Breast cancer is the most commonly occurring cancer and second leading cause of mortality in women. Metformin is a widely prescribed anti-hyperglycemic drug, which is emerging as a potential cancer preventative and treatment agent. However, the mechanisms underlying the suppressive effects of metformin on cancer cell growth and the induction of cancer cell apoptosis are not fully elucidated. The present study aimed to identify the pathways regulated by metformin in two breast cancer cell lines, MDA-MB-231 and MDA-MB-435. Cells were treated with various concentrations of metformin and then evaluated with respect to viability, proliferation, adenosine triphosphate (ATP) and reactive oxygen species (ROS) levels, mitochondrial membrane potential (∆ψm), and the expression of anti- and pro-apoptotic proteins. Metformin caused apoptosis in a concentration- and time-dependent manner, and decreased cell viability and ATP production. Furthermore, metformin induced the generation of ROS and decreased the ∆ψm. Moreover, metformin downregulated the expression of the anti-apoptotic proteins B-cell lymphoma 2 (BCL-2) and myeloid cell leukemia-1, and upregulated the expression of the pro-apoptotic BCL-2-associated X protein in MDA-MB-231 cells. These results demonstrate that the apoptotic and cytotoxic effects of metformin on breast cancer cells are mediated by the intrinsic mitochondria-mediated apoptosis pathway.
Breast cancer is the most common cancer among women worldwide and ranks first and second in cancer mortality rates among women in undeveloped and developed regions, respectively. According to GLOBOCAN 2012 estimates, 1.67 million women were diagnosed with breast cancer in 2012, representing an increase in breast cancer incidence of >20% since 2008 (
Apoptosis, also known as programmed cell death, is a process in living organisms that is necessary for the maintenance of proper development and the elimination of cell damage or excess. Apoptosis is characterized by distinct biochemical and morphological changes, including DNA fragmentation, plasma membrane blebbing and loss of cell volume. There are two major pathways by which apoptotic cell death can be induced: The intrinsic (or mitochondrial) pathway and the extrinsic (or death receptor) pathway. The intrinsic pathway regulates the activity of proteins of the survivin and B-cell lymphoma 2 (BCL-2) families. The latter family includes myeloid cell leukemia-1 (MCL-1), which plays an integral role in cell survival and apoptosis (
Metformin, an oral biguanide drug, has been used widely to treat type 2 diabetes and pre-diabetic conditions for >40 years due to its good tolerability profile and low cost. In addition to its anti-diabetic effect, epidemiological studies and basic research have suggested that metformin may reduce the risk of cancer in diabetic patients (
Metformin and propidium iodide (PI) were purchased from Sigma-Aldrich. (St. Louis, MO, USA). The fluorescent dyes JC-1 and dihydroethidium (DHE) were purchased from Nanjing KeyGen Biotech Co., Ltd. (Nanjing, China). Rabbit anti-MCL-1 (ab32087; 1:500) and anti-BCL-2 (ab32124; 1:1,000) monoclonal antibodies were obtained from Abcam (Cambridge, UK). Rabbit anti-BAX (5023; 1:1,000) monoclonal antibody was purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA), and rabbit anti-β-actin polyclonal antibody (sc-130657; 1:1,000) was obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (BL001A; 1:5,000) and goat anti-rabbit IgG (BL003A; 1:5,000) were purchased from Biosharp (Suzhou, China).
MDA-MB-231 and MDA-MB-435 breast cancer cells were purchased from Shanghai Cell Bank (Shanghai, China). The cells were inoculated in fresh Dulbecco's modified Eagle's medium containing 10% fetal calf serum (both Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 100 U/ml penicillin and 100 mg/l streptomycin (both Sigma-Aldrich). Cultures were maintained at 37°C in a 5% CO2 humidified atmosphere.
