The combination of sorafenib and radiation preferentially inhibits breast cancer stem cells by suppressing HIF-1α expression

Abstract

The importance of anticancer stem cell research for breast cancer lies in the possibility of providing new approaches for an improved understanding of anticancer activity and cancer treatment. In this study, we demonstrated that the preclinical therapeutic efficacy of combining the multikinase inhibitor sorafenib with radiation was more effective in hypoxia-exposed breast cancer stem cells. We assessed cell viability and Annexin V to evaluate the combined effect of sorafenib and radiation following exposure to hypoxia. Our results showed that the synergistic cytotoxicity increased tumor cell apoptosis significantly and reduced cell proliferation in MDA-MB-231 and MCF-7 cells under hypoxic conditions compared to sorafenib or radiation alone in vitro. Additionally, the combined treatment induced G2/M cell cycle arrest. Notably, the combination of sorafenib and radiation eliminated CD44+CD24-/low cells preferentially, which highly expressed hypoxia-inducible factor (HIF)-1α and effectively inhibited primary and secondary mammosphere formation in MDA-MB-231 cells. A combined effect on MDA-MB‑231 cells in response to hypoxia was shown by inhibiting angiogenesis and metastasis by suppression of HIF-1α and matrix metalloproteinase-2 (MMP-2). Collectively, these results indicate that the efficacy of sorafenib combined with radiation for treating human breast cancer cells is synergistic and suggest a new therapeutic approach to prevent breast cancer progression by eliminating breast cancer stem cells.

Figure 1

Combination of radiation and sorafenib induces synergistic apoptosis and G2/M cell cycle arrest in breast cancer cells. (A) MDA-MB-231 and MCF-7 cells (1×10⁴ cells/well) were seeded in 96-well microplates and treated with increasing doses of radiation and concentrations of sorafenib as indicated. After 72 h (radiation) and 48 h (sorafenib) of incubation, respectively, cell viability was assessed by the MTT assay, and the concentration that induced 50% growth inhibition (IC₅₀) was determined to be of ~10 Gy radiation and 5 μg/ml sorafenib for both cell lines. (B) Cells were treated with radiation (RT, 10 Gy), sorafenib (S, 5 μg/ml), radiation + sorafenib (RT + S, 10 Gy + 5 μg/ml) or DMSO. After 72 h, the percentage of Annexin V/PE-positive cells was quantified using flow assisted cell sorting. (C) Cells were treated with radiation (RT, 10 Gy), sorafenib (S, 5 μg/ml), radiation + sorafenib (RT + S, 10 Gy + 5 μg/ml) or DMSO for 24 h and analyzed by flow cytometry for the cell cycle analysis. Mean percentages ± standard deviation (SD) of cells in each cycle phase are shown. Data are means of three separate experiments ^*p<0.001, ^**p<0.01.

Figure 2

Combination of sorafenib and radiation inhibits the CD44⁺CD24^−/low cell population and mammosphere formation. (A) MDA-MB-231 and MCF-7 cells were treated with or without sorafenib (S, 5 μg/ml) combined with radiation (RT, 10 Gy) under 21% O₂ (normoxia) or 1% O₂ (hypoxia). After 72 h, a flow assisted cell sorting analysis was conducted using specific surface markers for basal (CD44-APC) and luminal (CD24-FITC) epithelial cells and the percentage of cells (CD44⁺CD24^−/low) of each cell line was evaluated in three independent experiments. (B) Cells were seeded to form primary mammospheres at a density of MCF-7 (5,000 cells/ml) and MDA-MB-231 (600 cells/ml) and secondary mammospheres at a density of MCF-7 (1,000 cells/ml) and MDA-MB-231 (300 cells/ml). Additionally, primary mammospheres were incubated with radiation (RT, 10 Gy), sorafenib (S, 5 μg/ml), radiation + sorafenib (RT + S, 10 Gy + 5 μg/ml) or DMSO for 7–10 days, whereas secondary mammospheres were not treated with radiation or sorafenib under hypoxia or normoxia for 7–10 days. White and black bars indicate MDA-MB-231 and MCF-7 cells, respectively. Data are means of three separate experiments; bar, standard deviation (SD). ^*p<0.001, ^**p<0.05. (C) Representative images from primary mammospheres.

Figure 3

Hypoxia inducible factor (HIF)-1α from CD44⁺CD24^−/low cells was highly induced and the combination of sorafenib and radiation blocked HIF-1α in MDA-MB-231 cells under hypoxic conditions. (A) CD44⁺CD24^−/low cells were sorted by flow cytometry. Then, sorted cells were cytospun, fixed, permeabilized and immunostained with antibodies to HIF-1α and vascular endothelial growth factor (VEGF). (B) Cells were treated with radiation (RT, 10 Gy), sorafenib (S, 5 μg/ml), radiation + sorafenib (RT + S, 10 Gy + 5 μg/ml) or DMSO for 72 h. Cells were lysed and protein expression was detected by western blotting. (C) Quantification of the results from western blotting shown in (B) (normalized by the β-actin value).

Figure 4

Combination of sorafenib and radiation highly reduced MMP-2 secretion from metastatic MDA-MB-231 rather than from non-metastatic MCF-7 breast cancer cells. (A) Cells were treated with radiation (RT, 10 Gy), sorafenib (S, 5 μg/ml), radiation + sorafenib (RT + S, 10 Gy + 5 μg/ml) or DMSO for 72 h. The cells were lysed and protein expression was detected by western blotting. (B) Quantification of the results from western blotting shown in (A) (normalized by the β-actin value).