Cytogenotoxic effects of Adenium obesum seeds extracts on breast cancer cells

Abstract

The extracts prepared from various areal parts of the Adenium obesum (Forssk.) Roem. & Schult. (Family: Apocynaceae) including leaves, fruit and seeds ethanolic extracts and seed aqueous extract were evaluated against MCF-7 cells in order to investigate its potential of cytogenotoxicity and induction of apoptosis. The ethanolic seeds extract had comparatively higher cytotoxicity (IC₅₀ ∼ 337 µg/ml). Further, apoptosis and DNA damaging potential of seeds ethanolic extract was analyzed by applying multiple sub-lethal concentrations and durations. Flow cytometry results revealed that maximum percentage of early apoptosis (37%) and late apoptosis (35%) were observed after 12 h exposure in concentrations 200 µg/ml and 300 µg/ml, respectively. Similarly, the higher effect of extract in terms of DNA damage by alkaline comet assay was registered after 12 h treatment at concentrations 200 and 300 µg/mL. The calculated total damage score (TDS) for these concentrations were 614 and 617, respectively. The above findings indicate that A. obesum ethanolic seeds extracts has cytogenotoxic properties that could be further explored for the potential source of chemotherapeutic lead against cancer.

Keywords: Adenium obesum; Apocynaceae; Apoptosis; Cancer cells; Comet assay; Cytotoxicity.

Fig. 1

(A) Comparison of MTT cell viability assays of MCF-7 cell treated with four extracts of A. obesum (ethanolic extract of leaves, fruits and seeds and aqueous extract of seeds) after 24 h of exposure. (B) MTT cell viability assay of MCF-7 cells treated with ethanolic seeds extract with indicated concentrations for 24 h. The IC₅₀ value was estimated as 337 µg/mL.

Fig. 2

(A) Photomicrograph of untreated MCF-7 cells. Cells are polygonal or spindle-shaped and tightly packed. MCF-7 cells treated for 24 h with ethanolic seeds extracts. (B) 100 µg/mL, more cells are detached (become round or irregular instead of spindle shape), there is increase in intercellular spaces and there is increase of cell-free area. (C) 200 µg/mL, there are many irregularly shaped cells and increase in the intercellular space. (D) 300 µg/mL, almost all cells are shrinked, irregular round, losing their normal spindle shape morphology.

Fig. 3

Apoptosis assay by flow cytometry of MCF-7 cells treated A. obesum ethanolic seeds extract for indicated cencentrations and durations. Representative figures showing population of viable (annexin V− PI−), early apoptotic (annexin V+ PI−), late apoptotic (annexin V+ PI + ) and necrotic (annexin V− PI+) cells.

Fig. 4

Showing the percentage of apoptotic cells relative to the viable cells of control in MCF-7 cells induced by A. obesum ethanolic seeds extract at concentrations 100, 200 and 300 µg/mL for 6, 12 and 24 h, respectively.

Fig. 5

Representative images of comets showing the genotoxic effect of A. obesum ethanolic seeds extract on MCF-7 cells for 12 h treatment analysed by comet assay. (A) 100 µg/mL (B) 200 µg/mL (C) 300 µg/mL. Numbers at or near each comet are the visual scoring of DNA damage according to the comet class. (D) Total damage score of comet assay by A. obesum ethanolic seeds extract on MCF-7 cells for three concentrations and durations (^*p < 0.01, ^**p < 0.05).