with overex-pression of E-cadherin with no sign of vimentin expression, and radioresistant T47D-RR cells showed a switch toward mixed epithelial and mesenchymal phenotype characterized by overexpression of both epithelial and mesenchymal markers. Parental Au565 cells and radioresistant Au565-RR cells were equal in their mesenchymal phenotype with vimentin overexpression and lack of E-cadherin expression. Cell treatment with simvastatin resulted in the time-dependent down-regulation of vimentin and up-regulation of E-cadherin in all investigated breast carcinoma cells. Even the cells lacking E-cadherin revealed a simvastatin-induced up-regulation of the epithelial marker. Epithelial-to-mesenchymal transition of radioresistant breast carcinoma cells was accompanied by morphological changes characterized by the membrane ruffling, filopodia and lamellipodia formation resulting in the increased cellular surfaces (Figure 4B).

Figure 4

As previously mentioned, radioresistant breast carcinoma cells are affected in the susceptibility to apoptosis caused by ionizing radiation. Since apoptosis represents only one type of cell death, we decided to be focused on the evaluation of the total treatment-induced cell killing using Nicoletti staining.

Cell death development was determined in all investigated breast cancer cells after their exposure to simvastatin alone, irradiation alone, and their combination. It was observed that among all investigated breast carcinoma cell lines only parental T47D cells were not sensitive to simvastatin (Figure 5). All other parental and radioresistant breast carcinoma cells demonstrated time-dependent cell death development in response to cell exposure to simvastatin at a clinically relevant dose of 8 μM. Thus, parental MDA-MB-231, radioresistant MDA-MB-231-RR and radioresistant T47D-RR cells showed equal cell death development with 46.35 ± 4.38%, 43.78 ± 3.19%, and 51.35 ± 2.96% at 96 hours, respectively. Parental and radioresistant Au565 breast carcinoma cells were less susceptible to simvastatin with cell death of 41.96 ± 9.05% for parental Au565 cells and 27.50 ± 6.03% for radioresistant Au565-RR cells at 96 hours after simvastatin treatment.

Figure 5

Breast cancer cell exposure to the clinically relevant single dose of irradiation of 2 Gy did not result in the substantial cell death in all investigated breast carcinoma cell lines. Parental and radioresistant Au565 cells were the most radiation sensitive among other cell lines, and radiation-caused cell death was 17.43 ± 2.58% in parental Au565 cells and 14.23 ± 1.38% in Au565-RR cells. Unfortunately, combination of simvastatin and irradiation did not lead to the enhancement of cell death compared to simvastatin alone in all parental and radioresistant breast carcinoma cells.

To understand which types of cell death are induced by simvastatin, we have performed Western blot analysis for the key regulators of caspase-dependent, caspase-independent apoptosis and autophagy (Figure 6). Interesting to note that parental and radioresistant triple-negative MDA-MB-231, hormone receptor-positive T47D, and Her2neu-positive Au565 breast carcinoma cells demonstrated different mechanisms of cell death development. Both intrinsic and extrinsic apoptosis pathways were implicated in simvastatin-triggered cell death in all investigated breast carcinoma cells. First, it was seen that initiator caspase-8 belonging to the extrinsic apoptosis pathway was activated in parental and radioresistant MDA-MB-231 cells independently from the kind of cell treatment, and both cleaved forms (43 kDa and 18 kDa) were observed. In contrast, all other cell lines were deficient in full caspase-8 cleavage. Thus, parental T47D cells demonstrated only one cleaved form (43 kDa) after treatment with simvastatin alone, irradiation alone, or combination treatment, whereas radioresistant T47D-RR cells possessed similar cleavage only after cell treatment with simvastatin alone at 48 and 72 hours, or with the combination treatment (simvastatin 48 hours + irradiation 24 hours). Parental Au565 breast carcinoma cells had full caspase-8 cleavage after cell exposure to simvastatin alone at 72 hours or irradiation alone at 24 hours. Radioresistant Au565-RR cells did not reveal a pronounced caspase-8 cleavage after any kind of treatment.

