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Abstract
Purpose
To analyze the effect of the growth control on human breast cancer cells with genistein treatment and to investigate the mechanism of genistein-induced G2/M arrest in T47D and MDA-MB231 breast carcinoma cells by Cdc25C expression.
Methods
We analysed the proliferartion of the two cell lines by using MTT proliferation assay, flow cytometric analysis, real-time quantitative RT-PCR and western blotting and investigated the effect of genistein on cell survival, cellular toxicity, cell cycle progression-related genes and their mRNA and protein alterations.
Results
The DNA flow cytometric analysis of both cell lines treated with genistein showed a dose-dependent growth inhibition and accumulation in the G2/M phase of cell cycle. The expression of p21 mRNA and protein increased in both cell lines following genistein treatment but p27 expression was unchanged. Furthermore, decreased Cdc25C expression with decreased polo-like kinase (PLK) 1 expression and increased PLK3 expression were observed after genistein treatment. The decreased level of Cdc25C in the nucleus was associated with decreased phosphorylation of Cdc25C by PLK1. The expression of PLK3 was increased with a dose-dependent and a time-dependent manner and was associated with decreased Cdc25C expression. Check point kinase (CHK) 1 and CHK2 revealed different expression patterns each other. The CHK1 expression was independent of the presence of genestein. CHK2 expression increased in MDA-MB231 cells associated with decreased Cdc25C expression but not in T47D.
Conclusion
These results suggest that genistein induces a G2/M arrest in human breast cancer cells, the mechanism of which is due, in part, to decreased in Cdc25C phosphatase by a regulatory effect of PLK1, PLK3, and CHK2 as well as increased expression of the cyclin dependent kinase inhibitor p21(WAF1/CIP1).
Figures and Tables
Figure 1
Concentration dependent growth inhibition of estrogen receptor (ER)-positive (T47D) and ER-negative (MDA-MB231) breast cancer cells by genistein. Twenty-four hours after inoculation, cells were cultured for 3 days in the absence or presence of genistein at concentratins ranging from 0.1 to 100 µM. The data represent the mean±SD of six independent experiments.
Figure 2
Changes of cell densitiy in T47D cells (A, C) and MDA-MB231cells (B, D). Each cells were treated with 0.1% DMSO (control) or 100 µM of genistein for 3 days. Genistein-treated cells are decreased in cellularity than control.
Figure 3
DNA-fluorescence histogram of T47D and MDA-MB231 cells after genistein treatment. T47D (A) and MDA-MB231 (B) cells were treated with 0.1% DMSO (CON) or 100 µM of genistein (GEN) for 72 hr. Three days after genistein treatment, there was 3.1-fold increase in the percent of T47D cells in G2/M and 4.2-fold increase in the percent of MDA-MB231cells in G2/M, respectively.
Figure 4
Real time quantitative analysis for p21 mRNA of genistein-treated T47D and MDA-MB231 cells. Each cell (5×105 cells) was treated with 0-100 µM genistein for 3 days or 100 µM of genistein for 0.5, 1, 2, 3 days.
Figure 5
Real time quantitative analysis for p27 mRNA of genistein-treated T47D and MDA-MB231 cells. Each cell (5×105 cells) treated with 0-100 µM genistein for 3 days or 100 µM of genistein for 0.5, 1, 2, 3 days.
Figure 6
Real time quantitative analysis for PLK1 mRNA and CHK1 mRNA of genistein-treated T47D and MDA-MB231 cells. Each cell (5×105 cells) treated with 100 µM of genistein for 0.5, 1, 2, 3 days.
Figure 7
Real time quantitative analysis for PLK3 of genistein-treated T47D and MDA-MB231 cells. Each cell (5×105 cells) treated with 0-100 µM genistein for 3 days or 100 µM of genistein for 0.5, 1, 2, 3 days.
Figure 8
Real time quantitative analysis for CHK2 mRNA of genistein-treated T47D and MDA-MB231 cells. Each cell (5×105 cells) treated with 0-100 µM genistein for 3 days or 100 µM of genistein for 0.5, 1, 2, 3 days.
Figure 9
Real time quantitative analysis for Cdc25C mRNA of genistein-treated T47D and MDA-MB231 cells. Each cell (5×105 cells) treated with 0-100 µM genistein for 3 days or 100 µM of genistein for 0.5, 1, 2, 3 days.
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