Abstract
Purpose
Despite a growing body of evidence indicating a potential efficacy of the anti-diabetic metformin as anti-cancer agent, the exact mechanism underlying this efficacy has remained largely unknown. Here, we aimed at assessing putative mechanisms associated with the ability of metformin to reduce the proliferation and migration of breast cancer cells.
Methods
A battery of in vitro assays including MTT, colony formation, NBT and scratch wound healing assays were performed to assess the viability, proliferation, anti-oxidative potential and migration of breast cancer-derived MCF-7, MDA-MB-231 and T47D cells, respectively. Reactive oxygen species (ROS) assays along with fluorescence microscopy were used to assess apoptotic parameters. Quantification of SOD, Bcl-2, Bax, MMPs, miR-21 and miR-155 expression was performed using qRT-PCR.
Results
We found that metformin inhibited the growth, proliferation and clonogenic potential of the breast cancer-derived cells tested. ROS levels were found to be significantly reduced by metformin and, concomitantly, superoxide dismutase (SOD) isoforms were found to be upregulated. Mitochondrial dysfunction was observed in metformin treated cells, indicating apoptosis. In metastatic MDA-MB-231 cells, migration was found to be suppressed by metformin through deregulation of the matrix metalloproteinases MMP-2 and MMP-9. The oncogenic microRNAs miR-21 and miR-155 were found to be downregulated by metformin, which may be correlated with the suppression of cell proliferation and/or migration.
Conclusions
Our data indicate that metformin may play a pivotal role in modulating the anti-oxidant system, including the SOD machinery, in breast cancer-derived cells. Our observations were validated by in silico analyses, indicating a close interaction between SOD and metformin. We also found that metformin may inhibit breast cancer-derived cell proliferation through apoptosis induction via the mitochondrial pathway. Finally, we found that metformin may modulate the pro-apoptotic Bax, anti-apoptotic Bcl-2, MMP-2, MMP-9, miR-21 and miR-155 expression levels. These findings may be instrumental for the clinical management and/or (targeted) treatment of breast cancer.
Electronic supplementary material
The online version of this article (10.1007/s13402-018-0398-0) contains supplementary material, which is available to authorized users.
Keywords: Apoptosis, Breast cancer, Metformin, MicroRNA, ROS, Superoxide dismutase
Introduction
Breast cancer is the second leading cause of mortality in females after lung cancer. Breast cancer is the most common cancer among females in the United States, with an estimated 246,660 new cases in 2016 (i.e., 29% of all cancer cases) [1, 2]. Cellular signaling pathways controlling metabolism and neoplasm have been shown to share intricate links, suggesting a close relationship between metabolic disorders and malignancies. Notably, several epidemiologic and case-control studies have indicated that type 2 diabetes mellitus (T2DM) is associated with a 10-20% increased breast cancer risk [3–5]. In addition, hyperinsulinemia and hyperglycemia have been shown to favor the growth of cancer cells in T2DM patients [6].
The biguanide metformin is the most widely used anti-diabetic medication. It exerts its systemic effects by inhibiting hepatic glucose production and lowering hyperinsulinemia, which is associated with insulin resistance and the uptake of circulating glucose in peripheral tissues [7]. Interestingly, an increasing body of evidence indicates a reduction in cancer incidence after metformin medication, including breast, prostate, colorectal and liver cancer [8, 9], but the precise mechanism underlying this anticancer action has remained unknown. The anti-neoplastic mode of action of metformin follows two distinct pathways: one involving inhibition of the mammalian target of rapamycin (mTOR) and the other involving activation of the “fuel gauge” adenosine monophosphate-activated protein kinase (AMPK), an enzyme that is involved in maintaining cellular and organismal energy balances [10, 11]. AMPK activation disrupts complex I of the mitochondrial respiratory chain, resulting in a decrease in ATP and an increase in AMP levels, leading to alterations in mitochondrial activity [12, 13]. As a result of a decrease in the ATP/AMP ratio and energy depletion, glycolysis becomes stimulated, thereby inducing uptake of glucose by muscles to maintain cellular metabolism [14].
Several drugs and compounds may disrupt the mitochondrial machinery by raising oxidative stress, a process that enhances the production of reactive oxygen species (ROS) [15]. Apoptosis is linked to increased mitochondrial oxidative stress, which causes cytochrome c release, caspase activation and, ultimately, cell death [16]. Still, however, the exact correlation between metformin and oxidative stress/ROS production is unclear. Since it has been reported that oxidative stress may modulate several intracellular signaling pathways related to proliferation and apoptosis, it is reasonable to hypothesize that metformin may interfere with cancer growth by affecting the cellular redox status.
In the present study, we examined the mechanism of action of metformin by analyzing oxidative stress and the expression of markers associated with apoptosis and metastasis. In particular, we used the enzyme superoxide dismutase (SOD) as basis for our model to confirm the involvement of ROS in these processes.
Materials and methods
Chemicals and reagents
Metformin (1, 1-dimethylbiguanide hydrochloride) was kindly supplied as a gift by Ranbaxy Research Laboratories Ltd. (Mumbai, Maharashtra, India). Gene and microRNA primers were designed using Primer 3 software and custom synthesized by Imperial Life Sciences Pvt. Ltd. (New Delhi, India). All the chemicals and reagents used were of the highest purity grades.
Cell culture
Human breast cancer-derived cell lines with variable genetic and hormonal backgrounds, i.e., MCF-7, MDA-MB-231 and T47D, were obtained from the cell repository of the National Centre for Cell Sciences (NCCS, Pune, India) and cultured in DMEM, L-15 and RPMI-1640 medium, respectively. The culture media were supplemented with 10% fetal bovine serum (FBS), 100 units/ml penicillin and 100 μg/ml streptomycin MCF-7 and T47D cells were incubated at 37°C in a humidified atmosphere containing 5% CO2 whereas MDA-MB-231 cells were incubated at 37°C in a humidified CO2-free atmosphere.
Cell proliferation and viability assays
The effect of metformin on cell proliferation was evaluated using a MTT assay [17]. To this end, cells were treated with increasing concentrations of metformin (1, 10 and 20 mmol/L) in triplicate and cultured for 24-72 hours. Next, the cells were treated with fresh MTT solution (0.5 mg/ml in media) and incubated at 37°C in the dark for 4 hours, after which the supernatants were discarded and the reduced MTT formazan crystals were solubilized in dimethyl sulfoxide (DMSO). Finally, the absorbance was measured at 570 nm using a microplate reader (Synergy H1, BioTek, USA). The number of proliferating cells was determined relative to the number of control (untreated) cells.
