Tumor Biol.

DOI 10.1007/s13277-015-4395-x

ORIGINAL ARTICLE

Mechanism of metformin action in MCF-7 and MDA-MB-231

human breast cancer cells involves oxidative stress generation,
DNA damage, and transforming growth factor β1 induction
Poliana Camila Marinello 1 & Thamara Nishida Xavier da Silva 2 &
Carolina Panis 3 & Amanda Fouto Neves 1 & Kaliana Larissa Machado 1 &
Fernando Henrique Borges 3 & Flávia Alessandra Guarnier 2 &
Sara Santos Bernardes 1 & Júlio Cesar Madureira de-Freitas-Junior 4 &
José Andrés Morgado-Díaz 4 & Rodrigo Cabral Luiz 1 & Rubens Cecchini 3 &
Alessandra Lourenço Cecchini 1

Received: 3 September 2015 / Accepted: 5 November 2015

# International Society of Oncology and BioMarkers (ISOBM) 2015

Abstract The participation of oxidative stress in the mecha- cytoplasmic extracellular signal-regulated kinases 1 and 2
nism of metformin action in breast cancer remains unclear. We (ERK1/2) and AKT and increased nuclear p53 and cytoplas-
investigated the effects of clinical (6 and 30 μM) and experi- mic transforming growth factor β1 (TGF-β1) in both cell
mental concentrations of metformin (1000 and 5000 μM) in lines. These findings suggest that metformin reduces cell sur-
MCF-7 and in MDA-MB-231 cells, verifying cytotoxicity, vival by increasing reactive oxygen species, which induce
oxidative stress, DNA damage, and intracellular pathways re- DNA damage and apoptosis. A relationship between the in-
lated to cell growth and survival after 24 h of drug exposure. crease in TGF-β1 and p53 levels and the decrease in ERK1/2
Clinical concentrations of metformin decreased metabolic ac- and AKT was also observed. These findings suggest the
tivity of MCF-7 cells in the MTT assay, which showed in- mechanism of action of metformin in both breast cancer cell
creased oxidative stress and DNA damage, although cell death lineages, whereas cell line specific undergoes redox changes
and impairment in the proliferative capacity were observed in the cells in which proliferation and survival signaling are
only at higher concentrations. The reduction in metabolic ac- modified. Taken together, these results highlight the potential
tivity and proliferation in MDA-MB-231 cells was present clinical utility of metformin as an adjuvant during the treat-
only at experimental concentrations after 24 h of drug expo- ment of luminal and triple-negative breast cancer.
sition. Oxidative stress and DNA damage were induced in this
cell line at experimental concentrations. The drug decreased Keywords Metformin . MCF-7 . MDA-MB-231 . Oxidative
stress . Breast cancer
Electronic supplementary material The online version of this article
(doi:10.1007/s13277-015-4395-x) contains supplementary material,
which is available to authorized users.
Abbreviations
* Alessandra Lourenço Cecchini ERK1/2 Extracellular signal-regulated kinases 1 and 2
[email protected] AKT Protein kinase B
TGF-β1 Transforming growth factor β1
1
Laboratory of Molecular Pathology, State University of Londrina, AMPK Adenosine-5′-monophosphate-activated protein
Rodovia Celso Garcia Cid, PR445, Km 380 Campus Universitário, kinase
Londrina CEP 86051-990, Paraná, Brazil mTOR Mammalian target of rapamycin
2
Laboratory of Pathophysiology and Muscle Adaptation, State HER-2 Human epidermal growth factor receptor 2
University of Londrina, Londrina, PR, Brazil TNBC Triple-negative breast cancer
3
Laboratory of Pathophysiology and Free Radicals, State University MTT 2-(3,5-Diphenyltetrazol-2-ium-2-yl)-4,5-
of Londrina, Londrina, PR, Brazil dimethyl-1,3-thiazole bromide
4
Brazilian National Cancer Institute, INCA, Rio de Janeiro, Brazil PBS Phosphate-buffered saline
 Tumor Biol.

