Damnacanthal is a potent inducer of apoptosis with anticancer activity by stimulating p53 and p21 genes in MCF-7 breast cancer cells

Abstract

Damnacanthal, an anthraquinone compound, is isolated from the roots of Morinda citrifolia L. (noni), which has been used for traditional therapy in several chronic diseases, including cancer. Although noni has long been consumed in Asian and Polynesian countries, the molecular mechanisms by which it exerts several benefits are starting to emerge. In the present study, the effect of damnacanthal on MCF-7 cell growth regulation was investigated. Treatment of MCF-7 cells with damnacanthal for 72 h indicated an antiproliferative activity. The MTT method confirmed that damnacanthal inhibited the growth of MCF-7 cells at the concentration of 8.2 μg/ml for 72 h. In addition, the drug was found to induce cell cycle arrest at the G1 checkpoint in MCF-7 cells by cell cycle analysis. Damnacanthal induced apoptosis, determined by Annexin V-fluorescein isothiocyanate/propidium iodide (PI) dual-labeling, acridine-orange/PI dyeing and caspase-7 expression. Furthermore, damnacanthal-mediated apoptosis involves the sustained activation of p21, leading to the transcription of p53 and the Bax gene. Overall, the present study provided significant evidence demonstrating that p53-mediated damnacanthal induced apoptosis through the activation of p21 and caspase-7.

Keywords: MCF-7; anticancer; apoptosis; caspase; damnacanthal; p21; p53.

Figure 1

Chemical structure of damnacanthal.

Figure 2

Effect of damnacanthal on growth of breast cancer MCF-7 cell line. Cells were seeded in 96-well plates and incubated with various concentrations of damnacanthal for 72 h at 37°C. Cell viabilities were determined by MTT assay. Data points are presented as the means ± standard deviation of triplicate experiments.

Figure 3

(A) Morphological assessment of MCF-7 cells stained with acridine orange (green) and propidium iodide (red). Cells were incubated with or without damnacanthal at IC₅₀ concentration for 72 h. Cells with intact membranes and stained green indicated viable cells; cells that showed chromatin condensation, nuclear genome fragmentation and membrane blebbing indicated early apoptosis; and cells that were stained orange and contained fragmented DNA represented secondary necrotic or late apoptotic cells (magnification, ×100; scale bar, 100 μm). (B) Population of viable, apoptotic and necrotic cells in vehicle-treated MCF-7 cells (control) and MCF-7 cells treated with damnacanthal for 72 h. ^**P<0.05 vs. vehicle-treated group.

Figure 4

Flow cytometry analysis of MCF-7 cells, vehicle-treated (control) or treated with damnacanthal for 72 h, stained with Annexin V-fluorescein isothiocyanate/propidium iodide. Data are presented as the means ± standard error of the mean for three assays, each in triplicate. ^**P≤0.05, vs. vehicle-treated group, determined by analysis of variance.

Figure 5

Flow cytometry analysis of MCF-7 cells treated with and without damnacanthal for 72 h stained with propidium iodide. DNA contents were analyzed by flow cytometry and apoptosis was measured by the accumulation of sub-G1 DNA contents in cells and compared with vehicle-treated MCF-7 cells. Results are representative of three independent experiments. Data are presented as the means ± standard deviation of the percentage of cells in individual phases of the cell cycle from four independent experiments.

Figure 6

Fold-change of gene expression in MCF-7 cells treated with and without damnacanthal for 72 h were analyzed by reverse transcription polymerase chain reaction. The control cells were treated with vehicle in RPMI medium. Results are representative of three independent experiments. Data are presented as the means ± standard deviation of four independent experiments. ^**P<0.05 vs. vehicle-treated group.

Figure 7

Flow cytometry analysis of MCF-7 cells treated with and without damnacanthal for 72 h were analyzed for the expression of Bcl-2, estrogen receptor-α, p53 and X-linked inhibitor of apoptosis proteins. The control cells were treated with vehicle in RPMI medium. Results are representative of three independent experiments. Data are presented as the means ± standard deviation of the percentage of protein expression from four independent experiments. ^**P<0.05 vs. vehicle-treated group.