To determine their viability, cells were exposed to culture medium without (control) and with (treatment) metformin at different concentrations (see above) for 48 hours. Next, the cells were stained with 0.4% (w/v) trypan blue dye for 15 minutes, after which both live (unstained) and dead (blue stained) cells were counted thrice using a Countess automated cell counter (Invitrogen, India) [17].
Colony formation assay
Colony formation assays were performed as reported before [18]. Briefly, cells were seeded in 6-well plates in triplicates at a density of ~800 cells/well in 2 ml medium containing 10% FBS. After 24 hours, the culture medium was replaced by fresh medium containing 0.5% FBS (control) or the same medium containing metformin (1, 10 and 20 mmol/L), after which the cells were allowed to grow for 3 weeks at 37°C in a humidified atmosphere. Next, cell colonies were fixed with methanol-acetic acid (3:1) for 5 minutes and stained for 15 minutes with a solution containing 0.5% crystal violet and 25% methanol. After removal of excess dye, the colonies were counted using ImageJ software (NIH, USA).
Intracellular ROS measurement
Intracellular ROS levels were measured using two different probes: 2’, 7’-dichlorodihydrofluorescein diacetate (DCFH-DA) and dihydroethidium (DHE). The cells were seeded in 6-well plates (1.5×105 cells/well) and cultured for 24 hours, after which they were treated with metformin (1, 10 and 20 mmol/L) for another 24 hours. Next, the cells were thoroughly washed with phosphate buffer saline (PBS) and incubated in 10 μmol/L DCFH-DA or DHE in a dark humidified chamber for 30 minutes at room temperature. After this, the cells were again washed with PBS and fluorescence was monitored using a confocal laser scanning microscope (FV1200, Olympus Japan). All images of the stained cells were taken at the same exposure time. In a separate experiment, fluorescence signals were evaluated using a microplate reader as reported before [19, 20].
Nitroblue tetrazolium (NBT) reduction assay
SOD activities were determined using a nitroblue tetrazolium (NBT) reduction assay as reported before [21]. Briefly, ~1×104 cells were seeded in 96-well plates and incubated for 24 hours. The next day, the cells were incubated with and without metformin (control) for another 24 hours, after which NBT (5 mg/ml) was added to each well and incubated for 3 hours at 37°C. The resulting nitroblue-formazan (black-blue/water-insoluble) deposits were solubilized in DMSO after which absorbance was measured at 550 nm. The results were transformed in % SOD activity compared to (untreated) control cells.
4, 6-Diamidino-2-phenylindole (DAPI) nuclear staining
Nuclei were stained with DAPI to assess morphological alterations typical of apoptosis [22]. To this end, cells were grown on sterile glass coverslips in a 12-well plate (~5×104/well) followed by treatment with metformin for 48 hours. Next, the cells were washed twice with ice-cold PBS and fixed with 3.7% paraformaldehyde in PBS for 20 minutes at room temperature. The fixed cells were permeabilized by incubation in 0.2% Triton X-100 for 5 minutes, washed with PBS and stained with DAPI solution (0.5 μg/ml) in the dark for 1 hour at 37°C, after which they were imaged using a fluorescence microscope (Olympus FSX-100). Cells with fragmented, condensed and/or bright nuclei were scored as apoptotic.
Acridine orange (AO)/Ethidium bromide (EtBr) dual staining
Another morphological assay to detect apoptosis was performed through Acridine orange/Ethidium bromide staining as reported before [23]. Briefly, cells were grown on sterile glass coverslips and treated with metformin for 48 hours, after which 10 μl AO/EtBr solution was added for 5 minutes. Next, the cells were washed twice with PBS and images were captured using a fluorescence microscope (see above).
Mitochondrial membrane potential (ΔΨm) assay
ΔΨm is an important parameter that can be used to distinguish healthy from apoptotic cells [24]. Metformin-treated cells (2×105/well in a 6-well plate) were incubated with 2 ml PBS containing 10 mg/ml JC1 dye for 15 minutes at 37°C, after which fluorescence was monitored using a confocal laser scanning microscope (see above). The results were transformed as the ratio of red to green fluorescence compared to the (untreated) control. In a separate experiment, fluorescence intensities were measured using a microplate reader.
Scratch wound healing assay
The migration of metastatic MDA-MB-231 cells was measured using a scratch wound healing assay as reported before [25]. Briefly, confluent monolayers of cells in 6-well plates were scratched across the centre using a sterile 200 μl pipette tip to create a wound. Next, the cultures were washed twice with PBS to remove the detached cells and photomicrographs of the initial wounds were taken (0 h). The cell cultures were subsequently incubated with fresh media containing different concentrations of metformin after which cell migration into the wound area was monitored and photographed at various time points over a 48 hour period using an inverted phase contrast microscope equipped with a digital camera at 100× magnification. To analyze migration rates, the wound areas were quantified using ImageJ software, after which the differences between the initial and final wound areas were calculated.
RNA isolation and quantitative RT-PCR
Following different treatments, mRNA expression levels were quantified using a SYBR green master mix in conjunction with an ABI Step One Plus Real-Time system (Applied Biosystems, India). Specific primers were designed for SOD 1, SOD 2, SOD 3, Bcl-2, Bax, MMP-2, MMP-9 and β-actin (listed in Supplementary Table). The fluorescence threshold cycle (CT) values for all mRNAs were normalized to the CT value of the endogenous control β-actin. MicroRNAs were extracted using a mirVana isolation kit (Ambion) and subsequently reverse transcribed using a miRNA first-strand cDNA synthesis kit (Ambion). Primers used for amplification were 5’-GCCGCTAGCTTATCAGACTGATGT-3’ and 3’-GTGCAGGGTCCGAGGT-5’ for miR-21; 5’-TTAATGCTAATCGTGATAGGGGT-3’ and 3’-GCTGTCAACGATACGCTACGTAACG-5’ for miR-155; 5’-GCTTCGGCAGCACATATACTAAAAT-3’ and 3’-CGCTTCACGAATTTGCGTGTCAT-5’ for SnU6. The CT values for mir-21 and miR-155 were normalized to the CT value of the endogenous control SnU6. Relative fold-changes in mRNA/microRNA expression levels compared to control samples were calculated using the 2-ΔΔCTmethod.
