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Antitumor Effects of Naturally Occurring Cardiac Glycosides Convallatoxin and Peruvoside on Human ER+ and Triple-Negative Breast Cancers

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Antitumor Effects of Naturally Occurring Cardiac Glycosides Convallatoxin and Peruvoside on Human ER+ and Triple-Negative Breast Cancers

Vivek Kaushik et al. Cell Death Discov.

Abstract

Breast cancer is second most prevalent cancer in women, and the second only to lung cancer in cancer-related deaths. It is a heterogeneous disease and has several subtypes based on the presence or absence of hormone receptors and/or human epidermal growth factor receptor 2 (HER2). Hormone receptor-positive and HER2-enriched cancers can be targeted using hormone and HER2-targeting therapies such as trastuzumab or lapatinib. However, triple-negative breast cancers (TNBCs) do not express any of the receptors and therefore are resistant to most targeted therapies, and cytotoxic chemotherapies are the only viable option available for the treatment of TNBCs. Recently, cardiac glycosides (CGs) have emerged as potential anticancer agents that impart their antiproliferative effect by targeting multiple pathways. In this study our aim was to evaluate anticancer effects of two naturally occurring CGs, Convallatoxin (CT) and Peruvoside (PS), on ER+ and TNBCs cells. CT and PS demonstrated dose- and time-dependent cytotoxic effect on MCF-7 cells, which was further supported by loss of colony formation on drug treatment. CT and PS arrested MCF-7 cells in the G0/G1 phase and reduced the viability of MCF-7-derived mammospheres (MMs). Interestingly, while CT and PS imparted cell death in TNBCs cells from both Caucasians (MDA-MB-231 cells) and African Americans (MDA-MB-468 cells) in a dose- and time-dependent manner, the drugs were much more potent in MDA-MB-468 as compared with TNBC MDA-MB-231 cells. Both drugs significantly inhibited migration and invasion of both MCF-7 and MDA-MB-468 cells. An assessment of intracellular pathways indicated that both drugs were able to modulate several key cellular pathways such as EMT, cell cycle, proliferation and cell death in both cell types. Our data suggest a promising role for CGs in breast cancer treatment specifically in targeting TNBCs derived from African Americans, and provides impetus for further investigation of the anticancer potential of this class of drugs.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
CT and PS inhibit the viability of ER+ MCF-7 breast cancer cells in a dose- and time-dependent manner. MCF-7 cells were incubated in serum-free media with indicated doses of CT and PS for 24 (left panel) or 72 h (right panel). Cell viability was assessed by the MTT assay. Plots show mean values±S.E. of quadruplicates determinations of three or more experiments at P<0.05.
Figure 2
Figure 2
CT and PS prevent MCF-7 colony formation and arrest cell cycle in the G0/G1 stage. MCF-7 cells were plated (200 cells/well), allowed to adhere overnight, treated with the indicated concentrations of drugs for 72 h in serum-free media. After treatment, cells were allowed to form colony in media containing serum (10%) for 7–10 days. Representative images of colonies (a) CT treatment, (b) PS treatment. (c) Dose–response curves for CT and PS. (d) Cells were synchronized in the G0/G1 phase by incubating in serum-free media for 72 h followed by drug treatments in complete media for 24 h. Cells were fixed with 70% ethanol for at least 24 h at −20 °C, stained with propidium iodide and analyzed by flow cytometry. (e) Quantitative representation of cell cycle data. (f) Western blot analysis of cell cycle regulating proteins CDK4 and Cyclin D1. Data are the mean values ±S.E. of at least three independent experiment performed in triplicates at P<0.05.
Figure 3
Figure 3
CT and PS decrease viability of breast cancer mammosphers (MSs). MSs were generated as described in the Material and Methods section. Representative images of treated MSs clearly demonstrates reduction of viability of MSs on drug treatment: (a) Untreated (Ntx), (b) CT 50 nM, (c) PS 50 nM. (d) Dose–response of CT and PS on MSs was measured by CCK-8 essay. Data are the mean values±S.E. of at least three independent experiment performed in triplicates at P<0.05.
Figure 4
Figure 4
CT and PS inhibit the viability of triple-negative breast cancer cells (TNBCs) MDA-MB-231 and MDA-MB-468 cells in a dose and time-dependent manner. TNBCs cells were incubated in serum-free media with indicated doses of CT and PS for 24 or 72 h, respectively, and cell viability was assessed by the MTT assay. (a) MDA-MB-468 cells and (b) MDA-MB-231 cells. Plots show mean values±S.E. of quadruplicates determinations of three or more experiments at P<0.05.
Figure 5
Figure 5
CT and PS inhibit migration of MCF-7 and MDA-MB-468 breast cancer cells. Representative images (×20) of migratory cells in (a) MCF-7 and (b) MDA-MB-468 cells after treatment with the indicated concentration of Convallatoxin (CT) or Peruvoside (PS). The plot shows the mean values±S.E. of migratory cells from five pictures each taken under a light microscope for two independent experiments at P<0.05.
Figure 6
Figure 6
CT and PS inhibit invasion of MCF-7 and MDA-MB-468 breast cancer cells. Representative images (×20) of invasive cells in (a) MCF-7 and (b) MDA-MB-468 cells after treatment with the indicated concentration of Convallatoxin (CT) or Peruvoside (PS). The plot shows the mean values ±S.E. of invasive cells from five pictures each taken under a light microscope for two independent experiments at P<0.05.
Figure 7
Figure 7
CT and PS modulate the expression of key signaling pathways. Cell lysates (30 μg) collected after 24 h of drug treatment were analyzed for the indicated proteins within the (a) EMT, (b) proliferation and (c) cell death pathways by western blotting. Blots were re-probed for β-actin to confirm equal loading of samples. Representative blots from three independent experiments are shown.

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