Figure 6

Activation of the intrinsic apoptosis pathway was characterized by caspase-9 cleavage. In comparison with parental breast carcinoma cells, their radioresistant counterparts revealed a more pronounced caspase-9 cleavage after simvastatin-based treatments. It was manifested either in a more pronounced expression of the cleaved form of 37 kDa in T47D-RR cells or in caspase-9 cleavage accompanied by formation of a 37 and 35 kDa form in MDA-MB-231-RR and Au565-RR cells. Additionally, release of cytochrome C correlated with caspase-9 activation in the investigated parental and radioresistant breast carcinoma cells. Thus, there were very weak differences in cytochrome C expression in parental and radioresistant MDA-MB-231 cells that resulted in the similar caspase-9 activation with slightly more pronounced cleavage in radioresistant cells. Very low constitutive cytochrome C expression in the parental T47D cells was upregulated at 24 hours after simvastatin treatment only. Radioresistant T47D-RR cells were characterized by more stable release of cytochrome C after treatment with simvastatin alone, irradiation alone, and especially after combination treatment. Although cytochrome C expressions were not altered by any kind of treatment in parental and radioresistant Au565 breast carcinoma cells, radioresistant Au565-RR cells demonstrated higher cytochrome C expression compared to their parental counterparts.

Simvastatin-induced activation of initiator caspases 8 and 9 resulted in the modulation of expression of the executioner caspases 3 and 7. Although both parental and radioresistant MDA-MB-231 cells showed high expression of the total caspase-3, they were deficient in caspase-3 cleavage after all kinds of cell treatment with simvastatin alone, irradiation alone or their combination. Parental T47D breast carcinoma cells revealed comparable results as parental and radioresistant MDA-MB-231 cells. In contrast, radioresistant T47D-RR cells, parental Au565 and radioresistant Au565-RR cells demonstrated very pronounced simvastatin-induced caspase-3 cleavage at 48 and 72 hours after treatment with simvastatin. Irradiation alone was unable to induce cleavage caspase-3, however combination of simvastatin and irradiation led to equal caspase-3 cleavage as observed in simvastatin-treated breast carcinoma cells. Another executioner caspase-7 was fully cleaved in parental and radioresistant MDA-MB-231 and Au565 cells after treatment with simvastatin alone or its combination with irradiation. Since neither caspase-3 nor caspase-7 cleavages were observed in the parental T47D cells, PARP-1 cleavage was also not observed in these cells after any kind of treatment. In contrast, activation of caspases executioners was accompanied by PARP-1 cleavage in parental and radioresistant MDA-MB-231 and Au565 cells, and radioresistant T47D-RR cells after application of simvastatin alone, irradiation alone, and their combination. It is necessary to note that PARP-1 was even cleaved in the cells treated with irradiation alone, though activation of caspases initiators and executioners was not detected. This led to the hypothesis that other mechanisms of cell death such as caspase-independent apoptosis and autophagy could be implicated in cell killing after cell exposure to irradiation alone or its combination with simvastatin.

Indeed, AIF and Smac/DIABLO expressions were modified by the treatment with simvastatin, irradiation, and their combination. Upregulation of AIF was more pronounced in radioresistant breast carcinoma cells than in their parental counterparts after administration of all treatments. Smac/DIABLO was weakly expressed in parental and radioresistant T47D cells, but it was increased by simvastatin, irradiation, or their combination in parental and radioresistant MDA-MB-231 and Au565 cells. Constitutive expression of the apoptosis inhibitor XIAP was enhanced in all radioresistant breast carcinoma cells. However, simvastatin alone or its combination with irradiation decreased XIAP expression. Although XIAP was up-regulated in parental and radioresistant MDA-MB-231 cells after their exposure to ionizing radiation, combination of simvastatin and irradiation did not result in the augmentation of XIAP expression.

Autophagy-related proteins beclin-1 and LC3 A/B were also analyzed for their constitutive and treatment-modulated expressions in parental and radioresistant breast carcinoma cells. Beclin-1 did not show any significant dysregulation in response to all treatment approaches. Although LC3 A/B was activated and cleaved after application of simvastatin alone or its combination with irradiation in parental and radioresistant MDA-MB-231 and Au565 cells, parental and radioresistant T47D cells were characterized by LC3 A/B cleavage after cell exposure to all kinds of cell treatment. It is necessary to note that the most prominent up-regulation of LC3 A/B expression and enhancement of its cleavage were detected in radioresistant MDA-MB-231-RR and T47D-RR breast carcinoma cells after simvastatin-containing treatment. Thus, LC3 A/B-II/LC3 A/B-I ratio in parental MDA-MB-231 and radioresistant MDA-MB-231-RR cells was ~ 0,6 and ~ 0,5 after simvastatin treatment at 72 hours, and ~ 0,6 and ~ 0,8 after combination treatment, respectively; for parental T47D and radioresistant T47D-RR cells: ~ 0.3 and ~ 0.8 after simvastatin treatment at 72 hours, and ~ 0,3 and ~ 0,8 after combination treatment, respectively; for parental Au565 and radioresistant Au565-RR cells: ~ 0.2 and ~ 0.4 after simvastatin treatment at 72 hours, and ~ 0,5 and ~ 0,4 after combination treatment, respectively.