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Radiol Oncol 2021; 55(3): 305-316. doi: 10.2478/raon-2021-0020
305
research article
Simvastatin is effective in killing the 
radioresistant breast carcinoma cells
Bertram Aschenbrenner1,2,3, Giulia Negro1,2,3, Dragana Savic1,2,3, Maxim Sorokin3,4,5,6,  
Anton Buzdin3,6,7,8, Ute Ganswindt1, Maja Cemazar3,9, Gregor Sersa9, Sergej Skvortsov1,2,  
Ira Skvortsova1,2,3 
1 Medical University of Innsbruck, Therapeutic Radiology and Oncology, Innsbruck, Austria
2 Tyrolean Cancer Research Institute, Innsbruck, Austria
3 EORTC PathoBiology Group
4 Institute of Personalized Medicine, Sechenov First Moscow State Medical University, Moscow, Russia
5 Omicsway Corp., Walnut, USA
6 Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Moscow, Russia
7 Oncobox ltd., Moscow, Russia
8  World-Class Research Center “Digital Biodesign and Personalized Healthcare”, Sechenov First Moscow State Medical 
University, Moscow, Russia
9 Institute of Oncology Ljubljana, Department of Experimental Oncology, Ljubljana, Slovenia
Radiol Oncol 2021; 55(3): 305-316.
Received 10 March 2021 
Accepted 2 April 2021
Correspondence to: Prof. Ira Skvortsova, Laboratory for Experimental and Translational Research on Radiation Oncology (EXTRO-Lab), 
Department of Therapeutic Radiology and Oncology, Medical University of Innsbruck, Anichstr. 35, A-6020 Innsbruck, Austria.  
E-mail: Ira.Skvortsova@i-med.ac.at
Disclosure: No potential conflicts of interest were disclosed. 
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
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 accom-
panied 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 simvasta-
tin 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.
Key words: breast cancer cells; cancer stem cells; radiotherapy; migration; simvastatin 
Introduction
Breast cancer is the most diagnosed malignant tu-
mor in women. It is estimated over 355,000 cases in 
the European Union will be registered in 2020 that 
corresponds to 13.3% of all diagnosed malignan-
cies.1 Therapeutic management of breast cancer is 
markedly changed during last 20 years. Thus, af-
ter breast-conserving surgery, all patients receive 
either partial or whole-breast radiation therapy.2 
This strategy allows to significantly reduce the risk 
of local breast cancer recurrences and breast can-
Radiol Oncol 2021; 55(3): 305-316.
Aschenbrenner B et al. / Simvastatin kills radioresistant breast carcinoma cells306
cer-related mortality.3,4 However, ipsilateral local 
recurrences still occur in locally and systemically 
treated breast cancer patients. Current clinical clas-
sification requires to determine whether an ipsilat-
eral relapsed tumor is a true recurrence or a second 
primary tumor.5,6 The relapsed tumors and sec-
ond primary tumors are distinct in their localiza-
tion and molecular features. It was also found that 
recurrence-free and overall survival rates for true 
recurrences are significantly shorter than for the 
second primary tumors.7 We, therefore, assumed 
that true recurrences usually occur from surviv-
ing breast carcinoma cells after primary treatment 
using radiotherapy with or without chemothera-
peutics, antihormonal or targeted agents. The 
cells recovered after their exposure to anti-cancer 
therapeutic approaches may possess a treatment 
resistant phenotype characterized by enhancement 
of pro-survival mechanisms protecting them from 
cytotoxic or cytostatic agents. It is logical to suggest 
that carcinoma cells comprising the tumor recur-
rences can demonstrate radiation resistance due 
to their abilities to survive and further proliferate 
within the irradiated field. To have a therapeu-
tic benefit from re-irradiation, the radiation dose 
should be markedly increased to effectively kill ra-
dioresistant breast carcinoma cells. Unfortunately, 
due to the problem of tissue tolerance, the required 
dose can usually not be achieved3, and a lower 
dose of re-irradiation is not able to successfully 
eradicate radioresistant carcinoma cells and can 
even increase the molecular characteristics under-
lying radiation resistance.
Since therapy resistant carcinoma cells can also 
possess an augmentation of their metastatic capaci-
ties, true local breast cancer recurrences are often 
accompanied by distant metastasis.8,9 These meta-
static lesions can also be treated by radiotherapy. 
