Rhein induces apoptosis in human breast cancer cells

Abstract

Human breast cancers cells overexpressing HER2/neu are more aggressive tumors with poor prognosis, and resistance to chemotherapy. This study investigates antiproliferation effects of anthraquinone derivatives of rhubarb root on human breast cancer cells. Of 7 anthraquinone derivatives, only rhein showed antiproliferative and apoptotic effects on both HER2-overexpressing MCF-7 (MCF-7/HER2) and control vector MCF-7 (MCF-7/VEC) cells. Rhein induced dose- and time-dependent manners increase in caspase-9-mediated apoptosis correlating with activation of ROS-mediated activation of NF-κB- and p53-signaling pathways in both cell types. Therefore, this study highlighted rhein as processing anti-proliferative activity against HER2 overexpression or HER2-basal expression in breast cancer cells and playing important roles in apoptotic induction of human breast cancer cells.

Figure 1

Expression of HER2/neu in human breast cancer cells. Vector control MCF-7 cells (MCF-7/VEC) and HER2-overexpressing MCF-7 cells (MCF-7/HER2) were harvested, and cell lysates were analyzed by Western blotting with anti-HER2 and anti-β actin antibodies.

Figure 2

Effect of anthraquinone derivatives on the growth of human breast cancer cells. MCF-7/VEC (a) and MCF-7/HER2 (b) cells were plated in 96-well plates (5 × 10⁴ cells/well) and then treated with serial dilution of each tested compound. After treatment for 48 h, cell growth was examined using MTT assay. OD_570-630 in each well was measured with a micro-ELISA reader. Survival rate (%) = ((A_control − A_experiment)/A_control) × 100. *P  value < 0.05; **P  value < 0.01 compared with untreated cells.

Figure 3

Cell cycle analysis of human breast cancer cells in response to rhein. MCF-7/VEC and MCF-7/HER2 cells were treated with serial dilution of rhein. After 24, 48, and 72 h incubation, cells were fixed by 70% ethanol, stained with PI, and analyzed using flow cytometry. Percentage of cells in sub G1 (apoptotic) (a), G1 (b), S (c), and G2 (d) phases were shown representing three independent studies. *P  value < 0.05; **P  value < 0.01 compared with untreated cells.

Figure 4

Western blotting analysis of caspase 9 in human breast cancer cells with/without rhein treatment. After 48 h incubation with rhein, MCF-7/VEC and MCF-7/HER2 cells were harvested, and lysates were analyzed by Western blotting with anticaspase-9 and anti-β actin antibodies.

Figure 5

Flow cytometric analysis of reactive oxygen species (ROS) in human breast cancer cells with/without rhein treatment. After 24 and 48 h incubation with rhein, MCF-7/VEC and MCF-7/HER2 cells were harvested, and then stained by DCFH-DA dye. The fluorescence intensity of stained cells was determined by flow cytometry. *P  value < 0.05; **P  value < 0.01 compared with untreated cells.

Figure 6

Western blotting analysis of ASK1 expression in human breast cancer cells with/without rhein treatment. After 48 h incubation with rhein, MCF-7/VEC and MCF-7/HER2 cells were harvested, and lysates were analyzed by Western blotting with anti-ASK1 and anti-β actin antibodies.

Figure 7

Effect of rhein on in vivo signal pathways in human breast cancer cells. MCF-7/VEC and MCF-7/HER2 cells were transiently co-transfected with cis-reporter plasmids (pAP1-Luc, pNF-κB-Luc, and p53-Luc) and an internal control reporter (pRluc-C1), and then treated with rhein for 24 h. Firefly and Renilla Luciferase enzymes were measured; the relative firefly luciferase activity was normalized by Renilla Luciferase. *P  value < 0.05; **P  value < 0.01 compared with untreated cells.

Figure 8

Western blotting analysis of p21 expression in human breast cancer cells with/without rhein treatment. After 48 h incubation with rhein, MCF-7/VEC and MCF-7/HER2 cells were harvested, and lysates were analyzed by Western blotting with anti-p21 and anti-β actin antibodies.