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. 2023 Aug 28;47(8):1835-1852.

Anti-proliferative effect of Annona extracts on breast cancer cells

Maria-Luisa Veisaga  1   2 , Mariam Ahumada  2 , Stacy Soriano  2 , Leonardo Acuna  3 , Wei Zhang  3 , Ivy Leung  2 , Robert Barnum  2 , Manuel A Barbieri  1   2   3   4   5
Affiliations

Anti-proliferative effect of Annona extracts on breast cancer cells

Maria-Luisa Veisaga et al. Biocell. .

Abstract

Backgorund: Fruits and seed extracts of Annona montana have significant cytotoxic potential in several cancer cells. This study evaluates the effect of A. montana leaves hexane extract on several signaling cascades and gene expression in metastatic breast cancer cells upon insulin-like growth factor-1 (IGF-1) stimulation.

Methods: MTT assay was performed to determine the proliferation of cancer cells. Propidium iodide staining and flow cytometry analysis of Annexin V binding was utilized to measure the progression of the cell cycle and the induction of apoptosis. Protein expression and phosphorylation were determined by western blotting analysis to examine the underlying cellular mechanism triggered upon treatment with A. montana leaves hexane extract.

Results: A. montana leaves hexane (sub-fraction V) blocked the constitutive stimulation of the PI3K/mTOR signaling pathways. This inhibitory effect was associated with apoptosis induction as evidenced by the positivity with Annexin V and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNNEL) staining, activation of caspase-3, and cleavage of PPAR. It also limited the expression of various downstream genes that regulate proliferation, survival, metastasis, and angiogenesis (i.e., cyclin D1, survivin, COX-2, and VEGF). It increased the expression of p53 and p21. Interestingly, we also observed that this extract blocked the activation of AKT and ERK without affecting the phosphorylation of the IGF-1 receptor and activation of Ras upon IGF-1 stimulation.

Conclusion: Our study indicates that A. montana leaves (sub-fraction V) extract exhibits a selective anti-proliferative and proapoptotic effect on the metastatic MDA-MB-231 breast cancer cells through the involvement of PI3K/AKT/mTOR/S6K1 pathways.

Keywords: AKT; Annona montana; Anti-proliferative; Apoptosis; Cancer cell lines; Cell cycle; mTOR.

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Conflict of interest statement

Conflicts of Interest: The authors declare no conflicts of interest to report regarding the present study.