However, an efficacy of this therapeutic approach 
might be diminished due to radiation resistance of 
breast carcinoma cells spreading to distant organs 
and tissues. Therefore, it is logical to suggest the 
use of systemic treatment to improve radiation re-
sponse and combat metastatic spread of radiore-
sistant breast carcinoma cells.
Systemic treatment of cancer can include a va-
riety of different agents. Our research group has 
focused on the lipid-lowering drug simvastatin, 
which is often used by breast cancer patients inde-
pendently from their cancer diagnose. Simvastatin 
as other statins is a competitive inhibitor of 3-hy-
droxy-3-methylglutharyl-coenzyme A reductase 
(HMGCR). Although anti-cancer activity of statins 
is described in literature10, there is still no agree-
ment whether statins generally and simvastatin 
particularly can be used to improve therapy re-
sponse of radioresistant breast carcinoma cells, 
and which molecular properties of carcinoma cells 
make them susceptible to simvastatin treatment. 
Therefore, the main aim of this study was to 
determine the sensitivities of radioresistant breast 
carcinoma cells to simvastatin and mechanisms un-
derlying the cellular responses to the drug alone or 
in combination with ionizing radiation.
Materials and methods
Cell culture and treatment with ionizing 
radiation
MDA-MB-231 (triple-negative type: estrogen, pro-
gesterone 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 strepto-
mycin (Thermo Fisher Scientific, Vienna, Austria), 
and 10% fetal bovine serum (FBS) (HyCloneTm) 
(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 ex-
posure to ionizing radiation (10 Gy) (16 MV x-rays) 
using an Elekta Precise Linear Accelerator (Elekta 
Oncology Systems, UK) at a dose rate of approxi-
mately 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 ioniz-
ing radiation independently from a number of pas-
sages.
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 paren-
tal and RR breast carcinoma cells, and at a single 
Radiol Oncol 2021; 55(3): 305-316.
Aschenbrenner B et al. / Simvastatin kills radioresistant breast carcinoma cells 307
clinically relevant dose of 2 Gy for all other experi-
ments using an Elekta Precise Linear Accelerator 
(Elekta Oncology Systems, UK).
3D tomographic microscopy
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 col-
lected.
Apoptosis assay 
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 centrif-
ugation at 300 g for 10 minutes at 4°C. The super-
natant 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 ana-
lyzed 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.
Cell death development and Sub-G1 
evaluation
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 pre-
treatment with simvastatin (8 μM) for 24 hours 
followed by irradiation (2 Gy). Cell death develop-
ment 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 solu-
tion (50 μg/mL propidium iodide (PI), 0.1% sodi-
um 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) analy-
sis. To determine DNA fragmentation, PI fluores-
cence of individual nuclei was evaluated with an 
excitation wavelength of 488 nm and an emission 
wavelength of 670 nm. Gating was done on sin-
gle 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 analysis 
Western blot was performed as published previ-
ously12,13 using E-cadherin, caspase-3, caspase-7, 
caspase-8, caspase-9, PARP-1, cytochrome C, 
XIAP, AIF, beclin-1, LC3 A/B rabbit monoclo-
nal 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 us-
ing 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 nor-
malized protein band densities in parental and ra-
dioresistant RR cells after background correction.
Migration assay
Scratch assay or wound healing assay was per-
formed to evaluate two-dimensional cancer cell 
migration. Parental and radioresistant breast car-
cinoma cells were grown to confluence in 6-well 
plates. A scratch was made on the monolayer us-
ing a sterile 200 μL-pipette tip. The monolayer 
was rinsed three times with PBS and placed in the 
appropriate complete medium with either simv-
astatin 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, 
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Aschenbrenner B et al. / Simvastatin kills radioresistant breast carcinoma cells308
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.
Statistical analysis
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) fol-
lowed by Bonferroni post-hoc comparisons to ana-
lyze the differences between each group. Statistical 
significance was defined as: * = P  ≤  0,05, ** = P ≤ 
0,01, *** = P ≤ 0.001 and **** = P ≤ 0.0001.
Results
Radiation resistance of breast carcinoma 
cells
To confirm that newly received breast carcinoma 
cells (RR cells) possess radiation resistance, paren-
tal and RR cells were exposed to different doses of 
ionizing radiation and their susceptibility to apop-
tosis 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 pa-
rental counterparts. Although there are no signifi-
cant differences in apoptosis development in pa-
rental 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.
