Genistein induces G2/M cell cycle arrest and apoptosis via ATM/p53-dependent pathway in human colon cancer cells

Abstract

Soybean isoflavones have been used as a potential preventive agent in anticancer research for many years. Genistein is one of the most active flavonoids in soybeans. Accumulating evidence suggests that genistein alters a variety of biological processes in estrogen-related malignancies, such as breast and prostate cancers. However, the molecular mechanism of genistein in the prevention of human colon cancer remains unclear. Here we attempted to elucidate the anticarcinogenic mechanism of genistein in human colon cancer cells. First we evaluated the growth inhibitory effect of genistein and two other isoflavones, daidzein and biochanin A, on HCT-116 and SW-480 human colon cancer cells. In addition, flow cyto-metry was performed to observe the morphological changes in HCT-116/SW-480 cells undergoing apoptosis or cell cycle arrest, which had been visualized using Annexin V-FITC and/or propidium iodide staining. Real-time PCR and western blot analyses were also employed to study the changes in expression of several important genes associated with cell cycle regulation. Our data showed that genistein, daidzein and biochanin A exhibited growth inhibitory effects on HCT-116/SW-480 colon cancer cells and promoted apoptosis. Genistein showed a significantly greater effect than the other two compounds, in a time- and dose-dependent manner. In addition, genistein caused cell cycle arrest in the G2/M phase, which was accompanied by activation of ATM/p53, p21waf1/cip1 and GADD45α as well as downregulation of cdc2 and cdc25A demonstrated by q-PCR and immunoblotting assay. Interestingly, genistein induced G2/M cell cycle arrest in a p53-dependent manner. These findings exemplify that isoflavones, especially genistein, could promote colon cancer cell growth inhibition and facilitate apoptosis and cell cycle arrest in the G2/M phase. The ATM/p53-p21 cross-regulatory network may play a crucial role in mediating the anticarcinogenic activities of genistein in colon cancer.

Figure 1.

Effect of genistein, daidzein and biochanin A on HCT-116/SW-480 human colon cancer cell proliferation. Cell survival was determined by the MTS assay and calculated as a ratio, comparing it to the vehicle-treated cells. (A–C) HCT-116 cell growth was inhibited in a time-dependent manner 24, 48 and 72 h after genistein, daidzein and biochanin A exposure. (D–F) Dose-dependent response in HCT-116 cells after compounds were treated at 24, 48 and 72 h. (G) Bar plot of HCT-116/SW-480 cells treated with genistein, daidzein or biochanin A at 48 h. HCT-116 cells showed more sensitivtity than SW-480 cells (^**p<0.01). Data are presented as mean ± SD from at least triplicate wells and 3 independent experiments.

Figure 2.

Genistein, biochanin A and daidzein promoted apoptosis in HCT-116/SW-480 cells. Data are counted as a ratio of total cells counted. (A) All compounds induced significant apoptosis in HCT-116 cells. Biochanin A was the most potent proapoptotic chemical among the compounds. (B) Genistein, biochanin A and daidzein induced more apoptotic cells in HCT-116 cells than in SW-480 cells (^**p<0.01 compared with each other).

Figure 3.

Effect of genistein, biochanin A and daidzein on cell cycle arrest in HCT-116/SW-480 cells. Data are presented as a ratio of total cells counted. (A) Genistein induces G2/M cell cycle arrest in both cell lines at higher doses (50 and 100 μM) in a dose-dependent way 48 h (^**p<0.01). (B) Biochanin A induces G2/M cell cycle arrest in SW-480 cells at doses (50 and 100 μM) (^**p<0.01). (C) Daidzein arrests HCT-116 cells at G2/M phase at higher doses (50 and 100 μM) (^**p<0.01).

Figure 4.

Relative expression of selected cell cycle arrest related genes examined by qPCR array. A human cell cycle RT² Profiler PCR array plate was used to assess the expression of cell cycle related genes. Representative upregulated and downregulated genes are shown. Data are expressed as mean ± SD, relative gene expression was normalized using β-actin as a control and calculated by the 2^−ΔΔct method (^*p<0.05).

Figure 5.

Genistein induces G2/M cell cycle arrest in both HCT-116 p53^+/+ and p53^−/− cells via a p53-dependent pathway. Genistein arrested more HCT-116 p53^+/+ cells in the G2/M phase than in the p53^−/− cells at concentrations (25 and 50 μM) (33.1 vs 25.8%, 45.6 vs 28%).

Figure 6.

Genistein induces G2/M phase arrest via p53/ATM-p21^waf1/cip1 activation and cdc2/cdc25A downregulation. Western blotting of p53, p21, GADD45α, ATM, cdc2, cdc25A and β-actin in HCT-116 p53^+/+ and p53^−/− cells were taken after treatment of genistein at the indicated doses (25, 50 and 100 μM).