In silico studies
SOD protein structures were downloaded from the Protein Data Bank (PDB) (www.rcsb.org) and processed for molecular docking. The PDB provides information on 3-dimensional structures of bio-macromolecules and their complexes based on X-ray crystallography and nuclear magnetic resonance spectroscopy data. A grid box was generated around the binding site of the co-crystallized ligand in SOD. Molecular docking experiments were performed using the GLIDE software tool developed by Schrödinger [26].
Statistical analysis
Statistical analyses were performed using Sigma Plot 11 software. The results are expressed as mean ± standard deviation (SD). Student’s t-test followed by one-way ANOVA was used to identify significant differences between two groups. The level of significance was set at p ≤ 0.05 (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001).
Results
Metformin inhibits the growth of breast cancer cells
When MCF-7, MDA-MB-231 and T47D cells were grown in the presence of increasing concentrations of metformin (1-20 mmol/L), a significant inhibition of proliferation was observed in all three cell lines using a MTT assay (Fig. 1a, b and c). The inhibitory action was higher after 72 hours exposure compared to that after 24 and 48 hours. At a 20 mmol/L concentration the growth of MCF-7, MDA-MB-231 and T47D cells was found to be inhibited to 77.2 %, 59.8% and 67.2% after 72 hours, respectively, suggesting that among the cells tested MCF-7 is most sensitive to metformin. Consistent with these results, a trypan blue dye-based viability assay revealed a metformin-induced cytotoxicity in the different breast cancer-derived cells tested, and that the MCF-7 cells showed the highest reduction in viability (Supplementary Fig. 1).
Fig. 1. Effect of metformin on viability and proliferation/colony formation of breast cancer cells.
a, b, c Viabilities of MCF-7, MDA-MB-231 and T47D cells treated with increasing concentrations of metformin for 24-72 hours, measured by MTT absorbance assay. d, e Colony forming abilities of MCF-7, MDA-MB-231 and T47D cells in the presence of metformin. All data are represented as mean ± SD, N = 3, *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001 versus control
We also examined the effect of metformin on the colony-forming capacity of these cells. We found that over a period of 3 weeks metformin reduced colony formation in a dose-dependent manner (Fig. 1d). Specifically, we found that cells treated with metformin gave rise to decreased numbers and smaller sizes of colonies compared to the (untreated) control cells. Metformin, at 20 mmol/L, reduced colony formation by 86%, 71% and 82% in MCF-7, MDA-MB-231 and T47D cells, respectively, compared to (untreated) controls (Fig. 1e).
Metformin reduces intracellular ROS generation
In order to assess whether metformin induces reactive oxygen species (ROS) production, we next set out to expose the different breast cancer-derived cells to increasing concentrations of metformin (1-20 mmol/L), after which fluorescent probes DCFH-DA and DHE were used to measure the levels of intracellular ROS. We found that ROS production was decreased by metformin treatment in a concentration-dependent manner (Fig. 2a, b). At a concentration of 20 mmol/L, metformin significantly reduced DCFH-DA fluorescence 5.41-, 24.3- and 8-fold in MCF-7, MDA-MB-231 and T47D cells, respectively (Fig. 2c). Similarly, we found that cells treated with metformin showed a significant and concentration-dependent decrease in DHE fluorescence. Metformin at a 20 mmol/L concentration attenuated ROS levels by 3-, 4.31- and 3.11-fold in MCF-7, MDA-MB-231 and T47D cells, respectively, compared to (untreated) controls (Fig. 2d).
Fig. 2. Effect of metformin on ROS generation in breast cancer cells.
a, b Representative pictures showing ROS levels in MCF-7, MDA-MB-231 and T47D cells after metformin treatment, assessed using DCFH-DA (a) and DHE (b). c, d DCFH-DA and DHE fluorescence intensities quantified spectrophotometrically using a microplate reader. Data are normalized to untreated controls. All data are represented as mean ± SD, N = 3, *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001 versus control
Metformin upregulates SOD expression and activity
The different breast cancer-derived cells were treated with metformin and 24 hours later the mRNA expression levels of all three isoforms of human superoxide dismutase (SOD) were determined using qRT-PCR, while SOD activity was determined using a NBT assay. We found that exposure to metformin (10 mmol/L) enhanced the expression of SOD 1, SOD 2 and SOD 3 by 0.76-, 1.49- and 2.36-fold in MCF-7 cells, by 3.60-, 3.37- and 0.765-fold in MDA-MB-231 cells and by 1.36-, 1.34- and 1.94-fold in T47D cells, respectively, compared to (untreated) control cells (Fig. 3a, b and c). Next, we confirmed these results by directly measuring SOD enzyme activities using a NBT reduction assay and found that the SOD activities increased significantly in all cells tested (Fig. 3d). Together, these findings indicate a key role of the SOD system in controlling ROS-mediated breast cancer cell growth.
Fig. 3. Effect of metformin on SOD isoform expression and SOD activity in breast cancer cells.
MCF-7, MDA-MB-231 and T47D cells were treated with increasing concentrations of metformin (1-20 mmol/L) for 24 hours. mRNA levels of SOD isoforms in MCF-7 (a), MDA-MB-231 (b) and T47D (c) cells, were measured by qRT-PCR. SOD activities upon treatment with metformin and SOD inhibitor, were determined by NBT assay (d, e). All data are represented as mean ± SD, N = 3, *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001 versus control
In order to confirm that metformin treatment enhances the SOD system, we employed a SOD inhibitor, silver diethyldithiocarbamate, as model to quantify SOD levels. Cells pre-treated with the SOD inhibitor were exposed to metformin after which alterations in SOD levels were assessed compared to SOD inhibitor-only treated cells. We found that the SOD activities were significantly suppressed in the presence of the SOD inhibitor (Fig. 3e). We also found that the SOD activities did not increase after subsequent metformin treatment, indicating successful blocking of the SOD system by the inhibitor. Furthermore, we found that the mRNA expression levels of the different SOD isoforms were suppressed after SOD inhibitor treatment (Supplementary Fig. 2). In SOD inhibitor-treated cells, we found an excessive increase in ROS generation and an extensive cell death as quantified by DCFH-DA and MTT assays, respectively (Supplementary Fig. 2). From these data, we conclude that the excessive cell death in inhibitor-treated cells may be related to ROS overproduction, thereby masking cell defense systems and making them incapable of establishing a state of homeostasis. Taken together, we found that metformin significantly induces the SOD enzymatic system in breast cancer-derived cells which, at least in part, may contribute to the reduction of ROS generation in these cells.