Breast cancer cells were cultured in a 96-well plate for 24 h at an initial density of 1×105 cells/well, prior to treatment with various concentrations (1.25, 2.5, 5, 10 and 20 mM) of metformin for 24, 48 or 72 h at 37°C. Subsequently, 20 µl 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) solution (5.0 mg/l; Sigma-Aldrich) was added to each well and the cells were incubated for a further 4 h. The medium was then removed by gentle aspiration and 150 µl dimethyl sulfoxide (Sigma-Aldrich) was added to each well to dissolve the resulting crystals. Absorbance was read at 490 nm using a microplate reader (Synergy HT; BioTek Instruments, Inc., Winooski, VT, USA). Cell viability was assessed by comparison with control cells treated with vehicle alone.
Colony-forming rates of the tumor cells were determined using a colony formation assay. The breast cancer cells were seeded at 500 cells/well in 6-well plates and incubated for 24 h at 37°C. The medium was removed and the cells were then treated with various concentrations of metformin (0, 0.1, 0.5 and 2.0 mM) using standard cell culture conditions under a 5% CO2 humidified atmosphere at 37°C. After 8 days, the dishes were washed twice with phosphate-buffered saline (PBS), fixed with paraformaldehyde (Sigma-Aldrich) at −20°C for 10 min, and then stained with crystal violet (Thermo Fisher Scientific, Inc.).
Cellular ATP levels were determined by a luciferase-based assay with an ATP Bioluminescence Assay kit (Merck Millipore, Darmstadt, Germany), according to the manufacturer's protocol. Briefly, breast cancer cells (2×106) were seeded in each well of a 24-well plate and allowed to reach the exponential growth phase prior to being treated with various concentrations of metformin (10, 20 and 40 mM) for 24 h at 37°C. After 5 h, the cells were harvested and centrifuged at 10,000 × g for 5 min at 4°C. Supernatants (100 µl) were mixed with 100 µl ATP detection working solution in a white 96-well plate. Measurements were obtained using a luminometer (GloMax® 96 Microplate Luminometer; Promega Corporation, Sunnyvale, CA, USA) at an emission maximum of ~560 nm for 300 sec.
The ∆ψm was assessed using the JC-1 Apoptosis Detection kit (Beyotime Institute of Biotechnology, Jiangsu, China). Briefly, cells were plated in a 12-well plate at a density of 2×106 cells/well and treated with metformin (5 mM) for 24 h at 37°C. Cells were washed once with PBS and incubated at 37°C for 30 min in medium containing 0.5 ml JC-1. Then, the supernatant was removed and cells were rinsed twice with JC-1 staining buffer. Fluorescence images were observed within 30 min using a fluorescent microscope (Eclipse Ti-U; Nikon Corporation, Tokyo, Japan). A decline in the ratio of red to green fluorescence indicated a loss of ∆ψm.
The determination of ROS levels was based on the oxidation of DHE. Breast cancer cells were seeded at a density of 2×105 cells/well in a six-well plate for 24 h and incubated with 20 mM metformin for 1, 3 and 6 h. The cells were then treated with DHE (5 mM, Beyotime Institute of Biotechnology) for 20 min at 37°C in the dark. The cells were then washed twice and harvested in PBS. The fluorescence of DHE was detected with a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) with excitation at 488 nm and emission at 530 nm.
Apoptosis of the breast cancer cells was determined using a PI apoptosis detection kit (Nanjing KeyGen Biotech Co., Ltd.) according to the manufacturer's protocol. Briefly, breast cancer cells (2×106) were plated in each well of a 24-well plate and allowed to reach the exponential growth phase before being treated with various concentrations of metformin (5, 10 and 20 mM) for 24 h. The cells were then harvested and collected by centrifugation at 1,500 × g for 10 min at 4°C, and resuspended in 200 µl ice-cold binding buffer. The cell suspensions were incubated with 5 µl PI for 10 min at room temperature in the dark, and the percentage of apoptotic cells was measured by flow cytometry (Accuri™ C6; BD Biosciences). For caspase inhibitor assays, the cells were pretreated with a pan-caspase inhibitor (z-VAD-FMK; Sigma-Aldrich, Shanghai, China; 20 µM) for 2 h and then treated with various concentrations of metformin (2.5, 5, 10 and 20 mM) for an additional 24 h. The extent of apoptosis was then determined using the MTT assay.