HMGCR expression in breast carcinoma 
cells
Since HMGCR is a target for simvastatin, its ex-
pression was evaluated in parental and RR breast 
carcinoma cells (Figure 2A). It was found that tri-
ple-negative MDA-MB-231-RR and hormone re-
ceptor 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 downregu-
lation of HMGCR compared to the parental Au565 
cells. 
Administration of simvastatin alone at a clini-
cally 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 treat-
ment. Simvastatin-pretreated parental and RR 
breast carcinoma cells were protected from radia-
tion-induced HMGCR upregulation, which did not 
significantly differ from those in cells treated with 
simvastatin alone.
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.
Radiol Oncol 2021; 55(3): 305-316.
Aschenbrenner B et al. / Simvastatin kills radioresistant breast carcinoma cells 309
Simvastatin-caused modulation of 
migratory abilities of breast carcinoma 
cells
Next, we have investigated whether RR breast 
carcinoma cells are altered in their migratory ca-
pacities (Figure 3A, 3B). Triple-negative MDA-MB-
231-RR and hormone receptor-positive T47D-RR 
breast carcinoma cells showed increased migra-
tory properties compared to their parental coun-
terparts. 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 
A
A
B
B
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 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.
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Aschenbrenner B et al. / Simvastatin kills radioresistant breast carcinoma cells310
after scratching, whereas radioresistant T47D-RR 
cells demonstrated only 22.97 ± 2.05% of the origi-
nal 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. 
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 radi-
oresistant 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 pa-
rental 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.
Simvastatin-regulated expression of 
epithelial and mesenchymal markers in 
breast carcinoma 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 mesen-
chymal phenotype compared to parental cells. 
Interesting to note that radioresistant MDA-MB-
231-RR cells lost E-cadherin expression accompa-
nied by up-regulation of vimentin. Parental T47D 
cells had a pure epithelial phenotype with overex-
pression of E-cadherin with no sign of vimentin ex-
pression, and radioresistant T47D-RR cells showed 
a switch toward mixed epithelial and mesenchy-
mal 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 vi-
mentin overexpression and lack of E-cadherin 
expression. Cell treatment with simvastatin re-
sulted 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-
A
B
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.
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Aschenbrenner B et al. / Simvastatin kills radioresistant breast carcinoma cells 311
to-mesenchymal transition of radioresistant breast 
carcinoma cells was accompanied by morphologi-
cal changes characterized by the membrane ruf-
fling, filopodia and lamellipodia formation result-
ing in the increased cellular surfaces (Figure 4B).
Cell death development in breast 
carcinoma cells treated with simvastatin 
and ionizing radiation
As previously mentioned, radioresistant breast 
carcinoma cells are affected in the susceptibility to 
apoptosis caused by ionizing radiation. Since ap-
optosis represents only one type of cell death, we 
decided to be focused on the evaluation of the total 
treatment-induced cell killing using Nicoletti stain-
ing. 
Cell death development was determined in all in-
vestigated breast cancer cells after their exposure to 
simvastatin alone, irradiation alone, and their com-
bination. It was observed that among all investigat-
ed 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 de-
velopment in response to cell exposure to simvas-
tatin at a clinically relevant dose of 8 μM. Thus, pa-
rental 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.
Breast cancer cell exposure to the clinically rel-
evant single dose of irradiation of 2 Gy did not re-
sult in the substantial cell death in all investigated 
breast carcinoma cell lines. Parental and radiore-
sistant Au565 cells were the most radiation sensi-
tive 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.
Simvastatin activates different types of 
cell death
To understand which types of cell death are in-
duced 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 pa-
rental 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 
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.
Radiol Oncol 2021; 55(3): 305-316.
Aschenbrenner B et al. / Simvastatin kills radioresistant breast carcinoma cells312
T47D cells demonstrated only one cleaved form (43 
kDa) after treatment with simvastatin alone, irra-
diation alone, or combination treatment, whereas 
radioresistant T47D-RR cells possessed similar 
cleavage only after cell treatment with simvasta-
tin alone at 48 and 72 hours, or with the combina-
tion 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. 
Activation of the intrinsic apoptosis pathway 
was characterized by caspase-9 cleavage. In com-
parison 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 
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 de-
ficient in full caspase-8 cleavage. Thus, parental 
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.
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Aschenbrenner B et al. / Simvastatin kills radioresistant breast carcinoma cells 313
of 37 kDa in T47D-RR cells or in caspase-9 cleav-
age 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 paren-
tal 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 cleav-
age 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, irradia-
tion alone, and especially after combination treat-
ment. 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 paren-
tal counterparts.