Figures

FIGURE 1.
FIGURE 1.
A dose-dependent inhibitory effect of Annona montana methanol extract on cell proliferation. (A) Cells (MCF-10A [●], MCF-7 [○], and MDA-MB-231 [◻]) were treated with several concentrations (0 to 600 μg/mL) of A. montana methanol extract for 8 h, respectively. Then, cellular proliferation was quantified spectroscopically using the MTT assay described in the Material and Methods. Dimethyl sulfoxide (0.01%) [◊] was used as a control with MCF-7 cells. IC50 was expressed as μg/mL. Data represent the mean ± SEM of three independent experiments. Results were expressed as a percentage of non-treated cells (control cells). *p < 0.05 and ***p < 0.001 as determined by two-way ANOVA followed by Tukey’s post hoc test. (B) Cells [MDA-MB-231(231), MCF-10A (10A), MCF-7 (7) PCS-600–100 (010)] were cultured in medium alone and containing either 0.01% dimethyl sulfoxide or 600 μg/mL of A. montana methanol extract for 8 h. Cell viability was determined by the LDH assay. All values are presented as the mean ± SEM of three experiments performed in triplicate. ns: it means not significant.
FIGURE 2.
FIGURE 2.
Effect of hexane, ethyl acetate, and water Anonna montana extracts on cell proliferation. Cells ((A) MDA-MB-231 (B): MCF-10) were treated with several concentrations (0 to 300 μg/mL) of hexane (○), ethyl acetate (□), and water (●) fractions of A. montana methanol extract for 8 h. Then, cellular proliferation was quantified spectroscopically using the MTT assay described in the Material and Methods section. IC50 was expressed as μg/mL. Data represent the mean ± standard error of three independent experiments’ mean (SEM). Results were expressed as a percentage of non-treated cells (control cells). *p < 0.05 and ***p < 0.001 as determined by two-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. (C) Cells (MDA-MB-231 and MCF-10A) were cultured in medium alone (−) and containing either 0.01% dimethyl sulfoxide (D) or 300 μg/mL of A. montana extracts (e.g., hexane-H, ethyl acetate-EA, water-W) for 8 h. Cell viability was determined by the LDH assay. All values are presented as the mean ± SEM of three experiments performed in triplicate. *p < 0.05 and ***p < 0.001 as determined by two-way ANOVA followed by Tukey’s post hoc test.
FIGURE 3.
FIGURE 3.
Annona montana hexane sub-fraction V suppressed cell proliferation. MDA-MB-231 cells were treated with 0.01% dimethyl sulfoxide (DMSO) alone (●), 20 μg/mL (□), and 40 μg/mL (○) of hexane sub-fraction V of A. montana several times (24, 48, and 72 h). Cell proliferation was quantified by spectrophotometric analysis using MTT assay as described in Material and Methods. Results were expressed as a percentage of 0.01% DMSO-treated cells (control cells). Data represent the mean ± SEM of three independent experiments. *p < 0.05 and ***p < 0.001 as determined by two-way ANOVA followed by Tukey’s post hoc test.
FIGURE 4.
FIGURE 4.
Histogram analysis of the cell cycle in MDA-MB-231 cells in the presence of Annona montana hexane subfraction. (A, B) MDA-MB-231 cells were treated with 0.01% DMSO alone (control) or 20 μg/mL of hexane sub-fraction V (hexane-V) of A. montana for 8 h. Then, the cells were fixed, stained with propidium iodide, and analyzed using a flow cytometer as described in the Material and Methods section. The data are representative of one of four independent experiments. Inset: Data represent the mean ± SEM of four independent experiments.
FIGURE 5.
FIGURE 5.
Annona montana hexane subfraction V induced apoptosis in MDA-MB-231 cells. MDA-MB-231 cells were treated with 0.01% DMSO alone (control) or 20 μg/mL of hexane sub-fraction V (hexane-V) of A. montana for 24 h. Then, the cells were fixed, stained with a FITC-conjugated Annexin V antibody (A, B), incubated using TUNEL reaction solution (C, D), and then analyzed using a flow cytometer as described in Material and Methods. The data are representative of one of four independent experiments.
FIGURE 6.
FIGURE 6.
Effect of Annona montana hexane subfraction V on migration, invasion, and colony formation. MDA-MB-231 cell migration was analyzed by wound healing assay (scale bar is 200 μm) (A) or by Transwell invasion assay (scale bar is 50 μm) (B) in the presence of 0.1% dimethyl sulfoxide (DMSO) alone (control) or 20 μg/mL of hexane subfraction V (hexane-V) of A. montana. (C) MDA-MB-231 cell invasion was examined by Transwell invasion assay in the presence of 0.01% DMSO alone (control) or 20 μg/mL of hexane subfraction V (hexane-V) of A. montana (scale bar is 50 μm). (D) MDA-MB-231 cell colony formation was detected following culturing with 30 μg/mL A. montana hexane subfraction V (hexane-V) of A. montana for 14 days (scale bar is 100 μm). (E) The expression of MMP-2 and MMP-9 was analyzed by western blotting t using A. montana hexane subfraction V (hexane-V) for 8 h. Then, equal amounts of lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted to a nitrocellulose membrane, and antibodies specific to MMP-2 and MMP-9 antibodies were used. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. Inset: the data are representative of one of four independent experiments. Western blotting was performed three times, and a representative image of the three independent experiments is shown. The results are expressed as the mean ± SEM of three independent experiments, and each was performed in triplicate. *p < 0.05 vs. DMSO-treated group (control). Hexane-V, A. montana hexane subfraction V; OD, optical density.
FIGURE 7.
FIGURE 7.