EB Ethidium bromide associated with poor prognosis due to its aggressiveness, the
AO Acridine orange absence of molecular treatment, and resistance against usual
MDA Malondialdehyde therapeutic methods [14]. Given this context, there is little
8-Oh-dG 8-Hydroxy-2-deoxyguanosine information available concerning the mechanisms of metfor-
ANOVA Analysis of variance min cytotoxicity in TNBC and none regarding the participa-
SOD Superoxide dismutase tion of oxidative stress.
OS Oxidative stress The proliferation and survival of breast cancer cells are
ROS Reactive oxygen species strongly associated with several signaling pathways, such
as the phosphatidylinositide 3-kinase/protein kinase B
(AKT), transforming growth factor β1 (TGF-β1), and
Introduction p53 pathways [15–17]. Most of these are modulated by
oxidative stress. With this in mind, we investigated the
Metformin is an anti-diabetic medication and a putative cytotoxic and anti-proliferative mechanisms of metformin
anti-neoplastic drug. Metformin use, at the time of breast in two distinct human breast cancer cell lines: a human
cancer diagnosis, has been associated with better clinical- breast cancer cell line positive for estrogen and progester-
pathological characteristics and a lower incidence of one receptors and negative for HER-2 receptor and a triple-
triple-negative and histological grade III tumors [1]. Met- negative cell line, MCF-7 and MDA-MB-231, respective-
formin affects cancer through direct and indirect mecha- ly. We evaluated the effects of the drug on oxidative stress,
nisms. The direct mechanisms are related to the capacity DNA damage, and several important signaling pathways,
of metformin to reduce the proliferation of many breast the extracellular signal-regulated kinases (extracellular
cancer subtypes in vitro [2]. One well-described mecha- signal-regulated kinases 1 and 2 (ERK1/2)), AKT,
nism for this anti-proliferative effect involves the activa- TGF-β1, and p53 pathways.
tion of adenosine-5′-monophosphate-activated protein ki-
nase (AMPK) [3], which inhibits the mammalian target of
rapamycin (mTOR) pathway, reducing translation initia- Materials and methods
tion and inhibiting cell growth [4]. The indirect mecha-
nisms for the effects of metformin on cancer include re- Cell culture and treatment
ductions in insulin levels and in the bioavailability of sex
hormones [5]. Numerous other studies have used breast MCF-7 (ATCC® HTB-22™; ATCC, Manassas, VA, USA) and
cancer cells to investigate the mechanisms of action of MDA-MB-231 cells (ATCC® HTB-26™; ATCC, Manassas,
metformin [3, 6–9]. VA, USA) were grown in high-glucose Dulbecco’s modified
Because oxidative stress modulates several intracellular Eagle’s medium (Gibco® Life Technologies, Carlsbad, CA)
pathways related to cellular growth and death [10], it is rea- supplemented with 10 % fetal bovine serum and a 1 %
sonable to hypothesize that the ability of metformin to inter- penicillin/streptomycin mixture. Cells were maintained in a
fere with breast cancer cell growth is related to its effect on the humidified atmosphere of 5 % CO2 at 37 °C (Sanyo
cellular redox status. Furthermore, our group has shown that CO2 Incubator; Sanyo, Kitanagoya, Aichi). The cytotox-
different oxidative stress statuses occur at distinct stages in icity, proliferation, and genotoxicity assays were per-
breast cancer patients [11]. Recently, it was demonstrated that formed by seeding 105 cells in 24-well plates and cultur-
metformin inhibited the proliferation of MCF-7 cells due to ing them in fresh culture media for 24 h. Cells were then
the promotion of cell cycle arrest and the induction of cell exposed to metformin (Santa Cruz Biotechnology, Dallas,
apoptosis and necrosis and was associated with increased cel- TX) at two clinically relevant concentrations (6 and
lular oxidative stress [12]. 30 μM) and two experimental concentrations (1000 and
However, despite the related involvement of oxidative 5000 μM) for 24 h or 48 h. Immunocytochemistry and
stress in metformin cytotoxicity to MCF-7 cells, it is known cell death assays were performed similarly: 105 cells were
that breast cancer is a heterogeneous disease, classified into cultured on circular glass coverslips distributed in 24-well
different subtypes according to the gene expression profile, plates. To analyze oxidative stress parameters, 106 cells
such as luminal, subtype with overexpression of human epi- were seeded in 25-cm2 cell culture flasks, allowed to
dermal growth factor receptor 2 (HER-2), triple-negative, bas- grow, and then exposed to metformin as described above.
al-like, and normal-like [13]. MCF-7 cells could correspond to All the experiments were performed in triplicate and re-
only one subtype of the disease, the luminal subtype, but it is peated thrice. The only technique performed with two
known that prognosis and treatment response differ between time points of metformin exposure (24 and 48 h) was
disease subtypes [13]. Triple-negative breast cancer (TNBC) the proliferation assay; all other analyses were performed
represents 10 to 20 % of all breast cancer in women, and it is after 24 h of metformin treatment.
Tumor Biol.