In addition, we performed in silico studies to confirm that distinct interactions may exist between metformin and the different SOD isoforms. SOD 1 is a Cu-Zn containing dismutase, whereas mitochondrial SOD 2 is a manganese-dependent superoxide dismutase (MnSOD). The selection of proteins from the PDB was done by resolution (1.82 Å). Binding cavities of SOD 1 and SOD 2 where the ligand can bind were predicted using sitemap. Metformin was docked into the Cu-Zn containing active site of SOD 1 (PDB ID: 1HL4) and the Mn-containing active site of SOD 2 (PDB ID: 1PM9) to investigate favorable binding patterns, as reported before [27, 28]. Favorable interactions between metformin and SOD 1/SOD 2 were scored using the GLIDE tool (Schrödinger MAESTRO 11.1). As there is no ligand in the active site of the protein, blind docking was performed in triplicate to reveal metformin binding to the same site. By doing so, putative interactions of metformin with SOD 1 were indeed noted at a site encompassing THR 137, SER 142 and ARG 143 (Fig. 8a), whereas putative interactions of metformin with SOD 2 were noted at a site encompassing LEU B:49, GLU A:42 and GLN A:46 (Fig. 8b).
Fig. 8. Interaction of metformin with SOD 1 and SOD 2.
Diagrams showing potential ligand binding of metformin to SOD 1 (a) and SOD 2 (b) at their respective binding sites
Metformin promotes apoptosis in breast cancer-derived cells
To gain insight into the mechanisms underlying the growth inhibitory role of metformin, we set out to assess the potential role of apoptosis-related processes, i.e., morphological changes and expression alterations in the pro-apoptotic (Bax) and anti-apoptotic (Bcl-2) genes. Using fluorescence microscopy-based analyses of DAPI stained cells, we found that untreated control cells showed large oval-shaped nuclei, whereas metformin treated cells showed chromatin fragmentation/condensation with nuclear shrinkage and rupture of nuclear membranes, indicative of apoptosis, in the breast cancer-derived cells tested (Fig. 4a). Compared to (untreated) control cells, the apoptosis-related features increased up to 9.5-, 8.4- and 8.9-fold in MCF-7, MDA-MB-231 and T47D cells, respectively, when treated with 10 mmol/L metformin (Supplementary Fig. 3). AO/EtBr dual staining showed early apoptotic features, i.e., densely greenish orange stained cells, after treatment with metformin (1 mmol/L) (Fig. 4b). A higher dose of metformin (10 mmol/L) resulted in late apoptotic features, indicated by orange-red highly condensed chromatin or fragmented nuclei. These results indicate metformin-induced cell death through apoptosis. In order to substantiate the putative role of metformin in apoptosis induction, the mRNA expression levels of pro-apoptotic Bax and anti-apoptotic Bcl-2 were quantified using qRT-PCR. We found that the Bax expression level increased significantly after metformin treatment (p ≤ 0.001) in a more or less dose-dependent manner (Fig. 4c) and that the Bcl-2 expression level decreased significantly (2.4-, 1.3- and 6.8-fold) after 20 mmol/L metformin treatment of MCF-7, MDA-MB-231 and T47D cells, respectively (Fig. 4d).
Fig. 4. Metformin treatment enhances apoptosis in breast cancer cells.
a, b Representative pictures showing cellular and nuclear morphologies of MCF-7, MDA-MB-231 and T47D cells after metformin treatment, assessed by DAPI staining (a) and Acridine orange/Ethidium bromide dual staining (b). Arrows indicate apoptotic cells. c Bax and d Bcl-2 mRNA levels measured by qRT-PCR. All data are represented as means ± SD, N = 3, *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001 versus control
Metformin activates mitochondrial pathways to induce apoptosis
As apoptosis is often associated with a collapse of mitochondrial membrane potential (ΔΨm), we next set out to assess the ability of metformin to depolarize mitochondrial membranes by JC-1 staining. By doing so, we observed a high degree of mitochondrial damage through ΔΨm loss (48%, 24% and 36%, respectively) in metformin-treated MCF-7, MDA-MB-231 and T47D cells compared to (untreated) control cells (Figs. 5a, b).
Fig. 5. Effect of metformin on mitochondrial membrane potential (ΔΨm) in breast cancer cells.
a Representative pictures showing alterations in mitochondrial membrane potential of MCF-7, MDA-MB-231 and T47D cells, assessed using the mitochondrial-selective dye JC-1 after metformin treatment. Images represent shifts in fluorescence from red (left) to green (right) due to △Ψm loss. b % ΔΨm loss calculated spectrophotometrically. All data are represented as mean ± SD, N = 3, *p ≤ 0.05 versus control
Metformin attenuates breast cancer cell migration
In order to investigate the effect of metformin on the migratory ability of metastatic MDA-MB-231 cells, a scratch wound healing assay was performed. Through this assay, a concentration-dependent migration inhibitory effect of metformin was observed (Fig. 6a). After the initial wound was created (0 hour), significant differences in wound closure were observed after 12, 24 and 48 hours, suggesting a prominent role of metformin in delaying this process (Fig. 6b). At concentrations of 1 and 10 mmol/L, metformin inhibited the migration of MDA-MB-231 cells up to 52% and 87%, respectively, after 48 hours treatment (p ≤ 0.05 and p ≤ 0.01).
Fig. 6. Effect of metformin on migration and MMP-2/MMP-9 expression in breast cancer cells.
a Representative pictures showing alterations in the migratory ability of MDA-MB-231 cells after metformin treatment over a time period of 0-48 hours. b Quantification of scratch wound healing by MDA-MB-231 cells through migration over different time periods. c MMP-2 and MMP-9 mRNA expression levels in MDA-MB-231 cells after metformin treatment, measured by qRT-PCR. All data are represented as means ± SD, N = 3, *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001 versus control
MMP-2 and MMP-9 are downregulated by metformin
To confirm the attenuation of migration observed in MDA-MB-231 cells, the effect of metformin on the expression of MMP-2 and MMP-9 was assessed by qRT-PCR analysis. We found that metformin treatment down-regulated MMP-2 and MMP-9 mRNA expression in a dose-dependent manner after 24 hours treatment (Fig. 6c). At a concentration of 20 mmol/L, metformin significantly reduced the MMP-2 and MMP-9 expression levels by 3.03- and 3.68-fold, respectively, compared to untreated control cells (p ≤ 0.001).
miR-21 and miR-155 are downregulated by metformin
To investigate possible mechanisms that may underlie the observed effects of metformin on migration and (putatively) metastasis, we decided to assess the expression of two well-known microRNAs, miR-21 and miR-155, whose aberrant expression has been reported to affect the invasive and metastatic properties of breast cancer cells. We found that metformin treatment significantly suppressed the expression levels of miR-21 and miR-155 in all three breast cancer-derived cell lines tested (Fig. 7a, b). We conclude that metformin not only promotes apoptotic processes in breast cancer cells, but also affects the migratory ability of metastatic breast cancer cells by downregulating the expression of MMP-2, MMP-9, miR-21 and miR-155.
