Genistein targets the cancerous inhibitor of PP2A to induce growth inhibition and apoptosis in breast cancer cells

Abstract

Genistein is a soy isoflavone with phytoestrogen and tyrosine kinase inhibitory properties. High intake of soy/genistein has been associated with reduced breast cancer risk. Despite the advances in genistein-mediated antitumor studies, the underlying mechanisms remain unclear. In the present study, we investigated genistein-induced regulation of the cancerous inhibitor of protein phosphatase 2A (CIP2A), a novel oncogene frequently overexpressed in breast cancer, and its functional impact on genistein-induced growth inhibition and apoptosis. We demonstrated that genistein induced downregulation of CIP2A in MCF-7-C3 and T47D breast cancer cells, which was correlated with its growth inhibition and apoptotic activities. Overexpression of CIP2A attenuated, whereas CIP2A knockdown sensitized, genistein-induced growth inhibition and apoptosis. We further showed that genistein-induced downregulation of CIP2A involved both transcriptional suppression and proteasomal degradation. In particular, genistein at higher concentrations induced concurrent downregulation of E2F1 and CIP2A. Overexpression of E2F1 attenuated genistein-induced downregulation of CIP2A mRNA, indicating the role of E2F1 in genistein-induced transcriptional suppression of CIP2A. Taken together, our results identified CIP2A as a functional target of genistein and demonstrated that modulation of E2F1-mediated transcriptional regulation of CIP2A contributes to its downregulation. These data advance our understanding of genistein-induced growth inhibition and apoptosis, and support further investigation on CIP2A as a therapeutic target of relevant anticancer agents.

Figure 1

Genistein-mediated downregulation of CIP2A is associated with its growth inhibition and apoptosis induction. (A) MCF-7, MCF-7-C3 and T47D cells were treated with genistein (GENI) at indicated concentrations for 48 h. Protein levels of CIP2A and PARP in all three cell lines, and caspase-3, cleaved caspase-3 (C-caspase-3) in MCF-7-C3 and T47D cells were detected with western blot analysis. (B) Genistein-induced growth inhibition in MCF-7, MCF-7-C3 and T47D cells assessed with MTT assays. The cells in 96-well plates were treated with genistein at indicated concentrations for 5 days, followed by MTT assays as detailed in Materials and methods. The data were based on samples of 6 replicates. (C) Histogram of cell cycle distributions in genistein treated MCF-7-C3 and T47D cells. The cells were treated with 60 μM genistein for 24 h, followed by flow cytometric analysis. (D) Cell cycle distributions in different phases were analyzed using ModFit software.

Figure 2

Overexpression of CIP2A attenuates genistein-induced growth inhibition. (A) CIP2A levels in control and CIP2A overexpressing MCF-7-C3 and T47D cells detected with western blot analysis. Cells from each line were infected with control and CIP2A encoding lentiviruses, respectively. Stable CIP2A overexpressing clones were pooled after puromycin selection. (B) CIP2A overexpression attenuates genistein-induced growth inhibition. Control and CIP2A overexpressing cells were treated with genistein (GENI) for 5 days, followed by MTT assays. (C) Cell cycle analysis of genistein-treated control and CIP2A overexpressing MCF-7-C3 and T47D cells. The cells were treated with 60 μM genistein for 24 h followed by flow cytometric analysis. ^*P<0.05; ^**P<0.01.

Figure 3

Overexpression of CIP2A reduces genistein-induced apoptosis. (A) Control (MCF-7-C3/CON; T47D/CON) and CIP2A overexpressing (MCF-7-C3/CIP2A; T47D/CIP2A) breast cancer cells were treated with 60 μM genistein (GENI) for 24 h. Relative apoptosis in individual samples was assessed with apoptosis ELISA assays. The data are presented as mean ± SEM based on triplicate samples. (B) Control and CIP2A overexpressing MCF-7-C3 and T47D cell lines were treated with genistein at indicated concentrations for 48 h, followed by western blot detection of CIP2A, PARP and β-actin. ^*P<0.05, ^**P<0.01.

Figure 4

CIP2A knockdown sensitizes MCF-7-C3 and T47D cells to genistein-induced growth inhibition. (A) CIP2A levels in control (siCIP2A −) and CIP2A knockdown (siCIP2A +) cells detected by western blot analysis. MCF-7-C3 and T47D cells were infected with control and CIP2A siRNA encoding lentivirus. Stable clones were collected after puromycin selection. (B) Control and CIP2A knockdown MCF-7-C3 and T47D cells were treated with genistein (GENI) at indicated concentrations for 5 days, followed by MTT assays. (C) Cell cycle analysis of genistein-treated control and CIP2A knockdown MCF-7-C3 and T47D cells. The cells were treated with 60 μM genistein for 24 h followed by flow cytometric analysis. ^*P<0.05; ^**P<0.01.

Figure 5

CIP2A knockdown enhances genistein-induced apoptosis. (A) Control (MCF-7-C3/siCON; T47D/siCON) and CIP2A knockdown (MCF-7-C3/siCIP2A; T47D/siCIP2A) cells were treated with 60 μM genistein (GENI) for 24 h. Relative apoptosis in individual samples was assessed with apoptosis ELISA assays. The data were presented as mean ± SEM based on triplicate samples. (B) Control and CIP2A knockdown MCF-7-C3 and T47D cells were treated with genistein at indicated concentrations for 48 h, followed by western blot detection of CIP2A, PARP and β-actin. ^*P<0.05, ^**P<0.01.

Figure 6

Genistein-induced downregulation of CIP2A involves both transcriptional and post-translational regulation. (A) CIP2A mRNA levels in control and genistein (GENI) treated MCF-7-C3 and T47D cells. The cells were treated with genistein at indicated concentrations for 16 h. Relative CIP2A mRNA levels were detected with quantitative RT-PCR. mRNA levels of GAPDH were used for normalization. (B) MG132 attenuates genistein-induced downregulation of CIP2A. MCF-7-C3 and T47D cells were treated with 60 μM genistein in the absence (−) or presence (+) of MG132 (1 μM) for 24 h, followed by western blot detection of CIP2A and β-actin. (C) Genistein inhibits Akt phosphorylation. MCF-7-C3 and T47D cells were treated with genistein as in Fig. 1A. Phospho-Akt and Akt were detected with western blotting. ^*P<0.05.

Figure 7

Genistein-induced downregulation of CIP2A involves the modulation of E2F1-mediated CIP2A transcription. (A) Genistein induces concurrent downregulation of E2F1 and CIP2A. MCF-7-C3 and T47D cells were treated with genistein (GENI) for 48 h. Protein levels of CIP2A and E2F1 were detected with western blot analysis. (B) Overexpression of E2F1 attenuates genistein-induced CIP2A downregulation. MCF-7-C3 and T47D cells were transiently transfected with control (−) and E2F1 encoding (+) plasmids. Twenty-four hours post-transfection, the cells were treated with 60 μM genistein for 48 h, followed by western blot detection of E2F1, CIP2A, c-Myc and actin. (C) CIP2A mRNA levels in control and E2F1 overexpressing MCF-7/C3 and T47D cells with different treatments. The cells were transiently transfected with control and E2F1 encoding plasmids followed by genistein (60 μM) treatment for 16 h. Relative CIP2A mRNA levels were detected with quantitative RT-PCR. mRNA levels of GAPDH were used for normalization. ^*P<0.05, ^**P<0.01.