Cytotoxicity and cell proliferation assays measured to determine the anti-oxidant profile of the cells.
Cellular enzyme activity was determined through hydrogen
Cytotoxicity assays peroxide decomposition, which was spectrophotometrically
followed at 240 nm for 3 min using quartz cuvettes. The
2-(3,5-Diphenyltetrazol-2-ium-2-yl)-4,5-dimethyl-1,3-thiazole catalase absorption velocity (Vabs) was calculated by the dif-
bromide (MTT) assay [18] and neutral red assay [19] were ference in absorbance values at 60 and 180 s, and the results
used to investigate the effect of metformin on cell viability were expressed as Vabs per minute per gram of total protein
by assessing alterations in mitochondrial and lysosomal func- [24]. Total thiol levels were determined using 5,5′-dithiobis
tion, respectively. For MTT assay, histidine (1 μM hydroxyl (2-nitrobenzoic acid) as previously described [25]. Total thiol
radical k=3.0×109 M−1 s−1; singlet oxygen k=5×107 M−1 s−1) groups were calculated using a calibration curve prepared by
[20, 21], superoxide dismutase (SOD) (50 U/mL, k =5.0 GSH (Sigma-Aldrich), and the results were expressed in mi-
105 M−1 s−1) [20], and trolox (200 μM, k=2.5 106 M−1 s−1) cromolar thiol/gram of protein.
[22] were used at metformin concentrations of 30 and Lipid peroxidation was estimated by chemiluminescence
5000 μM for MCF-7 cells and at 1000 and 5000 μM for stimulated by tert-butyl hydroperoxide as described by
MDA-MB-231 cells to verify the participation of these reactive Gonzalez-Flecha et al. (1991) [26]. Until the time of analysis,
oxygen species in metformin action on metabolic cellular the tubes containing cells were maintained in the dark and the
activity. reaction was assessed in a TD/20 20 luminometer (Turner
Designs, Sunnyvale, CA), with a response range of 300–
Cell proliferation assays 650 nm. A kinetic capture of chemiluminescence was per-
formed for 30 min, followed by linearization of the data and
Cells were treated as previously described. After 24- and 48-h correction per number of cells [26]. The results were
of treatment, the cells were washed with phosphate-buffered expressed in relative light units (URL)/number of cells. The
saline (PBS) and trypsinized. The cells were then suspended malondialdehyde (MDA) content was determined by high-
in trypan blue (0.05 %) and counted using a Neubauer cham- performance liquid chromatography [27]. The cell suspen-
ber. Cells were classified as viable (no staining) and unviable sions were incubated with perchloric acid and then with
(blue staining) to determine the percentage of viable cells and 0.5 M thiobarbituric acid at 100 °C for 30 min. The reaction
identify the cytostatic or cytotoxic effect of the treatment. was cooled in an ice bath and centrifuged at 5000×g at 4 °C.
The readings are taken at 535 nm during 11 min, and the
Cell death patterns—EB and AO staining results were expressed in nanomolar MDA/protein (g).