Fig. 7. Effect of metformin on miR-21 and miR-155 expression in breast cancer cells.
miR-21 (a) and miR-155 (b) expression levels in MCF-7, MDA-MB-231 and T47D cells after metformin treatment, measured by qRT-PCR. The experiments were repeated three times. All data are represented as mean ± SD, N = 3, *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001 versus control
Discussion
Breast cancer is a prevalent pathophysiological condition among women [29]. Its occurrence may be affected by several risk factors, including insulin resistance-associated hyperinsulinemia [30]. Epidemiological studies have indicated that metformin, a front-line therapy for type 2 diabetes mellitus (T2DM), decreases the cancer incidence in diabetic patients [31]. Others have reported that metformin exhibits an anti-proliferative effect in cancer cells in vitro and in vivo [32, 33]. It has been found that this anti-proliferative effect may act either through activating the “cellular energy sensor” adenosine monophosphate-activated protein kinase (AMPK) or through inhibiting the mammalian target of rapamycin (mTOR) signaling pathway [10, 34]. The exact mechanism underlying the anti-cancer action of metformin and its biological consequences, however, still remains to be resolved. Here, we investigated the inhibitory effects of metformin on the growth and invasion of MCF-7, MDA-MB-231 and T47D breast cancer-derived cells.
We found that metformin inhibited the growth and proliferation of the breast cancer-derived cells in a dose- and time-dependent manner. Metformin turned out to be cytotoxic at low micromolar concentrations. These results are in agreement with those from a previous study reporting significant cytotoxic effects of metformin at a wide range of concentrations (1 to 100 mmol/L) [35]. Additionally, we found that the growth inhibitory effect of metformin was not associated with increased ROS generation in the breast cancer-derived cells tested. These findings may provide insight into the mechanism underlying the anti-cancer activity of metformin. It has been reported that ROS-mediated signaling affects various aspects of tumor cell behavior, i.e., cell cycle progression, proliferation, survival, apoptosis, metabolism and angiogenesis [36]. Droge [37] has proposed that oxidative stress may act as a double-edged sword in T2DM, i.e., moderate ROS production may enhance insulin signaling, while high ROS production may be destructive. It has been suggested that the positive versus negative implications of oxidative stress may depend on ROS levels: low ROS levels may stimulate cellular proliferation/survival, whereas high ROS levels may lead to apoptosis [38]. Under normal physiological conditions, ROS activates several target proteins (tyrosine phosphatases/receptor tyrosine kinases/transcription factors) and redox-sensitive signaling pathways (MAP/Erk and PI3K/NF-κB pathways) [39, 40]. It has been reported that chronic ROS production enhances the growth and tumorigenic potential of breast cancer cells [41]. We found that incubating breast cancer-derived cells with metformin (1-20 mmol/L) decreased its ROS levels, indicating that metformin reduces ROS levels below the baseline required for cells to survive.
In order to resolve the mechanism underlying the ability of metformin to reduce ROS generation, we used SOD as a basis for our model. To maintain “cellular redox homeostasis”, an elaborate endogenous antioxidant defense system must be at work. One of the key members of this system is SOD [42]. SOD converts harmful superoxide radicals into hydrogen peroxide and molecular oxygen, which are subsequently catalyzed to water by catalase. In mammals, three isoforms of SOD do exist, namely cytosolic SOD 1 [(Cu/Zn)-SOD]; mitochondrial SOD 2 [(Mn)-SOD] and extracellular SOD 3 [(EC)-SOD], each with a distinct localization, but catalyzing similar reactions [43]. Previously, it has been shown that metformin may enhance the expression of (Mn)-SOD in MIA PaCa2 and Panc1 pancreatic cancer-derived cells [44]. Metformin is known to activate AMPK by inhibiting mitochondrial respiratory chain complex I, triggering decreases in ATP and increases in AMP levels [12]. Inhibition of complex I leads to superoxide anion generation and apoptosis [45]. To scavenge superoxide anions, cells have to overexpress SOD. We found that metformin increases SOD mRNA levels and activities in breast cancer-derived cells compared to untreated control cells. The increased SOD activities appear to be due to the binding of metformin to different protein residues of SOD isoforms as revealed by in silico analyses. To confirm the ability of metformin to modulate SOD activity for free radical quenching, we employed a SOD inhibitor. By doing so, we indeed found that SOD upregulation is intricately linked to the ability of metformin to decrease ROS. Exposure of SOD inhibitor-pretreated cells to metformin led to excessive ROS production and extensive cell death compared to metformin-only treated cells. Hence, we conclude that SOD activity is central to the ROS quenching capacity of metformin. A further understanding of metformin-related cellular signaling pathways may facilitate the design of targeted approaches to ameliorate human breast cancer.
Apoptosis induction is amply being explored as a therapeutic approach to treat cancer [46]. We assessed the potential effect of metformin on apoptosis in breast cancer-derived cells, i.e., alterations in chromatin structure, mitochondrial membrane potential and pro-apoptotic/anti-apoptotic gene expression levels. We found that metformin induced the downregulation of anti-apoptotic Bcl-2 and the upregulation of pro-apoptotic Bax. Mitochondrial membrane potential alterations were observed in all three cell lines tested, but with different kinetics, indicating a mitochondria-mediated apoptosis induction.