Annona montana hexane subfraction V suppressed PI3K/AKT/mTOR/S6K1 and MAPKs signaling pathways in breast cancer cells. (A–H) MDA-MB-231 cells were incubated with 0.01% DMSO or 20 μg/mL hexane subfraction of V (hexane-V) A. montana for 8 h as described in Material and Methods. Then, equal amounts of lysates were subset to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted to nitrocellulose, and antibodies specific to tyrosine-phosphorylated (p)-PI3K (Tyr458), PI3K, p-AKT (Ser473), AKT, p-mTOR (Ser2448), mTOR, p-S6K1 (Thr 421/Ser424), S6K1, p-ERK (Thr202/Tyr204), t-ERK, p-JNK (Thr183/Tyr185), JNK, p-p38 (Thr180/Tyr182), and p38. Relative levels of phosphorylated proteins were determined by densitometry as described in Material and Methods, and they were expressed as a ratio of phosphorylated/total proteins. The western blotting was performed three times, and a representative image of the three independent experiments is shown. Data represent the mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01 by student’s t-test compared to non-treated cells.
FIGURE 8.
FIGURE 8.
Annona montana hexane subfraction V decreases the expression of proteins involved in proliferation, anti-apoptotic, metastatic, and angiogenetic proteins. (A–H) MDA-MB-231 cells were treated with 0.01% DMSO or 20 μg/mL hexane subfraction (hexane-V) of A. montana for 8 h. Thereafter, equal amounts of lysates were analyzed by western blot analysis using antibodies against survivin, IAP-1, IAP-2, Cyclin D1, COX-2, VEGF, procaspase-3, cleaved caspase-3, and PARP. (I, J) MDA-MB-231 cells were treated with 0.01% DMSO or 20 μg/mL hexane subfraction V of A. montana for 8 h. After, equal amounts of lysates were analyzed by western blotting analysis using antibodies against p21 and p53. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. Relative levels of each protein were determined by densitometry as described in the Material and Methods section, and they were expressed as a ratio of each protein/GAPDH. The data represents the mean ± SEM of three independent experiments. The western blots were performed three times, and a representative image of the three independent experiments is shown. *p < 0.05, and **p < 0.01.
FIGURE 9.
FIGURE 9.
Annona montana hexane subfraction potentiated the apoptotic effects of pharmacological PI3K/AKT blockers and Ras inhibitors on the blockage of the Ras/RAF/ERK signaling pathway in MDA-MB-231 cells. (A) MDA-MB-231 cells were treated with 0.01% DMSO (Control) and the indicated combination of hexane sub-fraction V (H, 20 μg/mL) or wortmannin (W, 0.233 μM) for 8 h. Then, equal amounts of lysates were analyzed by western blotting using antibodies against p-AKT (Ser473), t-AKT, p-mTOR (Ser2448), t-mTOR, p-S6K1 (Thr421/Ser424), and t-S6K1. The western blots were performed three times, and a representative image of the three independent experiments is shown. (B–D) Relative levels of phosphorylated proteins (i.e., p-AKT, p-mTOR, p-S6K1) were determined by densitometry as described in the Material and Methods section. They were expressed as a ratio of phosphorylated/total proteins. Data represent the mean ± SEM of three independent experiments. *p < 0.05, and **p < 0.01 by student’s t-test compared to non-treated cells. (E) MDA-MB-231 cells were treated with 0.01% dimethyl sulfoxide (control), and the indicated combination of hexane subfraction V (H, 20 μg/mL), PF3758309 (PF, 20 μM), or FR180204 (FR, 50 μM) for 8 h. Then equal amounts of lysates were analyzed by western blotting using antibodies against p-RAF (Ser259), t-RAF, p-ERK (Thr202/Tyr204), and t-ERK. The western blots were performed three times, and a representative image of the three independent experiments is shown. (F, G) Relative levels of phosphorylated proteins (i.e., p-RAF, p-ERK) were determined by densitometry as described in the Material and Methods section. They were expressed as a ratio of phosphorylated/total proteins. Data represent the mean ± SEM of three independent experiments. *p < 0.05 by student’s t-test compared to non-treated cells. (H) MDA-MB-231 cells were treated with the indicated combination of hexane subfraction V (H, 20 μg/mL) or Ras Inhibitor-ISIS 2503 (IS). Then, equal amounts of lysates were analyzed by western blotting using antibodies against Ras. Western blotting was performed three times, and a representative image of the three independent experiments is shown. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. (I) Relative levels of Ras proteins were determined by densitometry as described in the Material and Methods section, and they were expressed as a ratio of Ras/GAPDH proteins. Data represent the mean ± SEM of three independent experiments. *p < 0.05 by student’s t-test compared to non-treated cells.
FIGURE 10.
FIGURE 10.
Annona montana hexane sub-fraction V suppressed AKT and ERK activities signaling in human breast cancer cells without affecting Ras activation upon IGF-1 stimulation. MDA-MB-231 cells were serum-starved for 16 h and then incubated in the presence of 0.01% dimethyl sulfoxide or 20 μg/mL hexane subfraction V (H) of A. montana for 8 h. Subsequently, cells were incubated in the presence of 2 ng/mL IGF-1 at 4°C for 90 min, washed with cold HBSS-BSA and incubated for 3 min at 37°C as described in the Material and Methods section. (A–C) Equal amounts of lysates were subset to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted to nitrocellulose, and antibodies specific to tyrosine-phosphorylated (p)-AKT (Ser473), total (t)-AKT, p-ERK (Thr202/Tyr204), t-ERK, p-IGF-1R (Tyr1131), and t-IGF-1R. Relative levels of phosphorylated proteins were determined by densitometry as described in the Material and Methods section, and they were expressed as a ratio of phosphorylated/total proteins. (D) After incubation, cells were washed with cold HBSS-BSA, lysed, and allowed to bind to glutathione beads in the presence of GST-RBD at 4°C for 60 min. Beads were washed with cold HBSS-BSA, and the presence of activated Ras (i.e., GTP-Ras) was analyzed by western blotting. Added GST-RBD and t-Ras proteins in the cell lysate were also evaluated by western blotting analysis. The Data represent the mean ± SEM of three independent experiments. *p < 0.05 by student’s t-test compared to non-treated cells.
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