Genistein decreases the breast cancer stem-like cell population through Hedgehog pathway

Abstract

Introduction: The existence of breast cancer stem-like cells (BCSCs) has profound implications for cancer prevention. Genistein, a predominant isoflavone found in soy products, has multiple robust anti-tumor effects in various cancers, especially in the breast and prostate cancer. In this study, we aimed to evaluate genistein inhibition of BCSCs and its potential mechanism by culturing MCF-7 breast cancer cells and implanting these cells into nude mice.

Methods: Cell counting, colony formation and cell apoptosis analysis were used to evaluate the effect of genistein on breast cancer cells’ growth, proliferation and apoptosis. We then used mammosphere formation assay and CD44CD24 staining to evaluate the effect of genistein on BCSCs in vitro. A nude mice xenograft model was employed to determine whether genistein could target BCSCs in vivo, as assessed by real-time polymerase chain reaction (PCR) and immunohistochemical staining. The potential mechanism was investigated utilizing real-time PCR, western blotting analysis and immunohistochemical staining.

Results: Genistein inhibited the MCF-7 breast cancer cells’ growth and proliferation and promoted apoptosis. Both in vitro and in vivo genistein decreased breast cancer stem cells, and inhibited breast cancer stem-like cells through down-regulation of the Hedgehog-Gli1 Signaling Pathway.

Conclusions: We demonstrated for the first time that genistein inhibits BCSCs by down-regulating Hedgehog-Gli1 signaling pathway. These findings provide support and rationale for investigating the clinical application of genistein in treating breast cancer, and specifically by targeting breast cancer stem cells.

Figure 1

Genistein inhibits breast cancer cells. (A) MCF-7 cells growing in the log phase were treated with increasing concentrations of genistein for 48 hours. The anti-proliferation effect of genistein was measured by Cell Counting Kit-8 (CCK-8;) assay. (B), (C) Genistein reduced the number of colonies in MCF-7 cells. Genistein’s effect on the surviving fraction on MCF-7 cells was detected by colony formation assay. The number of colonies was counted after 7 days. All data presented as mean ± standard deviation (n ≥3). (D) Genistein increased the percentage of late period apoptotic cells labeled with Annexin V– fluorescein isothiocyanate (FITC)/propidium iodide (PI) in MCF-7 cells. Experiments were repeated three times, and similar results were obtained. Representative scatter grams from flow cytometry profile represent Annexin V–FITC (AV) staining on the x axis and PI on the y axis. *P <0.05, Student’s t test. Each condition was repeated three times and error bars represent standard deviations.

Figure 2

Genistein decreases breast cancer stem cells in vitro. (A), (B) Genistein decreased the size (A) (×200, scale bar = 100 μm) and number (B) of mammospheres. MCF-7 cells were treated with different concentrations of genistein, and then were cultured in ultra-low-attachment plates with serum-free medium for 7 days. Mammospheres were collected and evaluated. (C), (D) The percentage of CD44⁺/CD24^– breast cancer stem-like cells was reduced by genistein treatment. The phenotype of CD44⁺/CD24^– cells was measured by flow cytometry. Similar results were obtained in three independent experiments, and the representative microscopic picture and flow cytometry pattern are shown. *P <0.05, **P <0.01, Student’s t test. FITC, fluorescein isothiocyanate; PE, phycoerythrin.

Figure 3

Genistein decreased breast cancer stem cells in vivo. (A) Genistein remarkably reduced the tumor volume and weight: 1 × 10⁶ MCF-7 cells were inoculated into the mouse mammary fat pad for 2 weeks, and then were treated daily with 20 and 50 mg/kg genistein. After 2 weeks of treatment, mice were sacrificed. Error bars represent standard deviations, n = 5. (B) Immunohistochemistry staining with aldehyde dehydrogenase isoform 1 (ALDH1) antibodies (brown in cytoplasm), ×200. (C) Real-time polymerase chain reaction analyses of ALDH1 in the mouse tumor tissues. (D) ALDH1 protein level before and after treatment with genistein. * P <0.05, **P <0.01, Student’s t test. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Figure 4

Hedgehog–Gli1 pathway inhibition by genistein in vitro. (A) Real-time polymerase chain reaction analysis of human Smoothened (SMO) and Gli1 in MCF-7 cells. The cells were incubated with different concentrations of genistein for 48 hours. (B) Protein levels of SMO and Gli1 were measured by western blot analysis in MCF-7 cells and accompanied by a quantitative bar chart. As an internal control, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used for normalization. *P <0.05, ^#P <0.05, **P <0.01, Student’s t test.

Figure 5

Hedgehog–Gli1 pathway inhibition by genistein in vivo. (A) Immunohistochemistry staining with human Smoothened (SMO) and Gli1 antibodies (brown in cytoplasm), ×200. (B) Real-time polymerase chain reaction analysis of SMO and Gli1 in mice tumors. (C) Smo and Gli1 protein level in tumor tissue and accompanied by a quantitative bar chart. Data presented as mean ± standard deviation, n = 5. *P <0.05, **P <0.01, Student’s t test. Each condition was repeated three times and error bars represent standard deviations. GAPDH, glyceraldehyde 3-phosphate dehydrogenase.