Since we also employed a metastatic cell line in our study (MDA-MB-231), we decided to assess the role of metformin on the migration, as well as the expression of the matrix metalloproteinases MMP-2 and MMP-9, and the microRNAs miR-21 and miR-155 in these cells. Despite recent advances, little improvements have been made in the survival of patients with metastasis [47]. We observed a significant time- and dose-dependent inhibition of MDA-MB-231 cell migration by metformin. MMPs are zinc-dependent proteases that are implicated in metastatic growth through the degradation of extracellular matrix components and basement membranes [48]. MMP-2 and MMP-9 are known to be intricately associated with the invasion and migration of tumor cells. We found that metformin significantly suppressed MMP-2 and MMP-9 expression in MDA-MB-231 cells, underscoring the anti-metastatic role of metformin. Further work is required to establish the signaling pathways involved in MMP expression modulation by metformin in breast cancer cells. MicroRNAs (miRNAs) are known to regulate gene expression in a sequence-specific manner [49] and they have been reported to play critical roles in many physiological processes. It has also been reported that miRNAs may be upregulated or downregulated during cancer development, leading to the categorization of miRNAs into oncogenic or tumor suppressive [50, 51]. We found that metformin downregulated the levels of two key oncogenic miRNAs, miR-21 and miR-155. Since it has been reported that aberrant overexpression of these miRNAs leads to metastasis, we conclude that metformin may, at least in part, inhibit breast cancer metastasis formation through their downregulation [52, 53]. Also this observation may open up new avenues for (targeted) therapeutic intervention.
Our results show that the growth-inhibitory effect of metformin on breast cancer-derived cells is associated with reduced ROS production. Metformin may decrease oxidative stress by increasing SOD scavenging activity. We also found that metformin may induce apoptosis in breast cancer-derived cells through ROS-independent mitochondrial dysfunction. In addition, our data show that metformin may modulate metastasis-related microRNA expression levels, thereby providing novel clues for the putative multi-dimensional mode of action of this drug. Further work is required to elucidate the various mechanistic aspects of metformin in more detail in order to transform the tag “antidiabetic” into “cancer therapeutic”.
Electronic supplementary material
Effect of metformin on BC cells viability. MCF-7, MDA-MB-231 and T47D cells were exposed to the culture medium (control) and metformin for 48 hours. Cell viability was estimated after staining with 0.4% (w/v) trypan blue dye. All data are represented as means ± SD, N = 3, *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001 versus control. (PNG 804 kb)
Effect of SOD inhibitor Silver diethyl thiocarbamate on the ROS levels, SOD expression levels, and growth of BC cells. (A) Representative pictures in panel display alterations in DCFH-DA fluorescence intensity after SOD inhibitor treatment. (B) DCFH-DA fluorescence intensities were quantified spectrophotometrically using microplate reader. (C) Silver diethyl thiocarbamate reduces SOD mRNA expression levels in cells. (D) Silver diethyl thiocarbamate reduces proliferation of BC cells. All data are represented as means ± SD, N = 3, *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001 versus control. (PNG 18884 kb)
Quantification of apoptotic nuclei in MCF-7, MDA-MB-231 and T47D BC cells. The apoptotic nuclei were quantified by fluorescence microscopic analysis. All data are represented as means ± SD, N = 3, *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001 versus control. (PNG 355 kb)
(DOCX 13 kb)
Acknowledgements
The authors express their sincere thanks to Ranbaxy Research Laboratories Ltd. (Mumbai, Maharashtra, India) for providing metformin as a gift sample. This research was funded by the Central University of Punjab, Bathinda as part of Ph.D Thesis. The authors remain indebted to journal editorial board for improving the quality of the manuscript. The authors would like to kindly acknowledge their efforts. We gratefully acknowledge Dr. Alpana Saini, Head, Department of classical and modern languages, Central University of Punjab, Bathinda, India, and Dr. Raju Gautam for their help and support in editing and polishing the language of the manuscript.
Compliance with ethical standards
Ethical approval
This sudy does not include human participants and/or animals.
Conflict of interest
The authors declare that they have no conflict of interest.
References
- 1.R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics 2016. CA Cancer J. Clin. 66, 7–30 (2016) [DOI] [PubMed] [Google Scholar]
- 2.R. Sharma, R. Sharma, T.P. Khaket, C. Dutta, B. Chakraborty, T.K. Mukherjee, Breast cancer metastasis: Putative therapeutic role of vascular cell adhesion molecule-1. Cell. Oncol. 40, 199–208 (2017) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.L. Vona-Davis, D.P. Rose, Type 2 diabetes and obesity metabolic interactions: Common factors for breast cancer risk and novel approaches to prevention and therapy. Curr. Diabetes Rev. 8, 116–130 (2012) [DOI] [PubMed] [Google Scholar]
- 4.P. Ferroni, S. Riondino, O. Buonomo, R. Palmirotta, F. Guadagni, Roselli, Type 2 Diabetes and breast cancer: The interplay between impaired glucose metabolism and oxidant stress. Oxidative Med. Cell. Longev. 2015, 183928 (2015) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.L.A. Flores-López, M.G. Martínez-Hernández, R. Viedma-Rodríguez, M. Díaz-Flores, L.A. Baiza-Gutman, High glucose and insulin enhance uPA expression, ROS formation and invasiveness in breast cancer-derived cells. Cell. Oncol. 39, 365–378 (2016) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.S. Suh, K.W. Kim, Diabetes and cancer: Is diabetes causally related to cancer? Diabetes Metab. J. 35, 193–198 (2011) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.I. Ben Sahra, Y. Le Marchand-Brustel, J.F. Tanti, F. Bost, Metformin in cancer therapy: a new perspective for an old antidiabetic drug? Mol. Cancer Ther. 9, 1092–1099 (2010) [DOI] [PubMed] [Google Scholar]