The rates of cellular apoptosis and necrosis were assessed by

ethidium bromide (EB)/acridine orange (AO) staining. After DNA damage
treatment, adherent cells were washed with PBS and incubat-
ed with 10 μL PBS containing 10 μg/mL of EB and 3 μg/mL The comet assay
of AO, as described by Sun et al. [23]. The stained cells were
immediately visualized under an Olympus BX3-URA Fluo- Single-cell gel electrophoresis (Comet assay) was performed
rescence System Microscope (Olympus Corporation, Tokyo). according to the procedure described by Tice et al. [28]. After
The differentiation between viable, apoptotic, and necrotic treatment, the cells were harvested, suspended in low-melting-
cells is based on the difference of dye permeability to intact point agarose (0.5 %), and deposited on pre-gelatinized slides
cell membrane. Cells stained green represent viable cells and (1.5 % agarose). Subsequently, the slides were placed in an
are stained only by AO, whereas yellow staining represents electrophoresis buffer (pH 13.0) for 20 min for DNA denatur-
apoptotic cells and is stained by AO and EB (with moderate ation. The slides were then submitted to alkaline electropho-
alteration in membrane permeability), and orange staining resis (pH 13.0; 25 V; 300 mA; 4 °C). All steps were performed
represents necrotic cells, which is stained by EB. Twenty under indirect light. Finally, the slides were treated with pH-
photos were taken of randomly selected areas of the coverslips neutralizing buffer, fixed with ethanol, and stained with
to ensure that the data obtained were representative. GelRed (33 %). For each slide, 100 random nucleoids were
blindly examined at a magnification of ×400, using a fluores-
Oxidative stress parameters cence microscope (excitation filter 420–490 nm; emission fil-
ter 520 nm) connected to an image capture system. To deter-
For the determination of oxidative stress parameters, cultured mine the % DNA in the tail of each nucleoid, CometScore™
cells were treated as previously described. After 24 h of treat- freeware (version: 1.5; TriTek, Sumerduck, VA) was used.
ment, the cells were washed with PBS, trypsinized, and col- Apoptotic/necrotic nucleoids with extensive DNA damage
lected. Cellular catalase activity and total thiol levels were were not included in the analysis. The mean value of the %
 Tumor Biol.

DNA in the tail was considered an index of DNA damage Results

[29].
Cytotoxic effect of metformin
Determination of free 8-Oh-dG
To investigate the cytotoxic effect of metformin, four doses
Oxidative DNA damage was investigated by determining of metformin were tested in vitro using MCF-7 and MDA-
free 8-hydroxy-2-deoxyguanosine (8-Oh-dG), in culture MB-231 cells. The 6- and 30-μM concentrations are clini-
medium samples without fetal bovine serum, using the 8- cally relevant, corresponding to the plasma concentration
Oh-dG EIA Kit (Cayman, Ann Arbor, MI). The experimen- range observed with a metformin dosage of 850 mg/day.
tal procedures were conducted following the manufac- The 1000- and 5000-μM concentrations were used to in-
turer’s protocol. vestigate the mechanisms of metformin-driven cytotoxicity.
The cellular metabolic activity was investigated through the
MTT and neutral red assay. The MTT assay measures cel-
Immunocytochemistry labeling for p53, ERK1/2, AKT, lular metabolic activity by NAD(P)H-dependent oxidore-
and TGF-β1 ductases. It predominantly not only occurs in mitochondria,
but may also occur in the presence of non-mitochondrial
Immunocytochemistry analysis was performed on cells using pyridine nucleotide-dependent enzymes [18]. The basis of
the labeled streptavidin-biotin method, with a LSAB KIT the neutral red method is that the dye diffuses through the
(DAKO Japan, Kyoto). The sections were incubated with plasma membrane and concentrates in the lysosomes,
0.1 % Triton X-100 solution for 1 h, washed three times in which measures lysosomal function based on the ability
PBS, and treated for 40 min at room temperature with 10 % of viable cells to incorporate and bind the supravital dye
bovine serum albumin. Coverslips were then incubated over- neutral red in the lysosomes [19].
night at 4 °C with the primary antibodies (anti-p53, anti- In the MTT assay, metformin significantly reduced the cel-
ERK1/2, anti-AKT, and anti-TGF-β1, diluted 1:400; Santa lular metabolic activity of MCF-7 cells at concentrations equal
Cruz Biotechnology, Dallas, TX). After secondary antibody to or greater than 30 μM and had no effect at 6 μM (Fig. 1a);
treatment (2 h at room temperature), horseradish peroxidase the same effect was observed in MDA-MB-231 cells at 1000
activity was visualized by treatment with H2O2 and 3,3′-di- and 5000 μM (Fig. 1a). In the neutral red assay, a reduction in
aminobenzidine for 5 min. During the last step, the sections MCF-7 metabolism was observed with 1000 and 5000 μM of
were weakly counterstained with Harris’ hematoxylin (Merck, metformin (Fig. 1b) and with 5000 μM, but not 1000 μM, of
Darmstadt). For each case, the primary antibody was omitted metformin in MDA-MB-231 cells (Fig. 1b). The proliferation
as a negative control. The intensity and localization of the curves demonstrated that 24 h of exposure to 1000 or
immunoreactivity were examined using a photomicroscope 5000 μM metformin interfered with cell proliferation in both
(Olympus BX41; Olympus Corporation). Color images of cell lines (Fig. 1c, d), and after 48 h of exposure, the same
representative areas (×400) were digitally acquired for analy- profile was observed in MCF-7 cells (Fig. 1c), but in MDA-
sis. The images were processed using the ImageJ program MB-231, all the metformin concentrations significantly re-
(National Institutes of Health, Bethesda, MD, USA). After duced cell proliferation (Fig. 1d). The concomitant evaluation
color deconvolution and thresholding, the total labeled area of cell viability using trypan blue exclusion showed that met-
was calculated and corrected for the total number of cells. For formin had a cytotoxic effect on proliferation in both cell lines
p53 analysis, the percentage of cells with a labeled nucleus after 24 h at a concentration of 5000 μM (Fig. 1e, f). After
was calculated. 48 h, the cytotoxic effect of metformin was observed at con-
centrations of 1000 and 5000 μM in MCF-7 cells (Fig. 1e) and
Statistical analysis at concentrations equal to or greater than 30 μM in MDA-
MB-231 cells (Fig. 1f).
Data, expressed as the mean±standard error of the mean,
were analyzed using one-way analysis of variance
(ANOVA). Intergroup differences were analyzed by Cell death induction by metformin
Bonferroni’s test, and p<0.05 was considered statistically
significant (*p<0.05, **p<0.001, and ***p<0.0001). Data As demonstrated by EB/AO staining, metformin significantly
was analyzed using GraphPad Prism (version 5; San Diego, increased apoptosis in MCF-7 and in MDA-MB-231 cells at
CA, USA), and only the differences between control and 1000 and 5000 μM (Fig. 2a). The staining also demonstrated
metformin-treated groups were indicated in the graphics. that metformin treatment did not alter the necrosis rate in
Two-way ANOVA was used to analyze the chemilumines- MCF-7 cells (Fig. 2b) but induced necrosis in MDA-MB-
cence, proliferation, and cell viability curves. 231 cells at 1000 and 5000 μM (Fig. 2b).
Tumor Biol.

