Triple-negative MDA-MB-231 and luminal MCF-7 breast cell lines were purchased from the American Type Culture Collection (ATCC). Cells were cultured in RPMI-1640 media supplemented with 10% FBS and 1% penicillin/streptomycin in a humidified incubator with 5% CO₂ at 37˚C. DMSO was used as a solvent to prepare amlodipine stocks (Tocris Bioscience), with a final DMSO concentration of <0.1% in all experiments.

To evaluate the effects of amlodipine on breast cancer cell viability, the colorimetric MTT cell proliferation assay (ATCC) was performed as described previously (16). Briefly, 1x10⁴ cells/well were cultured in a 96-well plate and incubated overnight. Then, cells were treated with several concentrations of amlodipine or with DMSO as a control. After 48 h of treatment, cells were incubated at 37˚C for 4 h with MTT solution at a final concentration of 500 µg/ml. To solubilize formazan crystals, 100 µl DMSO was added to each well. The optical density was measured at 490 nm on a microplate reader (BioTek Instruments, Inc.). The results are expressed as a percentage of viable cells normalized to vehicle-treated cells using the following equations: % Of viable cells in each well=(Absorbance _treatment/Average of Absorbance _{vehicle in 4 replicates})x100; and % of viable cells for each treatment concentration=Average of normalized % of viable cells in 4 treatment replicates.

An Apo-ONE^® homogeneous caspase-3/7 assay (Promega Corporation) was used to assess the effects of amlodipine on the induction of caspase-3/7 activities in MDA-MB-231 cells as described previously (17). Briefly, cells were plated at a density of 1x10⁴ cells/well in a 96-well black plate. After attachment, cells were treated with several concentrations of amlodipine or with DMSO as a control. After 48 h of treatment, the caspase-3/7 reagent was added to each well in a 1:1 ratio with the sample volume at room temperature. After 3 h of incubation, enzyme activity was analyzed using a synergy 2 multi-mode microplate reader (Biotek Instruments, Inc.) at excitation and emission wavelengths of 499 nm and 521 nm, respectively.

Assessing anchorage-dependent growth of breast cancer cells was performed using colony formation assays as previously described (18,19). MCF-7 cells were plated at a low density (2x10³ cells/flask) in T25 flasks and incubated for 24 h, then treated with amlodipine or DMSO as a control. Culture media was replaced every 3 days. Following 3 weeks of incubation, PBS was used to wash the cells before fixing them with pre-cooled (1:1) methanol/acetone at -20˚C for 15 min. After staining with 0.1% crystal violet for 5 min at room temperature, the colonies that had formed were visualized using a light microscope (x4 magnification).

Invasion assays were performed using Corning BioCoat Matrigel Invasion Chambers (Corning Inc.) as described previously (20,21). Cells were resuspended in serum-free media with various concentrations of amlodipine (0, 5 or 10 µM), and a chemotactic serum gradient was generated by placing media supplemented with 10% FBS in the bottom chambers. After 24 h, the invading cells were fixed with ice-cold ethanol at -20˚C for 15 min, stained with 0.1% crystal violet for 5 min at room temperature, visualized using a light microscope (x4 magnification) and counted using ImageJ (version 1.53q; National Institutes of Health). The results are expressed as a percentage of invading cells in treatment groups relative to the control group.

To assess the effects of amlodipine on the protein expression levels of downstream targets, western blotting was performed as previously described (21). Briefly, cells were plated at a density of 5x10⁴ cells/well into a 6-well plate. The following day, the cells were treated with amlodipine (1-25 µM) or DMSO as a control for 48 h. After cell washing with ice-cold PBS, cells were lysed in RIPA buffer (Thermo Fisher Scientific, Inc.) containing protease inhibitor (150 µl/well for 30 min on ice). Cell lysates were transferred into Eppendorf tubes and then centrifuged at 13,000 x g for 5 min at 4˚C. Proteins were quantified in all collected supernatants using a BCA assay. Protein samples were loaded in equal amounts (35 µg per lane) with one lane for 10 µl of the protein ladder into 10% polyacrylamide gels in tris-glycine buffer and run at 200 mv for 40 min at room temperature. After the proteins had been resolved, they were transferred to nitrocellulose membranes, incubated with primary antibodies (all 1:1,000 dilution) for 2 h at room temperature against phospho (p-)ERK1/2 (Cell Signaling Technology, Inc.; cat. no. 5726), ERK1/2 (Cell Signaling Technology, Inc.; cat. no. 9102), Bcl-2 (Cell Signaling Technology, Inc.; cat. no. 3498), and integrin β1 (Cell Signaling Technology, Inc.; cat. no. 4706). GAPDH was used as the loading control (Cell Signaling Technology, Inc.; cat. no. 5174). After washing with TBST buffer, membranes were incubated with the secondary horseradish peroxidase-conjugated antibodies (1:1,000) for 1 h at room temperature [anti-rabbit IgG (cat. no. 7074) or anti-mouse IgG (cat. no. 7076)]. An enhanced chemiluminescent detection kit was used to visualize the immunoreactive protein bands using the Montreal Biotech Fusion Pulse 6 imaging system (Montreal Biotech Inc.). All experiments were repeated three times.