- 8.A. Malki, A. Youssef, Antidiabetic drug metformin induces apoptosis in human MCF breast cancer via targeting ERK signaling. Oncol. Res. 19, 275–285 (2011) [DOI] [PubMed] [Google Scholar]
- 9.K.H. Stopsack, D.R. Ziehr, J.R. Rider, E.L. Giovannucci, Metformin and prostate cancer mortality: A meta-analysis. Cancer Causes Control 27, 105–113 (2016) [DOI] [PubMed] [Google Scholar]
- 10.B. Viollet, B. Guigas, N. Sanz Garcia, J. Leclerc, M. Foretz, F. Andreelli, Cellular and molecular mechanisms of metformin: an overview. Clin. Sci. 122, 253–270 (2012) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.K.Y. Hur, M.S. Lee, New mechanisms of metformin action: Focusing on mitochondria and the gut. J. Diabetes Investig. 6 600–609 (2015) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 12.W.W. Wheaton, S.E. Weinberg, R.B. Hamanaka, S. Soberanes, L.B. Sullivan, E. Anso, A. Glasauer, E. Dufour, G.M. Mutlu, G.S. Budigner, N.S. Chandel, Metformin inhibits mitochondrial complex I of cancer cells to reduce tumorigenesis. Elife 3, e02242 (2014) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.S.E. Weinberg, N.S. Chandel, Targeting mitochondria metabolism for cancer therapy. Nat. Chem. Biol. 11, 9–15 (2015) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.I. Pernicova, M. Korbonits, Metformin: Mode of action and clinical implications for diabetes and cancer. Nat. Rev. Endocrinol. 10, 143–156 (2014) [DOI] [PubMed] [Google Scholar]
- 15.E. Birben, U.M. Sahiner, C. Sackesen, S. Erzurum, O. Kalayci, Oxidative Stress and Antioxidant Defense. World Allergy Organ J. 5 9–19 (2012) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.G. Barrera, Oxidative stress and lipid peroxidation products in cancer progression and therapy. ISRN Oncol. 2012, 137289 (2012) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.B. Singh, M. Rani, J. Singh, L. Moudgil, P. Sharma, S. Kumar, G.S.S. Saini, S.K. Tripathi, G. Singh, A. Kaura, Identifying the preferred interaction mode of naringin with gold nanoparticles through experimental, DFT and TDDFT techniques: insights into their sensing and biological applications. RSC Adv. 6, 79470–79484 (2016) [Google Scholar]
- 18.N.A. Franken, H.M. Rodermond, J. Stap, J. Haveman, C. van Bree, Clonogenic assay of cells in vitro. Nat. Protoc. 1, 2315–2319 (2006) [DOI] [PubMed] [Google Scholar]
- 19.J. Zielonka, B. Kalyanaraman, Hydroethidine- and Mito-SOX-derived red fluorescence is not a reliable indicator of intracellular superoxide formation: Another inconvenient truth. Free Radic. Biol. Med. 48, 983–1001 (2010) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.B.S. Gill, P. Sharma, K.S. Navgeet, Chemical composition and antiproliferative, antioxidant, and proapoptotic effects of fruiting body extracts of the Lingzhi or Reishi medicinal mushroom, Ganoderma lucidum (Agaricomycetes), from India. Int. J. Med. Mushrooms 18, 599–607 (2016) [Google Scholar]
- 21.N. Jambunathan, Determination and detection of reactive oxygen species (ROS), lipid peroxidation, and electrolyte leakage in plants. Methods Mol. Biol. 639, 292–298 (2010) [DOI] [PubMed]
- 22.B. Chazotte, Labeling nuclear DNA using DAPI. CSH Protoc. 2011, 5556 (2011) [DOI] [PubMed] [Google Scholar]
- 23.S. Kasibhatla, G.P. Amarante-Mendes, D. Finucane, T. Brunner, E. Bossy-Wetzel, D.R. Green, Acridine orange/ethidium bromide (AO/EB) staining to detect apoptosis. CSH Protoc. 2006, pii (2006) [DOI] [PubMed]
- 24.A. Cossarizza, M. Baccaranicontri, G. Kalashnikova, C. Franceschi, A new method for the cytofluorometric analysis of mitochondrial membrane potential using the J-aggregate forming lipophilic cation 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolcarbocyanine Iodide (JC-1). Biochem. Biophys. Res. Commun. 197, 40–45 (1993) [DOI] [PubMed] [Google Scholar]
- 25.C.C. Liang, A.Y. Park, J.L. Guan, In vitro scratch assay: a convenient and inexpensive method for analysis of cell migration in vitro. Nat. Protoc. 2, 329–333 (2007) [DOI] [PubMed] [Google Scholar]
- 26.K.E. Hevener, W. Zhao, D.M. Ball, K. Babaoglu, J. Qi, S.W. White, R.E. Lee, Validation of molecular docking programs for virtual screening against dihydropteroate synthase. J. Chem. Inf. Model. 49, 444–460 (2009) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.R.W. Strange, S. Antonyuk, M.A. Hough, P.A. Doucette, J.A. Rodriguez, P.J. Hart, L.J. Hayward, J.S. Valentine, S.S. Hasnain, The structure of holo and metal-deficient wild-type human Cu, Zn superoxide dismutase and its relevance to familial amyotrophic lateral sclerosis. J. Mol. Biol. 328, 877–891 (2003) [DOI] [PubMed] [Google Scholar]
- 28.A.S. Hearn, L. Fan, J.R. Lepock, J.P. Luba, W.B. Greenleaf, D.E. Cabelli, J.A. Tainer, H.S. Nick, D.N. Silverman, Amino acid substitution at the dimeric interface of human manganese superoxide dismutase. J. Biol. Chem. 279, 5861–5866 (2004) [DOI] [PubMed] [Google Scholar]
- 29.Y. Huang, Q. Jin, M. Su, F. Ji, N. Wang, C. Zhong, Y. Jiang, Y. Liu, Z. Zhang, J. Yang, L. Wei, T. Chen, B. Li, Leptin promotes the migration and invasion of breast cancer cells by upregulating ACAT2. Cell. Oncol. 40, 537–547 (2017) [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
- 30.P.J. Wysocki, B. Wierusz-Wysocka, Obesity, hyperinsulinemia and breast cancer: novel targets and a novel role for metformin. Expert. Rev. Mol. Diagn. 10, 509–519 (2010) [DOI] [PubMed] [Google Scholar]
- 31.G.W. Landman, N. Kleefstra, K.J. van Hateren, K.H. Groenier, R.O. Gans, H.J. Bilo, Metformin associated with lower cancer mortality in type 2 diabetes: ZODIAC-16. Diabetes Care 33, 322–326 (2010) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.K. Kato, J. Gong, H. Iwama, A. Kitanaka, J. Tani, H. Miyoshi, K. Nomura, S. Mimura, M. Kobayashi, Y. Aritomo, H. Kobara, H. Mori, T. Himoto, K. Okano, Y. Suzuki, K. Murao, T. Masaki, The antidiabetic drug metformin inhibits gastric cancer cell proliferation in vitro and in vivo. Mol. Cancer Ther. 11, 549–560 (2012) [DOI] [PubMed] [Google Scholar]