Fig. 1 Cytotoxic effect of

metformin in human breast cancer
cells. Evaluation of metformin
cytotoxicity at different
concentrations (6, 30, 1000, and
5000 μM) in MCF-7 and MDA-
MB-231 cells. a MTT assay. b
Neutral red assay. c Proliferation
curve of MCF-7 cells. d
Proliferation curve of MDA-MB-
231 cells. e Viability curve
obtained by trypan blue exclusion
in MCF-7 cells. f Viability curve
obtained by trypan blue exclusion
in MDA-MB-231 cells. All the
experiments were performed in
triplicate and repeated thrice.
Statistical analyses were
performed. *p<0.05, **p<0.001,
***p<0.0001 vs. the MCF-7 cell
control group. +p<0.05, ++
p<0.001, +++p<0.0001 vs. the
MDA-MB-231 cell control group

Metformin induces oxidative stress and DNA damage Moreover, treatment increased the activity of the anti-
in human breast cancer cells oxidant enzyme catalase at 1000 and 5000 μM metformin in
MCF-7 cells and at 5000 μM in MDA-MB-231 cells
Quantification of total thiol levels demonstrated that metfor- (Fig. 3b). The chemiluminescence curves all differed, but the
min reduced cellular anti-oxidant defenses at all concentra- increase in lipid peroxidation was only statistically significant
tions used in MCF-7 cells and at concentrations equal to or after treatment with 1000 and 5000 μM of metformin in MCF-
greater than 30 μM in MDA-MB-231 cells (Fig. 3a). 7 cells (Fig. 3c) and with 5000 μM in MDA-MB-231 cells

Fig. 2 Metformin induces cell death in human breast cancer cells. Apoptosis rate. b Necrosis rate. All the experiments were performed in
Evaluation of cell death patterns in MCF-7 and MDA-MB-231 cells triplicate and repeated thrice. *p<0.05, **p<0.001, ***p<0.0001 vs. the
exposed to different metformin concentrations (6, 30, 1000, and MCF-7 cell control group. +p<0.05, ++p<0.001, +++p<0.0001 vs. the
5000 μM) for 24 h by ethidium bromide and acridine orange staining. a MDA-MB-231 cell control group
 Tumor Biol.

