The selective anti-proliferative and pro-apoptotic effect of A. cherimola on MDA-MB-231 breast cancer cell line

Abstract

Background: Herbal medicines have been a major target for numerous studies through the past years as an alternative treatment for cancer, mainly due to their minimal effects on normal healthy cells. Annona cherimola, popularly known as Cherimoya, is an edible natural fruit rich in phytochemical components and known to possess various biological activities. Previous studies have reported the anti-cancerous effect of A. cherimola ethanolic leaf extract (AELE) on leukemia. This study aims at studying the potential anti-cancer activity of this extract in vitro in two different breast cancer cell lines, namely MDA-MB-231 and MCF-7, in addition to investigating its toxicity on normal mesenchymal stem cells.

Methods: The anti-proliferative effect of AELE was evaluated via cell viability assay. Propidium iodide staining, Cell Death Detection ELISA and flow cytometry analysis of Annexin V binding were used to assess cell cycle progression, DNA fragmentation and apoptosis induction, respectively. Protein expression was determined via Western Blot analysis to decipher the underlying apoptotic molecular mechanism induced upon AELE treatment.

Results: The anti-proliferative effect of the extract was found to be selective on the triple-negative breast cancer cell line (MDA-MB-231) in a time- and dose-dependent manner with an IC₅₀ of 390.2 μg/mL at 48 h, with no cytotoxic effects on normal murine mesenchymal stem cells. The pro-apoptotic effect was confirmed by the increase in cellular and DNA fragmentation, flipping of the phosphatidylserine moiety to the outer leaflet, and the increase in Annexin V binding. The underlying molecular mechanism revealed the involvement of the mitochondrial pathway, as shown by alterations in mitochondrial permeability and the upregulation of cytochrome c expression.

Conclusion: All the data presented in our study suggest that AELE exhibits a selective anti-proliferative and pro-apoptotic effect on the chemo-resistant MDA-MB-231 breast cancer cells, providing evidence for the anti-tumor effects of A. cherimola.

Keywords: Annona cherimola; Apoptosis; Breast Cancer; MDA-MB-231; Medicinal foods.

Fig. 1

The selective effect of AELE on breast cancer cell proliferation using MTS assay. Percent proliferation of cells upon increasing AELE treatment. Bar graph showing the percent proliferation of MDA-MB-231 (a), MCF-7 (b) and Mesenchymal Stem Cells (MSCs) (c) treated with AELE. AELE showed a significant dose- and time- dependent inhibition of MDA-MB-231 cell proliferation and no inhibitory effect on MSCs isolated from rat bone marrow upon increasing AELE concentration. Dunnett’s test was used to compare the different concentrations to the control, and Sidak’s test was used to compare different time points of each concentration. Two-way ANOVA was used to determine the significant differences reported with * indicating a p-value: 0.01 < p < 0.05, ** indicating a p- value: 0.001 < p < 0.01, and *** indicating a p-value: p < 0.001

Fig. 2

The effect of AELE on DNA fragmentation in MDA-MB-231 cells using cell cycle analysis. Cell cycle analysis of MDA-MB-231 cells treated with increasing concentrations of AELE and a positive control treated with topotecan for 24 h, shows a dose-dependent increase in the pre-G₀/G₁ stage. Dunnett’s test was used to compare the different concentrations to the control. Two-way ANOVA was used to determine the significant differences reported with * indicating a p-value: 0.01 < p < 0.05, ** indicating a p- value: 0.001 < p < 0.01, and *** indicating a p-value: p < 0.001

Fig. 3

The quantitative effect of AELE on induction of apoptosis using Cell Death ELISA. A significant increase in DNA fragmentation is reported in MDA-MB-231 cells after 24 h of AELE treatment and topotecan which was used as a positive control. Plot 03 displays an overlay of the different treatments used (control in grey, 261 μg/mL in yellow, 522 μg/mL in pink, and the positive control in blue). Dunnett’s test was used to compare the different concentrations to the control. Ordinary one-way ANOVA was used to determine the significant differences reported with * indicating a p-value: 0.01 < p < 0.05, ** indicating a p- value: 0.001 < p < 0.01, and *** indicating a p-value: p < 0.001

Fig. 4

The qualitative assessment of apoptosis induced by AELE using Annexin V staining. Annexin V staining of MDA-MB-231 cells treated for 24 h with increasing concentrations of AELE and a positive control treated with topotecan shows a gradual increase in the number of Annexin-positive cells between the control group and the treated cells

Fig. 5

Flow cytometric analysis of Annexin V staining of MDA-MB-231 cells treated with AELE. Topotecan was used as a positive control. A significant increase in Annexin-V binding was reported after 24 h of treatment with increasing AELE concentrations. Dunnett’s test was used to compare the different concentrations to the control. Two-way ANOVA was used to determine the significant differences reported with * indicating a p-value: 0.01 < p < 0.05, ** indicating a p- value: 0.001 < p < 0.01, and *** indicating a p-value: p < 0.001

Fig. 6

The effect of AELE on the expression of pro- and anti-apoptotic proteins. The results of Western Blot analysis of apoptosis-regulating proteins in MDA-MB-231 cells upon AELE treatment show an increased Bax-Bcl2 ratio, and upregulated pro-apoptotic proteins cytochrome c and p21. Dunnett’s test was used to compare the different concentrations to the control. Ordinary one-way ANOVA was used to determine the significant differences reported with * indicating a p-value: 0.01 < p < 0.05, ** indicating a p- value: 0.001 < p < 0.01, and *** indicating a p-value: p < 0.001