MDA-MB-231 cells treated with metformin (5 mM) were collected at various time points (0, 6, 16 and 24 h), washed twice with ice-cold PBS, and incubated in radioimmunoprecipitation assay protein lysis buffer (Beyotime Institute of Biotechnology) for 30 min at 4°C. The lysates were centrifuged at 13,000 × g for 10 min at 4°C. The concentrations of total lysate protein were detected by a standard Bradford assay (Bio-Rad Laboratories, Inc., San Diego, CA, USA), resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Beyotime Institute of Biotechnology), and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Inc.). The nitrocellulose membranes were blocked with 5% non-fat milk at room temperature for 1 h and then incubated overnight at 4°C with rabbit anti-BCL-2 (1:1,000), anti-MCL-1 (1:500), anti-BAX (1:1,000) and anti-β-actin (1:1,000) primary antibodies. After washing the membranes three times for 10 min each with Tris-buffered saline containing Tween-20, they were incubated with HRP-conjugated secondary antibodies (1:5,000 dilution). Proteins were visualized using an Enhanced Chemiluminescence-Plus kit (Nanjing KeyGen Biotech Co., Ltd.).
All experiments were performed at least in triplicate. Data are expressed as the means ± standard error of the mean (SEM). Statistical analyses were performed using one-way analysis of variance with SPSS software version 20.0 (IBM SPSS; Armonk, NY, USA). Differences were considered statistically significant at P<0.05.
MDA-MB-231 and MDA-MB-435 cells were treated with metformin at different concentrations (1.25, 2.5, 5, 10 or 20 mmol/l) for 24, 48 or 72 h. MTT and colony formation assays were then conducted to assess cell proliferation. As shown in
The ∆ψm and its loss are near-universal hallmarks of, and critical steps for, subsequent cell death (
To study the effect of metformin on cellular ATP production, the two breast cancer cell lines were treated with different concentrations of metformin (10, 20 and 40 mM) for 24 h. The ATP levels of metformin-treated MDA-MB-231 and MDA-MB-435 cells were reduced compared with those in the respective control group. The lowest level was observed at a metformin concentration of 40 mM in MDA-MB-435 cells (
As ROS generation is a common after-effect of a reduction in ∆ψm (
The type of cell death induced in breast cancer cells treated with metformin was determined by PI staining. Following incubation with metformin for 24 h, the extent of apoptosis in MDA-MB-231 cells markedly increased from 7.0% with 5 mM metformin to 39.6% with 20 mM metformin. MDA-MB-435 cells were less sensitive to metformin, as the percentage of cells undergoing apoptosis only increased from 2.9 to 17.7% (
To confirm whether the metformin-induced cell death observed in MDA-MB-231 cells was a result of apoptosis, the pan-caspase inhibitor z-VAD-FMK was used to examine the role of caspases in the process. The results of the MTT assay revealed that the viability of MDA-MB-231 cells was significantly attenuated by z-VAD-FMK, confirming that the process is caspase-dependent (
To confirm whether metformin-induced apoptosis activates the mitochondrial pathway, MDA-MB-231 cells were incubated in the absence or presence of metformin and then harvested for western blot analyses. Since the mitochondrial pathway appeared to be involved in the induction of intrinsic apoptosis, the levels of anti- and pro-apoptotic proteins that dysregulate the mitochondrial balance were measured. Incubation of cells with metformin time-dependently upregulated the levels of the pro-apoptotic protein BAX and downregulated the levels of the anti-apoptotic proteins BCL-2 and MCL-1 (
Recent studies have shown that metformin exerts antitumor effects
Apoptosis suppresses cell proliferation (
Metformin is a partial inhibitor of complex 1 of the mitochondrial electron transport chain (
The potency of metformin against cancer cells has been linked to the generation of ROS (
In conclusion, the present study demonstrated that metformin treatment effectively reduced cell viability and induced apoptotic cell death in human breast cancer cells. Metformin also induced activation of the caspase-dependent pathway, accumulation of ROS, and a reduction in ΔΨm and ATP production in human breast cancer cells. These results suggest that metformin-induced apoptosis is mediated by the accumulation of ROS via the mitochondria-mediated apoptotic pathway. This study provides further support for the development of metformin as a novel therapeutic agent for the treatment of human breast cancer.