Fig. 3 Metformin induces

oxidative stress in human breast
cancer cells. Evaluation of
oxidative stress parameters in
MCF-7 and MDA-MB-231 cells
exposed to different metformin
concentrations (6, 30, 1000, and
5000 μM) for 24 h. a Total thiol
levels. b Catalase activity. c Tert-
butyl hydroperoxide-stimulated
chemiluminescence curves in
MCF-7 cells. d Tert-butyl
hydroperoxide-stimulated
chemiluminescence curves in
MDA-MB-231 cells. e
Malondialdehyde levels. All the
experiments were performed in
triplicate and repeated thrice.
*p<0.05, **p<0.001,
***p<0.0001 vs. the MCF-7 cell
control group. +p<0.05, ++
p<0.001, +++p<0.0001 vs. the
MDA-MB-231 cell control group

(Fig. 3d). However, the results of the MDA quantification and 1000 and 5000 μM for MDA-MB-231). The cells were
showed that metformin increased MDA in a dose-dependent, stimulated with metformin in the presence or absence of dif-
statistically significant manner, starting at a concentration of ferent specific ROS scavengers: SOD, histidine, and trolox
30 μM in MCF-7 cells and of 1000 μM in MDA-MB-231 (superoxide anion, singlet oxygen and hydroxyl radicals,
cells (Fig. 3e). and peroxyl radical scavengers, respectively). In MCF-7 cells,
The comet assay demonstrated that metformin increased at both concentrations of the drug, the presence of all three
DNA lesions at concentrations equal to or greater than scavengers restored cell metabolic activity to control levels
30 μM in MCF-7 cells and at 5000 μM in MDA-MB-231 (Fig. 5a, b). In MDA-MB-231 cells, the use of SOD and
cells (Fig. 4a). The determination of free 8-Oh-dG demonstrat- histidine did not alter the effects of metformin at a concentra-
ed that metformin increased oxidative DNA damage at con- tion of 1000 μM, while trolox did alter them (Fig. 5c). All
centrations equal to or greater than 30 μM in MCF-7 cells and three scavengers restored cellular activity to control levels at a
at 1000 and 5000 μM in MDA-MB-231 cells (Fig. 4b). concentration of 5000 μM (Fig. 5d).

Role of ROS in metformin cytotoxicity Metformin increases nuclear p53 and cytoplasmic
TGF-β1 and reduces the cytoplasmic ERK1/2 and AKT
To investigate which reactive species were involved in the
oxidative stress-driven cytotoxic mechanism of metformin in Metformin exposure increased the nuclear p53 at 1000 and
MCF-7 and MDA-MB-231 cells, two concentrations of the 5000 μM in MCF-7 cells and at 5000 μM in MDA-MB-231
drug that significantly reduced the cellular metabolic activity cells (Fig. 6a). The drug increased the cytoplasmic TGF-β1, at
in the MTT assay were tested (30 and 5000 μM for MCF-7 all the concentrations tested, in MCF-7 cells and, at
Tumor Biol.

Fig. 4 Metformin induces oxidative DNA damage in human breast assay. b Determination of free 8-hydroxy-2-deoxyguanosine. All the
cancer cells. Evaluation of oxidative DNA damage after metformin experiments were performed in triplicate and repeated thrice. *p<0.05,
treatment at different concentrations (6, 30, 1000, and 5000 μM) in **p<0.001, ***p<0.0001 vs. the MCF-7 cell control group. +p<0.05,
MCF-7 cells and MDA-MB-231 cells after 24 h of exposure. a Comet ++p<0.001, +++p<0.0001 vs. the MDA-MB-231 cell control group

concentrations equal to or greater than 30 μM, in MDA-MB- highlighted in numerous types of cancer [30–32]. However,
231 cells (Fig. 6b). At 5000 μM, a significant reduction in the involvement of oxidative status underlying the mecha-
cytoplasmic AKT was observed in both cell lines (Fig. 6c). nisms of the effects of metformin in different cellular lineages
Cytoplasmic ERK 1/2 decreased significantly with metformin of breast cancer is still poorly understood. Thus, herein, two
concentrations of 5000 μM in MCF-7 cells and 1000 and different cell lines (MCF-7 and MDA-MB-231) of human
5000 μM in MDA-MB-231 cells (Fig. 6d). An illustrative panel breast cancer were used to verify the effects of metformin.
showing the results of the immunocytochemistry analysis of Analysis of our results showed that metformin induced the
MCF-7 and MDA-MB-231 cells is available in the Supplemen- same response pattern in both cell lines, but in different con-
tary Fig. 1 (Supplementary Fig. 1 a, b, respectively). centrations, suggesting some particularity in the use of met-
formin. In this report, we investigated the effects of several
concentrations of metformin, and for this, we chose to work
Discussion with distant ranges of concentrations to verify whether the
effects of metformin could be achieved using clinically rele-
The anti-neoplastic activity of metformin and its possible use vant concentrations or only at higher concentrations, so-called
as an adjuvant in traditional cancer therapies have been experimental concentrations. The intensity of the cell response

