Corema album Leaves Mediate DNA Damage in Triple-Negative Breast Cancer Cells

Abstract

Corema (C.) album is a shrub endemic to the Atlantic coast and has been described as yielding beneficial effects for human health. Nevertheless, studies concerning the bioactivity of C. album leaves are scarce. This study aims at investigating the anticancer potential and mode of action, of an hydroethanolic extract of C. album leaves (ECAL) on triple-negative breast cancer. This is a poor survival breast cancer subtype, owing to its high risk of distant reappearance, metastasis rates and the probability of relapse. The ECAL ability to prevent tumor progression through (i) the inhibition of cell proliferation (cell viability); (ii) the induction of apoptosis (morphological changes, TUNEL assay, caspase-3 cleaved) and (iii) the induction of DNA damage (PARP1 and γH2AX) with (iv) the involvement of NF-κB and of ERK1/2 pathways (AlphaScreen assay) was evaluated. ECAL activated the apoptotic pathway (through caspase-3) along with the inhibition of ERK and NF-κB pathways causing DNA damage and cell death. The large polyphenolic content of ECAL was presumed to be accountable for these effects. The extract of C. album leaves can target multiple pathways and, thus, can block more than one possible means of disease progression, evidencing the anticancer therapeutic potential from a plant source.

Keywords: ERK 1/2 pathway; NF-κB pathway; anticancer; maritime plant; natural product.

Figure 1

Inhibition of cell proliferation induced by hydroethanolic extract of C. album leaves (ECAL) in MDA-MB-231 and MCF-12A cells. Cells were treated with increasing concentrations of ECAL (1.7–1730 μg/mL) for (A) 24 h, (B) 48 h and (C) 72 h. Label-free kinetic live monitoring of cell proliferation was performed using a LionheartFX automated microscope with the direct image acquisition of cells in microplates at 0, 24, 48 and 72 h of the post-addition of the tested compounds.

Figure 2

Morphological changes induced by increasing concentrations of the hydroethanolic extract of C. album leaves (ECAL) (1.7–1730 μg/mL) incubated for 24 h in (A) MDA-MB-231 cells or (B) MCF-12A cells. Upper panel—representative images from five independent experiments obtained under an objective lens of a phase-contrast of the Lionheart microscope; lower panel—representative images from five independent experiments with nuclei stained with Hoechst 33258 (blue) obtained under an objective lens of a Lionheart microscope. Scale bar = 100 μM.

Figure 3

DNA damage induced by the hydroethanolic extract of C. album leaves (ECAL). (A) TUNEL staining for apoptosis in MDA-MB-231 cells incubated for 24 h. (A1) with the increasing concentrations of ECAL (1.7–1730 μg/mL); (A2) with DNAse (3000 U/mL); (A3) without treatment (control). Blue nuclear stain and green TUNEL stain. TUNEL-positive apoptotic cells (arrows) are those in which the green and blue colocalize to the same cell. Scale bar = 100 μM; (B) immunoblot images of proteins γH2AX, PARP1 and Caspase-3 cleaved (histone H3, as the internal control) wherein the fold changes have been presented below each panel.

Figure 4

Hydroethanolic extract of C. album leaves (ECAL) effects on ERK and NF-kB pathways in MDA-MB-231 cells. (A) Phosphorylated ERK1/2 and (B) Phosphorylated NF-κB, measured in the presence of negative control, positive control (angiotensin II (Ang II), 300 nM) and with increasing concentrations of ECAL (1.7–1730 μg/mL). Significant differences from the values obtained in control: * p < 0.05 (One-way ANOVA followed by post hoc Dunnett’s multicomparison t-test.