- 33.T. Zhang, P. Guo, Y. Zhang, H. Xiong, X. Yu, S. Xu, X. Wang, D. He, X. Jin, The antidiabetic drug metformin inhibits the proliferation of bladder cancer cells in vitro and in vivo. Int. J. Mol. Sci. 14, 24603–24618 (2013) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 34.V. Nair, S. Sreevalsan, R. Basha, M. Abdelrahim, A. Abudayyeh, A. Rodrigues Hoffman, S. Safe, Mechanism of metformin-dependent inhibition of mammalian target of rapamycin (mTOR) and Ras activity in pancreatic cancer: role of specificity protein (Sp) transcription factors. J. Biol. Chem. 289, 27692–27701 (2014) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 35.I.N. Alimova, B. Liu, Z. Fan, S.M. Edgerton, T. Dillon, S.E. Lind, A.D. Thor, Metformin inhibits breast cancer cell growth, colony formation and induces cell cycle arrest in vitro. Cell Cycle 8, 909–915 (2009) [DOI] [PubMed] [Google Scholar]
- 36.D. Zhou, L. Shao, D.R. Spitz, Reactive oxygen species in normal and tumor stem cells. Adv. Cancer Res. 122, 1–67 (2014) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 37.W. Droge, Oxidative aging and insulin receptor signaling. J. Gerontol. A Biol. Sci. Med. Sci. 60, 1378–1385 (2005) [DOI] [PubMed] [Google Scholar]
- 38.I. Afanas’ev, Reactive oxygen species signaling in cancer: comparison with aging. Aging Dis. 2, 219–230 (2011) [PMC free article] [PubMed] [Google Scholar]
- 39.Y. Son, Y.K. Cheong, N.H. Kim, H.T. Chung, D.G. Kang, H.O. Pae, Mitogen-activated protein kinases and reactive oxygen species: How can ROS activate MAPK pathways? J. Signal Transduct. 2011, 792639 (2011) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 40.A. Jezierska-Drutel, S.A. Rosenzweig, C.A. Neumann, Role of oxidative stress and the microenvironment in breast cancer development and progression. Adv. Cancer Res. 119, 107–125 (2013) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 41.P.K.S. Mahalingaiah, K.P. Singh, Chronic oxidative stress increases growth and tumorigenic potential of MCF-7 breast cancer cells. PLoS One 9, e87371 (2014) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 42.B. Poljsak, D. Suput, I. Milisav, Achieving the balance between ROS and antioxidants: when to use the synthetic antioxidants. Oxidative Med. Cell. Longev. 2013, 956792 (2013) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 43.T. Fukai, M. Ushio-Fukai, Superoxide dismutases: Role in redox signaling, vascular function, and diseases. Antioxid. Redox Signal. 15, 1583–1606 (2011) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 44.G. Cheng, S. Lanza-Jacoby, Metformin decreases growth of pancreatic cancer cells by decreasing reactive oxygen species: Role of NOX4. Biochem. Biophys. Res. Commun. 465, 41–46 (2015) [DOI] [PubMed] [Google Scholar]
- 45.N. Li, K. Ragheb, G. Lawler, J. Sturgis, B. Rajwa, J.A. Melendez, J.P. Robinson, Mitochondrial complex I inhibitor rotenone induces apoptosis through enhancing mitochondrial reactive oxygen species production. J. Biol. Chem. 278, 8516–8525 (2003) [DOI] [PubMed] [Google Scholar]
- 46.M. Hassan, H. Watari, A. AbuAlmaaty, Y. Ohba, N. Sakuragi, Apoptosis and molecular targeting therapy in cancer. Biomed. Res. Int. 2014, 150845 (2014) [DOI] [PMC free article] [PubMed] [Google Scholar] [Retracted]
- 47.K. Zibara, Z. Awada, L. Dib, J. El-Saghir, S. Al-Ghadban, A. Ibrik, E.N. Zein, E.M. Sabban, Anti-angiogenesis therapy and gap junction inhibition reduce MDA-MB-231 breast cancer cell invasion and metastasis in vitro and in vivo. Sci. Rep. 5, (12598) (2015) [DOI] [PMC free article] [PubMed]
- 48.K. Kessenbrock, V. Plaks, Z. Werb, Matrix metalloproteinases: Regulators of the tumor microenvironment. Cell 141, 52–67 (2010) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 49.W. Wang, Y. Luo, MicroRNAs in breast cancer: oncogene and tumor suppressors with clinical potential. J. Zhejiang Univ. Sci. B 16, 18–31 (2015) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 50.G.S. Markopoulos, E. Roupakia, M. Tokamani, E. Chavdoula, M. Hatziapostolou, C. Polytarchou, K.B. Marcu, A.G. Papavassiliou, R. Sandaltzopoulos, E. Kolettas, A step-by-step microRNA guide to cancer development and metastasis. Cell. Oncol. 40, 303–339 (2017) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 51.G.C. Guo, J.X. Wang, M.L. Han, L.P. Zhang, L. Li, microRNA-761 induces aggressive phenotypes in triple-negative breast cancer cells by repressing TRIM29 expression. Cell. Oncol. 40, 157–166 (2017) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 52.S. Jiang, H.W. Zhang, M.H. Lu, X.H. He, Y. Li, MicroRNA-155 functions as an OncomiR in breast cancer by targeting the suppressor of cytokine signaling 1 gene. Cancer Res. 70, 3119–3127 (2010) [DOI] [PubMed] [Google Scholar]
- 53.S. Mattiske, R.J. Suetani, P.M. Neilsen, D.F. Callen, The oncogenic role of miR-155 in breast cancer. Cancer Epidemiol. Biomark. Prev. 21, 1236–1243 (2012) [DOI] [PubMed] [Google Scholar]
Associated Data
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Supplementary Materials
Effect of metformin on BC cells viability. MCF-7, MDA-MB-231 and T47D cells were exposed to the culture medium (control) and metformin for 48 hours. Cell viability was estimated after staining with 0.4% (w/v) trypan blue dye. All data are represented as means ± SD, N = 3, *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001 versus control. (PNG 804 kb)
Effect of SOD inhibitor Silver diethyl thiocarbamate on the ROS levels, SOD expression levels, and growth of BC cells. (A) Representative pictures in panel display alterations in DCFH-DA fluorescence intensity after SOD inhibitor treatment. (B) DCFH-DA fluorescence intensities were quantified spectrophotometrically using microplate reader. (C) Silver diethyl thiocarbamate reduces SOD mRNA expression levels in cells. (D) Silver diethyl thiocarbamate reduces proliferation of BC cells. All data are represented as means ± SD, N = 3, *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001 versus control. (PNG 18884 kb)
Quantification of apoptotic nuclei in MCF-7, MDA-MB-231 and T47D BC cells. The apoptotic nuclei were quantified by fluorescence microscopic analysis. All data are represented as means ± SD, N = 3, *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001 versus control. (PNG 355 kb)
(DOCX 13 kb)