Data were analyzed using GraphPad Prism version 9 (GraphPad Software, Inc.). A one-way ANOVA followed by a Tukey's multiple comparison test was used to compare the difference between multiple groups. The half-maximal inhibitory concentration (IC₅₀) values were obtained by applying a nonlinear regression curve fit analysis. P<0.05 was considered to indicate a statistically significant difference. Data are presented as the mean ± SEM.

The in vitro biological effects of amlodipine treatment on MDA-MB-231 and MCF-7 cell proliferation are shown in Fig. 1. Amlodipine treatment reduced MDA-MB-231 and MCF-7 cell viability in a dose-dependent manner. In MDA-MB-231 cells, treatment with 2.5-50 µM amlodipine significantly reduced cell viability compared with the control-treated cells (P<0.05, Fig. 1A). In MCF-7 cells, 5-50 µM amlodipine significantly reduced cell viability compared with the control-treated cells (P<0.05, Fig. 1B). The IC₅₀ values for amlodipine in MDA-MB-231 and MCF-7 cells were 8.66 and 12.60 µM, respectively. These findings suggest a cytotoxic effect of amlodipine on breast cancer cells.

Since amlodipine reduced breast cancer cell viability, the potential underlying mechanisms of the growth suppression were assessed by analyzing caspase-3/7 activity, which is a well-established marker of apoptosis (22). The results revealed that amlodipine treatment (2.5-10 µM) significantly increased caspase-3/7 activity compared with the control treatment in MDA-MB-231 cells (P<0.05, Fig. 1C) and thus highlighted caspase activation as a potential mechanism underlying the cytotoxic effects of amlodipine. However, caspase-3/7 activity was not assessed in the MCF-7 cells since they do not express caspase-3, and thus caspase activation may be underestimated (23,24).

To further determine the anticancer effects of amlodipine in cell proliferation, whether amlodipine could modulate anchorage-dependent growth was assessed using colony formation assays. Although it may be considered a limitation of the present study for MCF-7 cells to have a high capacity to form colonies compared to MDA-MB-231 cells (25), the MCF-7 clonogenic ability was still assessed following amlodipine treatment. The effect of amlodipine on colony formation of MCF-7 cells is shown in Fig. 2A and B. Amlodipine markedly inhibited colony formation in a dose-dependent manner in MCF-7 cells. Treatment with 2.5-10 µM amlodipine significantly decreased the colony size compared with the control (P<0.05). These findings indicate the ability of amlodipine to suppress the clonogenic proliferation of breast cancer cells over a prolonged period of time.

To mimic the in vivo process of cell invasion through the extracellular matrix, the effects of amlodipine on the invasive abilities of MDA-MB-231 cells were evaluated using Matrigel invasion chambers. As shown in Fig. 2C and D, amlodipine significantly suppressed MDA-MB-231 cell invasion in a dose-dependent manner. Treatment with 5 and 10 µM amlodipine significantly reduced MDA-MB-231 invasiveness by >60 and 90% compared with the control-treated cells, respectively. Since MCF-7 cells are not highly invasive cells (25), invasion assays were not performed using these cells, which is considered a limitation of our study. These results provide robust evidence of the anti-invasive effects of amlodipine on breast cancer cells in vitro.

To further shed light on the potential signaling molecules driving the anticancer effects of amlodipine on breast cancer cells, the expression levels of key proteins involved in cell proliferation, apoptosis, and invasion were evaluated using western blotting. The results indicated that amlodipine reduced the protein expression levels of the anti-apoptotic protein Bcl-2, in both MDA-MB-231 and MCF-7 cells compared with the control (Fig. 3). As MCF-7 cells are not highly invasive cells, p-ERK1/2 and integrin β1 protein expression levels were only assessed in MDA-MB-231 cells. Amlodipine treatment reduced ERK1/2 phosphorylation in MDA-MB-231 cells compared with the control treatment. Moreover, amlodipine reduced the protein expression levels of integrin-β1 in MDA-MB-231 cells compared with the control treatment. These findings may partially explain the possible molecular drivers of the anti-cancer effects of amlodipine.