Fig. 5 Oxidative stress mediates

metformin cytotoxicity in human
breast cancer cells. Evaluation of
cytotoxicity in human breast
cancer cells by MTT assay after
metformin treatment in the
presence or absence of specific
ROS scavengers: SOD (50 U/
mL), histidine (1 μM), and trolox
(200 μM). a MTT assay of MCF-
7 cells treated with 30 μM of
metformin. b MTT assay of
MCF-7 cells treated with
5000 μM of metformin. c MTT
assay of MDA-MB-231 cells
treated with 1000 μM of
metformin. d MTT assay of
MDA-MB-231 cells treated with
5000 μM of metformin. All the
experiments were performed in
triplicate and repeated thrice.
*p<0.05, **p<0.001,
***p<0.0001 vs. the MCF-7 cell
control group. +p<0.05, ++
p<0.001, +++p<0.0001 vs. the
MDA-MB-231 cell control group
 Tumor Biol.

Fig. 6 Metformin increases

nuclear p53 levels and
cytoplasmic TGF-β1 levels and
reduces the expression of ERK1/2
and AKT in human breast cancer
cells. Immunocytochemistry
analysis of MCF-7 cells and
MDA-MB-231 cells exposed to
different metformin
concentrations (6, 30, 1000, and
5000 μM) for 24 h. a Quantitative
results for p53. b Quantitative
results for TGF-β1. c
Quantitative results for AKT. d
Quantitative results for ERK1/2.
All the experiments were
performed in triplicate and
repeated thrice. *p<0.05,
**p<0.001, ***p<0.0001 vs. the
MCF-7 cell control group. +
p<0.05, ++p<0.001, +++
p<0.0001 vs. the MDA-MB-231
cell control group

toward different concentrations of metformin was different, At concentrations of metformin equal to or greater than
although the response pattern was similar. 30 μM, a dose-dependent reduction in anti-oxidants and an
After 24 h, MCF-7 cells were more sensitive to metfor- increase in MDA levels were observed in MCF-7 cells, indi-
min effects on cellular metabolic activity than MDA-MB- cating the occurrence of oxidative stress (OS). In MDA-MB-
231 cells. When reducing cellular metabolism, metformin 231 cells, the same parameters were increased from a concen-
had a greater action on the MTT results, which predomi- tration of 1000 μM; however, despite that it was possible to
nantly reflects mitochondrial function, than on the neutral detect OS at this concentration, other analyzed parameters
red results, which reflects lysosomal function. In fact, the (catalase activity and lipid peroxidation estimated by chemi-
concentrations that significantly reduced cell activity in the luminescence) are increased only at 5000 μM. The levels of
neutral red assay in MCF-7 cells and in MDA-MB-231 free 8-Oh-dG, which is DNA damage by reactive oxygen and
were higher than the concentrations that reduced cell activ- nitrogen species, increased after metformin treatment in both
ity in the MTT assay. In both cell lines, the observed reduc- cell lines at the same concentrations that we detected OS,
tion in the cellular metabolism by MTT assay occurred indicating oxidative DNA damage. However, in MDA-MB-
through an oxidative stress-dependent mechanism, which 231 cells, in despite of the increase of free 8-Oh-dG levels
has not been previously described. The activation of AMPK with metformin at 1000 μM, treatment increased DNA strand
is known to support the in vitro anti-proliferative action of breaks, observed in the comet assay, only at 5000 μM. This
metformin in cancer cells [33]. However, this study dem- could happen because OS in these cells was more intense with
onstrated that metformin cytotoxicity in MCF-7 and MDA- 5000 μM of metformin, with a greater increase in MDA
MB-231 cells is related to increased oxidative stress and levels. It is known that MDA is a reactive aldehyde that inter-
DNA damage. acts with intracellular proteins and DNA bases to induce mu-
Despite the reduction in cellular metabolic activity, the pro- tagenic lesions and cell death [20]. Thus, this reactive metab-
liferative capacity of both cell lines was only impaired at ex- olite, derived from the lipid peroxidation process, could be
perimental concentrations after 24 h of drug exposure, via a affecting the DNA structure in breast cancer cells during met-
cytotoxic rather than a cytostatic effect. However, after 48 h, formin exposure. The participation of OS in the process of
MDA-MB-231 was more sensitive to metformin effects since metformin-induced cytotoxicity to MCF-7 cells became clear
6 μM of metformin was cytostatic and 30 μM was cytotoxic. once the use of different scavengers recovered cell metabolic
Relevant comparisons between the results obtained herein and activity to control levels. However, in the triple-negative cell
the results available in the literature are difficult because pre- line, this was observed only with metformin at 5000 μM. At
vious research evaluating the impact of metformin on the vi- 1000 μM, only trolox was able to recover MDA-MB-231
ability of these cell lines frequently used higher concentrations vitality, which indicated that trolox protected cells against ox-
of the drug or increased periods of exposure [34–36]. idative membrane damage in both metformin 1000- and
Tumor Biol.