Simvastatin-induced activation of initiator cas-
pases 8 and 9 resulted in the modulation of expres-
sion 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, irra-
diation 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 demon-
strated very pronounced simvastatin-induced cas-
pase-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 irradia-
tion. Since neither caspase-3 nor caspase-7 cleav-
ages were observed in the parental T47D cells, 
PARP-1 cleavage was also not observed in these 
cells after any kind of treatment. In contrast, acti-
vation of caspases executioners was accompanied 
by PARP-1 cleavage in parental and radioresistant 
MDA-MB-231 and Au565 cells, and radioresist-
ant 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 irra-
diation 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 counter-
parts 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 pa-
rental and radioresistant MDA-MB-231 and Au565 
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.
Radiol Oncol 2021; 55(3): 305-316.
Aschenbrenner B et al. / Simvastatin kills radioresistant breast carcinoma cells314
cells. Constitutive expression of the apoptosis in-
hibitor 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 af-
ter their exposure to ionizing radiation, combina-
tion 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 simvasta-
tin alone or its combination with irradiation in pa-
rental and radioresistant MDA-MB-231 and Au565 
cells, parental and radioresistant T47D cells were 
characterized by LC3 A/B cleavage after cell expo-
sure 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 simvas-
tatin-containing treatment. Thus, LC3 A/B-II/LC3 
A/B-I ratio in parental MDA-MB-231 and radiore-
sistant 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, respective-
ly; 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.
Discussion
Radiotherapy is an important therapeutic approach 
used in the management of breast cancer. Although 
radiotherapy techniques are markedly improved, 
the problem of radiation resistance of the primary 
or secondary (relapsed) tumors still exists.4,14 It is 
currently known that radiation resistance can be 
supported by activated pro-survival intracellular 
pathways and microenvironmental factors.15,16 A 
Western diet containing a lot of fat links not only 
increased levels of cholesterol in blood but can also 
be associated with the formation of more aggres-
sive malignant breast tumors in women.15,17 Tumor 
aggressiveness is characterized by reduced cancer 
cell sensitivity to currently existing therapeutic ap-
proaches and inclination for metastatic spread.18-20 
Since high cholesterol levels could be related to the 
diminished therapy response and metastatic pro-
gression of breast cancer21-23, it is assumed that ad-
ministration of statins can help to improve tumor 
sensitivity to anti-cancer treatment and attenuate 
the metastatic cancer cell capabilities.
Simvastatin is one of the widely used statins 
inhibiting the HMGCR and reducing hypercho-
lesterolemia in patients. Since cholesterol levels 
can be regulated by the HMGCR, and cholester-
olemia can be linked to more aggressive behavior 
of breast tumors, it is hypothesized that HMGCR 
can be upregulated in breast cancers with unfa-
vorable clinical outcomes. Indeed, analysis of cor-
relation between HMGCR expression and overall 
survival in breast cancer patients24 has shown that 
HMGCR overexpression can be associated with 
reduced overall survival in breast cancer patients 
independently from the molecular subtypes of 
the tumors (Figure 7). Accordingly, our data have 
clearly demonstrated that breast carcinoma cells 
with confirmed radiation resistance possessed an 
augmentation of HMGCR expression. It was previ-
ously established that HMGCR is implicated in ra-
diation response of melanoma cells, and lipophilic 
HMGCR inhibitor pitavastatin (Livalo) causes a 
delay in DNA repair resulting in the persistence 
of double strand breaks and development of se-
nescence in malignant cells.23 Although radiosen-
sitizing and additional anti-tumor effects of the 
lipophilic statins are previously described22,23,25-29, 
there is only one report demonstrating that simv-
astatin can sensitize esophageal carcinoma cells to 
ionizing radiation via inhibition of PI3/Akt path-
way.30 To our knowledge, there are no publications 
showing a simvastatin efficacy on cell death and 
survival of radioresistant breast carcinoma cells. 
In this study, we have found that simvastatin-
caused cytotoxic effects were observed in breast 
carcinoma cells expressing HMGCR, and the levels 
of their radiation responses did not play the sig-
nificant roles. While irradiation alone resulted in 
the HMGCR up-regulation in all parental and ra-
dioresistant breast carcinoma cells independently 
from the constitutive HMGCR levels, combination 
treatment with simvastatin and ionizing radiation 
was not accompanied by enhancement of HMGCR 
in the majority of the investigated carcinoma cells. 
