Calcium channel upregulation has been implicated in cancer cell proliferation and progression including in breast cancer. Fortunately, the function of calcium channels can be manipulated pharmacologically using calcium channel blockers (CCBs). Amlodipine, a dihydropyridine CCB, has been demonstrated to exert cytotoxic effects in several types of cancers. The present study evaluated the effects of amlodipine on proliferation, caspase activation, colony formation, and invasion of human breast cancer cells. Cell viability was assessed using a colorimetric MTT assay. An Apo-ONE® caspase-3/7 assay was used to measure caspase-3/7 levels. Cell invasion was evaluated using Matrigel invasion chambers. The expression of phospho-(p-)ERK1/2, Bcl-2, and integrin β1 proteins were analyzed using western blotting. A one-way ANOVA with a post-hoc Tukey's multiple comparison tests was used for statistical analysis. Amlodipine significantly inhibited the growth of both MDA-MB-231 and MCF-7 human breast cancer cells in a dose-dependent manner and inhibited colony formation of MCF-7 cells, and this was accompanied by the downregulation of p-ERK1/2 in MDA-MB-231 cells. In addition, treatment with amlodipine resulted in increased caspase-3/7 levels in MDA-MB-231 cells, which was accompanied by the downregulation of the anti-apoptotic protein, Bcl-2. Moreover, amlodipine impaired the invasive abilities of MDA-MB-231 cells, and integrin β1 expression was concurrently downregulated. The present study illustrates the anticancer effects of amlodipine on breast cancer proliferation, colony formation, and invasion
Globally, breast cancer is the most prevalent cancer, accounting for 11.7% of all cancer cases and 6.9% of cancer deaths (
Calcium channel blockers (CCB) have been implicated as anti-cancer molecules in several types of human cancers. For example, amlodipine, a dihydropyridine CCB, has been shown to induce apoptosis, resulting in cell cycle arrest, and suppress the proliferation of cancerous cells in several studies (
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 (
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 (
Assessing anchorage-dependent growth of breast cancer cells was performed using colony formation assays as previously described (
Invasion assays were performed using Corning BioCoat Matrigel Invasion Chambers (Corning Inc.) as described previously (
To assess the effects of amlodipine on the protein expression levels of downstream targets, western blotting was performed as previously described (
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
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 (
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 (
To mimic the
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 (
Voltage-activated calcium channels are widely distributed in all types of human cells (
To ascertain the underlying mechanism(s) of amlodipine-induced growth suppression, breast cancer cellular apoptosis was assessed by measuring caspase-3/7 activity. The results showed that amlodipine induced caspase-3/7 activity in MDA-MB-231 cells, which may contribute to caspase-dependent apoptosis. Although this finding was limited by a lack of flow cytometry analysis to confirm the occurrence of apoptosis, activation of caspase-3/7 pathways was accompanied by downregulation of the anti-apoptotic protein Bcl-2, which strongly indicated that caspase-dependent apoptosis occurred in the breast cancer cells following amlodipine treatment. The Bcl-2 gene promotes cell survival and protects cells against apoptosis. High expression of Bcl-2 is associated with lower apoptosis-mediated death and contributes to resistance to chemotherapy. Moreover, Bcl-2 protein expression is typically altered in breast cancer cells (
To further illustrate the antiproliferative effect of amlodipine, the tumorigenic ability of breast cancer cells was assessed using colony formation assays whilst being treated with amlodipine. The inhibitory effects of amlodipine on colony formation were notable in MCF-7 cells and in agreement with previous findings in gastric cancer (
Previous studies have implicated calcium channels in breast cancer cell adhesion and invasion (
In conclusion, the results of the present study showed that amlodipine exerted anticancer effects on cell proliferation, colony formation, and invasion, and they were, at least in part, achieved by the inhibition of p-ERK1/2, integrin β1, and Bcl-2 expression and activation of caspase-3/7, indicating the induction of caspase-dependent apoptosis. This study highlights amlodipine as a potential therapeutic agent for the management of breast cancer and may provide novel insights for future research on the effects of amlodipine in the sensitization of breast cancer cells to chemotherapy.
We would like to acknowledge Jordan University of Science and Technology for providing sabbatical leave to Dr. Mohammad A. Y. Alqudah. We are grateful to Dr. Moh'd Shara for his kind revision of the language proficiency of this manuscript and to Prof. Omar Khabour for providing us with the total ERK1/2 antibody (Jordan University of Science and Technology, Jordan).
The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.
MAYA was involved in the study conception and design. MAYA, RAS and MA performed the experiments. MAYA, RAS, and KHA conducted the data analysis. MAYA and RAS wrote the first draft of the manuscript. All authors revised the manuscript. All authors read and approved the final manuscript.
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The authors declare that they have no competing interests.
Effect of amlodipine on cell proliferation and caspase-3/7 activity in breast cancer cells. Percentage cell viability of (A) MDA-MB-231 and (B) MCF-7 cells after 48 h of amlodipine treatment. (C) Percentage of caspase-3/7 activity in in MDA-MB-231 cells after 48 h of amlodipine treatment. Data are presented as the mean ± SEM of three independent experiments. *P<0.05, **P<0.01.
Effect of amlodipine on colony formation and invasion of breast cancer cells. (A) Representative images of colony formation and (B) quantitative analysis of the area of the colonies formed by MCF-7 cells following treatment with amlodipine (2.5, 5 or 10 µM) compared with the control treated cells. Scale bar, 100 µm. (C) Quantitative analysis of cell invasion percentage and (D) representative microscopic images for cell invasion by MDA-MB-231 cells treated with amlodipine (5 or 10 µM) compared with the control treated cells. Scale bar, 200 µm. **P<0.01.
Effects of amlodipine treatment on the protein expression levels of Bcl-2, p-ERK1/2 and integrin β1 in breast cancer cells. Western blotting was used to examine the changes in the expression levels of the relevant proteins in MCF-7 and MDA-MB-231 cells following 48-h treatment with 1-10 µM amlodipine. Western blot analyses and quantifications with statistical analysis are shown. *P<0.05, **P<0.01. p-, phospho.