5000-μM concentrations. The decrease in cellular vitality increased cytoplasmic TGF-β1 in MCF-7 cells, and concen-
measured by the MTT assay in MDA-MB-231 indicates that trations equal to or greater than 30 μM produced the same
at metformin 5000 μM, there is a more pronounced reduction effect in MDA-MB-231. The role of TGF-β1 in breast cancer
in whole cell activity and, consequently, in the production of is paradoxical. Although its action as a tumor suppressor in the
superoxide anion, singlet oxygen, and hydroxyl radicals, sug- breast is unquestionable, TGF-β1 is frequently identified as a
gesting that one of the mechanisms of cellular response to malignant molecule that drives breast cancer [15]. TGF-β1 is
metformin action is dependent on these oxygen species and a major regulator of the response to DNA damage. Thus, loss
that, at this concentration, the scavengers are more efficient. of responsiveness to this growth factor could increase geno-
Queiroz et al. (2014) studied the effects of metformin on mic instability through an ineffective DNA repair response
MCF-7 cells and demonstrated that 10,000 μM of metformin and checkpoint failure, leading to the apoptosis of aberrant
increased cellular total reactive oxygen species after 72 h of cells [42]. Analysis of the results of this study suggests that
drug exposure [12]. The authors also demonstrated that the the OS response of the cells to increased concentrations of
same concentration increased cellular apoptosis and that OS metformin is related in some degree with TGF-β1 induction.
was related to metformin-dependent apoptosis induction. In conclusion, our data suggest the pro-apoptotic effect of
However, at this concentration (10,000 μM), other mecha- metformin in breast cancer cells, whereas cell line specific is
nisms were associated with metformin effect, since percentage related to an increase in OS, which probably involves DNA
of dead cells with the association of metformin and anti- damage and, consequently, the induction of p53. Moreover,
oxidant enzymes was still higher than the control group [12]. the metformin-induced increase in TGF-β1 probably contrib-
Our results show that incubating the cells for 24 h with a utes to the induction of apoptosis in response to DNA damage.
metformin concentration of 30 μM, an increase in the forma- Further studies are required to understand the effects of met-
tion of MDA was verified and that a significant increase in p53 formin in vivo and to investigate the potential clinical utility of
nuclear levels was observed after treatment with experimental metformin as an adjuvant during breast cancer treatment.
concentrations of metformin, in both cell lines. p53 is a well-
known transcription factor that inhibits tumorigenesis in mam- Acknowledgments The authors are grateful to J.A. Vargas and P.S.R.
Dionízio-Filho, from the Department of General Pathology of the State
mals by regulating the transcription of a large number of genes
University of Londrina, for their excellent technical assistance.
responsible for the control of cell cycle arrest, apoptosis, cell
growth, and protein translation [37]. Furthermore, p53 has Compliance with ethical standards
transcription-independent activities, including the initiation
of cellular tumor suppression programs, such as apoptosis or Conflicts of interest None
cell cycle arrest, in response to intracellular genotoxic stresses,
such as DNA damage, hypoxia, and oncogene activation [38].
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