Only parental hormone receptor-dependent T47D 
cells lacking constitutive HMGCR expression did 
not show simvastatin-dependent downregulation 
of radiation-caused HMGCR expression in the ir-
Radiol Oncol 2021; 55(3): 305-316.
Aschenbrenner B et al. / Simvastatin kills radioresistant breast carcinoma cells 315
radiated cells. We assume that simvastatin was not 
able to prevent radiation caused HMGCR expres-
sion due to the constitutive lack of the target for 
simvastatin.
Interesting to note, there were no significant 
differences in cytotoxic cell responses to simvasta-
tin alone and its combination with a clinically rel-
evant dose of irradiation of 2 Gy. Simvastatin alone 
was equally effective as a combination treatment 
in both parental and radioresistant breast cancer 
cells if they expressed HMGCR. However, radia-
tion-caused HMGCR up-regulation in the parental 
T47D cells did not influence the cytotoxic activities 
of simvastatin in combination with irradiation. We 
also cannot exclude that combination of simvasta-
tin with a higher dose of irradiation can more effec-
tively kill breast cancer cells than drug or ionizing 
radiation alone. 
As expected, radioresistant breast carcinoma 
cells revealed an enhancement of metastasis-as-
sociated properties, such as increased migratory 
abilities and acquisition of more mesenchymal 
phenotype. Simvastatin treatment caused pheno-
typic transition of tumor cells between mesenchy-
mal and epithelial states, which was accompanied 
by the reduction in migratory capabilities of breast 
carcinoma cells. Hence, it is possible to speculate 
that metastatic potential of radioresistant cells can 
be affected by the use of simvastatin. 
Although there are several publications report-
ing on the inhibitory activities of statins on the 
epithelial-to-mesenchymal transition (EMT)30-34, 
our data additionally provide new evidence that 
simvastatin can effectively kill radioresistant breast 
carcinoma cells possessing a mesenchymal pheno-
type. We, therefore, hypothesize that radioresistant 
cells can be eliminated by simvastatin from local 
and/or distant recurrences. This assumption agrees 
with a clinical observation that statins cannot pre-
vent breast cancer formation but can reduce can-
cer-related mortality in metastatic breast cancer pa-
tients.33 It was also shown that lipophilic statins, in-
cluding simvastatin, can improve a recurrence-free 
survival in breast cancer patients.33,34 We, therefore, 
suppose that statins alter therapy-resistant breast 
carcinoma cells and affect carcinoma cell recovery 
after treatment.
In our study, simvastatin induced cell death in 
HMGCR-expressing breast carcinoma cells, and 
we have detected activation of extrinsic, intrinsic, 
and caspase-independent apoptotic pathways, and 
autophagy. Although HMGCR-positive breast car-
cinoma cells demonstrated comparable simvasta-
tin-induced cell death development, apoptosis was 
differently regulated either with more pronounced 
involvement of caspase-dependent or caspase-in-
dependent pathways. Interesting to note that sim-
vastatin enhanced the expression of autophagy-
related proteins beclin-1 and LC3 in HMGCR-
negative and simvastatin-resistant parental T47D 
cells. However, LC3 activation and cleavage was 
not very pronounced in parental cells as it was 
observed in radioresistant T47D cells. Therefore, 
we suppose that parental cells can be protected 
from cell death via weak activation of autophagy.35 
Since we have observed a variety of mechanisms 
regulating different types of cell death in simvas-
tatin-treated carcinoma cells, it was impossible to 
detect any unique scenario of breast cancer cell kill-
ing after simvastatin treatment. In our opinion, it 
opens the wider perspectives to use simvastatin as 
a therapeutic approach to treat breast carcinomas 
possessing different capabilities for activation of 
apoptosis or autophagy.
We conclude, radioresistant breast carcinoma 
cells possessing HMGCR expression accompanied 
by EMT activation can be successfully killed by 
simvastatin via mobilizing of a variety of pathways 
involved in apoptosis and autophagy.
Acknowledgement
This study was supported by Austrian Science 
Fund (FWF P29457; FWF I4140), Anniversary Fund 
of Austrian National Bank (ÖNB 17620), Ingrid 
Shaker-Nessmann Cancer Research Foundation. 
Anton Buzdin and Maksim Sorokin were financed 
by the Russian Foundation for Basic Research 
Grant 19-29-01108. The research was supported al-
so by the Slovenian Research Agency (ARRS) grant 
number P3-0003. 
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