The pro-apoptotic effect of a Terpene-rich Annona cherimola leaf extract on leukemic cell lines

Abstract

Background: The edible fruit Annona cherimola has previously shown many nutritional and medicinal properties. The current study evaluates the anti-cancer and anti-proliferative properties of Annona cherimola ethanolic leaf extract (AELE) on Acute Myeloid Leukemia (AML) cell lines cultured in vitro (Monomac-1 and KG-1).

Methods: The anti-proliferative effect of A. cherimola ethanolic leaf extract was evaluated via cell viability assay. Its pro-apoptotic effect was assessed through Cell Death ELISA and dual Annexin V/PI staining. To further investigate the molecular mechanism by which the extract promoted apoptosis and inhibited the proliferation of the AML cells used, apoptotic protein expression was determined through western blots. Extract composition was elucidated by Gas Chromatography-Mass Spectrometry (GC-MS).

Results: Our results showed that the treatment with A. cherimola ethanolic leaf extract exhibited an inhibitory effect on the proliferation of both cancer cell lines used in a dose- and time-dependent manner, with no toxic effects on normal mononuclear cells (MNCs) isolated from human bone marrow. This effect was mediated by DNA fragmentation and apoptosis, as revealed by Cell Death ELISA and dual Annexin V/PI staining. Western blot analysis revealed a Bax/Bcl2 dependent mechanism of apoptosis, as well as PARP cleavage, confirming the apoptotic results observed previously. These effects may be attributed to the presence of terpenes which constitute a large component of the leafy extract, as revealed via GC-MS.

Conclusion: All the data presented in our study show that the terpene-rich A. cherimola ethanolic leaf extract exhibits an anti-proliferative and pro-apoptotic effect on the AML cell lines used.

Keywords: Acute myeloid leukemia; Annona cherimola; Apoptosis; Cancer; Terpenes.

Fig. 1

The effect of AELE on cell proliferation using MTS assay. Proliferation of Monomac-1(a) and KG-1 (b) cells after 24, 48, and 72 h of treatment with increasing concentrations of AELE. The absorbance was measured at 492 nm. A significant dose and time-dependent decrease in proliferation of AML cells was observed upon increasing concentrations of AELE. The IC50 s were reached at 333.4 μg/mL for Monomac-1 and 254.5 μg/mL for KG-1 at 24 h. A time-dependent decrease in the IC₅₀ s was observed for both cell lines at 48 and 72 h. (* indicates a p-value: 0.01 < p < 0.05, ** indicates a p- value: 0.001 < p < 0.01, and **** indicates a p-value: p < 0.0001)

Fig. 2

The effect of AELE on MNCs isolated from Human Bone Marrow. AELE showed no inhibitory effect on Mononuclear Cells (MNCs) isolated from Human Bone Marrow

Fig. 3

The quantitative effect of AELE on induction of apoptosis using Cell Death ELISA. Cell Death ELISA on Monomac-1 (a) and KG-1 (b) cells, treated with the two concentrations of AELE closest to the IC50 (173 and 346 μg/mL), as well as a positive control treated with etoposide for 24 h. A significant dose-dependent increase in enrichment factor is noted for AML cells upon treatment with two increasing doses of AELE for 24 h. (** indicates a p- value: 0.001 < p < 0.01, *** indicates a p-value: 0.0001 < p < 0.001 and **** indicates a p- value: p < 0.0001)

Fig. 4

The quantitative assessment of apoptosis induced by AELE using Annexin V/PI. Monomac-1 (a) and KG-1 (b) were treated with the two concentrations of AELE within which the IC50 falls (173 and 346 μg/mL), followed by staining with Annexin V/PI, and analysis using flow cytometry. A shift from double-negative staining, to Annexin V-positive and PI-negative staining, an early apoptotic marker, upon treatment with AELE was observed. A slight increase in double positive stained cells was also observed

Fig. 5

The effect of AELE on the expression of pro- and anti-apoptotic proteins. Western blot analysis and quantification of apoptosis-regulating proteins in Monomac-1 cells treated with AELE for 24 h. Significant upregulation of cleaved PARP-1, and Bax/Bcl-2 ratio was observed between Monomac-1 control cells and cells treated with 173 μg/ml or 346 μg/ml of AELE for 24 h. Representative blots from three different experiments were cropped and are shown in the figure. The full-length blots are reported in the Additional file 1. (** indicates a p-value: 0.001 < p < 0.01, *** indicates a p-value: 0.0001 < p < 0.001 and **** indicates a p-value: p < 0.0001)

Fig. 6

Extract composition elucidation by GC-MS analysis