Dillenia Suffruticosa extract inhibits proliferation of human breast cancer cell lines (MCF-7 and MDA-MB-231) via induction of G2/M arrest and apoptosis

Abstract

The present research was designed to evaluate the anticancer properties of Dillenia suffruticosa extract. Our focus was on the mode of cell death and cell cycle arrest induced in breast cancer cells by the active fractions (designated as D/F4, D/F5 and EA/P2) derived from chromatographic fractionation of D. suffruticosa extracts. The results showed that the active fractions are more cytotoxic towards MCF-7 (estrogen positive breast cancer cells) and MDA-MB-231 (estrogen negative breast cancer cells) as compared to other selected cancer cell lines that included HeLa, A459 and CaOV3. The induction of cell death through apoptosis by the active fractions on the breast cancer cells was confirmed by Annexin V-FITC and PI staining. Cell cycle analysis revealed that D/F4 and EA/P2 induced G2/M phase cell cycle arrest in MCF-7 cells. On the other hand, MDA-MB-231 cells treated with D/F4 and D/F5 accumulated in the sub-G1 phase without cell cycle arrest, suggesting the induction of cell death through apoptosis. The data suggest that the active fractions of D. suffruticosa extract eliminated breast cancer cells through induction of apoptosis and cell cycle arrest. The reason why MCF-7 was more sensitive towards the treatment than MDA-MB-231 remains unclear. This warrants further work, especially on the role of hormones in response towards cytotoxic agents. In addition, more studies on the mechanisms underlying the induction of apoptosis and cell cycle arrest by the plant extract also need to be carried out.

Figure 1

Morphological changes of (A) MCF-7 and (B) MDA-MB-231 cells treated with different concentrations of active fraction of D/F4, D/F5 and EA/P2 of D. suffruticosa for 72 h viewed under an inverted light microscope (200× magnification). Reduce in cell population was noted with the increase in the concentration of the treatment as compared to the control (untreated cells).

Figure 2

Close-up views of MCF-7(B, C and D) and MDA-MB-231(F and G) cells treated with (B and F) 12.5 µg/mL of D/F4, (C and G) 12.5 µg/mL of D/F5 and (D) 50.0 µg/mL of EA/P2 of D. suffruticosa for 72 h viewed under an inverted light microscope. The cells showed characteristics of apoptosis such as nuclear compaction (NC), apoptotic bodies (AB) and membrane blebbing (MB). A and E shows the untreated MCF-7 and MDA-MB-231, respectively (400× magnification).

Figure 3

The dot plots (A) and the percentage (%) of cell distribution (B) of MCF-7 and the dot plots (C) and the percentage (%) of cell distribution (D) of MDA-MB-231 after treated with the active fractions of D. suffruticosa (D/F4, D/F5 and EA/P2) as determined by Annexin V-PI staining. The cells were treated with different concentrations of the active fractions for 24 hours. * p < 0.05 as compared to the untreated control cells. The data is the average value of three independent experiments ± SD.

Figure 4

Effects of (A) D/F4, (B) D/F5 and (C) EA/P2 on the cell cycle of MCF-7 and effects of (D) D/F4 and (E) D/F5 on the cell cycle of MDA-MB-231 analyzed by flow cytometer. The cells were treated with different concentrations of D/F4 for 24 h. * p < 0.05 as compared to the untreated control cells. The data is the average value of three independent experiments ± SD.

Figure 5

Chromatographic fractionation of DCM and EtOAc extract of D. suffruticosa roots. D/F4 and D/F5 represent the active fractions obtained from fractionation of DCM extract using hexane: EtOAc gradient as mobile phase. EA/P2 represents the active fraction obtained from fractionation of EtOAc extract using hexane: EtOAc: MeOH as mobile phase.