The present study was supported by a grant from the Natural Science Foundation of China (grant no. 81272739).
Metformin-induced cytotoxicity of breast cancer cells. (A) MDA-MB-231 and MDA-MB-435 cells were treated with metformin (1.25, 2.5, 5, 10 or 20 mmol/l) for 24, 48 or 72 h. Cell viability was analyzed using the MTT assay. The results revealed that metformin inhibited the growth of breast cancer cells in a dose- and time-dependent manner. Data are expressed as the mean ± standard error of the mean. *P<0.05 vs. the control (0 mmol/l metformin). (B) MDA-MB-231 and MDA-MB-435 cells were cultured with various concentrations of metformin (0, 0.1, 0.5 and 2 mmol/l) for 24 h and cell colony formation was analyzed. Colony formation by MDA-MB-231 and MDA-MB-435 cells was inhibited by metformin in a dose-dependent manner. MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
Metformin downregulated the mitochondrial membrane potential (∆ψm) and ATP production. (A) MDA-MB-231 and MDA-MB-435 cells were treated with 5 mmol/l metformin for 24 h. Changes in ∆ψm were monitored by loading with the fluorescent probe JC-1 and analyzed by fluorescence microscopy. Images of the cells were then captured under a light microscope (magnification, ×200). Metformin increased the green to red fluorescence intensity ratio, suggesting that ∆ψm was reduced in metformin-treated breast cancer cells. (B) MDA-MB-231 and MDA-MB-435 cells were incubated with various concentrations of metformin (10, 20 or 40 mmol/l) for 24 h. Absorbance was measured using a luminometer. Metformin reduced ATP levels in MDA-MB-435 cells in a concentration-dependent manner. In MDA-MB-231 cells, ATP levels were maximally reduced at 20 mmol/l metformin and were not further decreased by 40 mmol/l. Data are expressed as the mean ± standard error of the mean. *P<0.05 vs. the control.
ROS production and apoptosis in MDA-MB-231 and MDA-MB-435 cells treated with metformin. (A) ROS were measured in MDA-MB-231 and MDA-MB-435 cells treated with metformin (20 mmol/l) for 1, 3 and 6 h. Cells were loaded with DHE (5 mmol/l) and viewed using fluorescence microscopy. In the graph, cell number is presented on the y-axis and fluorescence intensity on the x-axis. The fluorescence intensity gradually increased over time, suggesting significant generation of intracellular ROS in metformin-treated cells compared with untreated control cells. (B) MDA-MB-231 and MDA-MB-435 cells were cultured with various concentrations of metformin (0, 5, 10 and 20 mmol/l) for 24 h, then loaded with propidium iodide before flow cytometric analysis to assess apoptosis. ROS, reactive oxygen species; DHE, dihydroethidium.
Metformin-induced apoptosis occurs via the mitochondria-mediated apoptotic pathway. (A) MDA-MB-231 cells were treated with metformin (0, 2.5, 5, 10 and 20 mmol/l) with or without z-VAD-FMK (20 µM) for 24 h. Viability was then assessed using the MTT assay. Data are expressed as the means ± standard error of the mean (*P<0.05 vs. the control). (B) Whole-cell lysates from MDA-MB-231 cells treated with 5 mmol/l metformin for 6, 16, and 24 h were used to measure apoptosis-related proteins by western blot analyses. The blots shown are representative of three independent experiments. BAX protein levels were time-dependently increased. BCL-2 and MCL-1 levels were time-dependently decreased. BAX, BCL-2-associated X protein; BLC-1, B-cell lymphoma 2; MCL-1, myeloid cell leukemia-1.