Induction of Cell Cycle Arrest and Apoptosis by Physcion, an Anthraquinone Isolated From Rhubarb (Rhizomes of Rheum tanguticum), in MDA-MB-231 Human Breast Cancer Cells

Abstract

Background: Physcion is an anthraquinone from rhubarb (rhizomes of Rheum tanguticum) and has been reported to have anti-inflammatory, hepatoprotective, antifungal, and anti-cancer activities. However, the growth inhibitory activity against human cancer cells and the underlying molecular mechanisms have been poorly determined. This study was designed to investigate the anti-proliferative activity of physcion by induction of cell cycle arrest and apoptosis in human MDA-MB-231 triple negative breast cancer cell line.

Methods: MDA-MB-231 cells were treated with physcion, and the anti-proliferative activity was evaluated by the sulforhodamine B assay. The mechanisms of action for the growth inhibitory activity of physcion were evaluated by flow cytometry for cell cycle distribution, and by Western blot for the assessment of potential target proteins.

Results: Physcion showed a significant anti-proliferative activity against MDA-MB-231 human breast cancer cells. Flow cytometric analysis indicated that physcion markedly induced the accumulation of cells in the G0/G1 phase and the increase of cell population in the sub-G1 phase. The G0/G1 cell cycle arrest by physcion was associated with the down-regulation of Cyclin D1, Cyclin A, CDK4, CDK2, c-Myc and phosphorylated Rb protein expressions. The increase of sub-G1 peak by physcion was closely correlated with the induction of apoptosis, which was confirmed by the induction of cleaved poly-(adenosine diphosphate ribose) polymerase, activation of Caspases, and suppression of Bid and Bcl-2 expression.

Conclusions: The induction of G0/G1 cell cycle arrest and apoptosis might be one of the plausible mechanisms of actions for the anti-proliferative activity of physcion in human breast cancer cells.

Keywords: Apoptosis; Breast neoplasms; Cell cycle arrest; Physcion; Rhubarb.

Figure 1.

Chemical structure of physcion.

Figure 2.

Effect of physcion on the proliferation of human breast cancer MDA-MB-231 cells. MDA-MB-231 cells were plated at 10,000 cells in 96-well plate in RPMI supplemented with 10% fetal bovine serum, and incubated with the test compound as the indicated concentrations for 72 hours. Anti-proliferative activity was determined using the sulforhodamine B assay. The values (% of control) are calculated by the mean absorbance of samples/absorbance of vehicle- treated control. Data are represented as the mean ± standard deviation (n = 3).

Figure 3.

Morphological changes mediated by physcion in MDA-MB-231 cells. Cells were treated with various concentrations of physcion for 24 hours. Cells were photographed by inverted microscopy (×100).

Figure 4.

Effect of physcion on the cell cycle progression in the MDA-MB-231 cells. Cells were treated with vehicle or various concentration of physcion (35, 70, and 140 μM) for 24 hours. The cell cycle distribution was analyzed by flow cytometry as described in MATERIALS AND METHODS.

Figure 5.

Effect of physcion on the expression of cell cycle regulatory proteins (A) and apoptosis related proteins (B) in the MDA-MB-231 cells. Cells were treated with the indicated concentrations of physcion for 24 hours. The expression level of proteins was analyzed by Western blot.