Silencing NOTCH signaling causes growth arrest in both breast cancer stem cells and breast cancer cells

Abstract

Background: Breast cancer stem cells (BCSCs) are characterized by high aldehyde dehydrogenase (ALDH) enzyme activity and are refractory to current treatment modalities, show a higher risk for metastasis, and influence the epithelial to mesenchymal transition (EMT), leading to a shorter time to recurrence and death. In this study, we focused on examination of the mechanism of action of a small herbal molecule, psoralidin (Pso) that has been shown to effectively suppress the growth of BSCSs and breast cancer cells (BCCs), in breast cancer (BC) models.

Methods: ALDH(-) and ALDH(+) BCCs were isolated from MDA-MB-231 cells, and the anticancer effects of Pso were measured using cell viability, apoptosis, colony formation, invasion, migration, mammosphere formation, immunofluorescence, and western blot analysis.

Results: Psoralidin significantly downregulated NOTCH1 signaling, and this downregulation resulted in growth inhibition and induction of apoptosis in both ALDH(-) and ALDH(+) cells. Molecularly, Pso inhibited NOTCH1 signaling, which facilitated inhibition of EMT markers (β-catenin and vimentin) and upregulated E-cadherin expression, resulting in reduced migration and invasion of both ALDH(-) and ALDH(+) cells.

Conclusion: Together, our results suggest that inhibition of NOTCH1 by Pso resulted in growth arrest and inhibition of EMT in BCSCs and BCCs. Psoralidin appears to be a novel agent that targets both BCSCs and BCCs.

Figure 1

Breast cancer stem cells exhibit aggressive growth and resistance to chemotherapeutic agents: (A) Aldefluor assay showing the percentage of ALDH⁻ and ALDH⁺ cells population in MDA-MB-231 cells by flow cytometry analysis. (B) To check cell proliferation, ALDH⁻ and ALDH⁺ 25 × 10³ cells were plated in 24-well plates and allowed to grow for 24 h followed by harvesting and counting of viable cells using a trypan blue exclusion assay. (C) To assess anchorage-independent growth, 5 × 10³ cells (ALDH⁻ and ALDH⁺ cells) were grown in soft agar for 10 days. Colonies were stained with crystal violet and counted manually. (D) A mammosphere assay was performed using 4 × 10³ cells 2 ml⁻¹ of DMEM/Mammocult media in ultra-low attachment plates. The cells were allowed to grow for 2 weeks, and then mammospheres were counted. (E) ALDH⁻, ALDH⁺, and commercial BCSCs were plated in six-well plates and grown until confluent. A uniform wound was created in the center of the monolayer. The wound gap was photographed in a Biostation CT programmed to take pictures every 2 h. The distance between the edges of wound was measured in μM using NIS-Element software, and statistical analysis was performed. (F) A transwell invasion assay was performed with ALDH⁻ cells, ALDH⁺ cells, and commercial BCSCs using Boyden chambers. The invasive cells were stained with crystal violet and counted. Data are expressed as mean±s.e.m. of two independent experiments. Student's t-test was used to calculate statistical significance. *P<0.05, **P<0.005, and ***P<0.0001.

Figure 2

Breast cancer stem cells show resistance to chemotherapeutic agents. (A–C) ALDH⁻ and ALDH⁺ cells were exposed to different concentrations of chemotherapeutic agents (doxorubicin, docetaxel, or 5-FU) for 24 h. Cell viability was measured by MTT assay. Data are expressed as mean±s.e.m. of three independent experiments. Student's t-test was used to calculate statistical significance. *P<0.05, **P<0.005, and ***P<0.0001.

Figure 3

Psoralidin inhibits cell growth, mammosphere formation and induces apoptosis in BC and BCSCs. (A) ALDH⁻ cells, ALDH⁺ cells, commercial BCSCs, and normal breast epithelial cells (MCF-12A) were treated with vehicle (DMSO) or the indicated dose of Pso for 24 h. Cell viability was determined using a trypan blue exclusion assay. Data are expressed as mean±s.e.m. of two independent experiments done in triplicates. (B) Anchorage-independent growth of ALDH⁻ and ALDH⁺ cells was determined by assessing the colony-forming ability of these cells. Approximately, 5 × 10³ cells were treated with Pso at the IC₅₀ value specific to the cell type. Cells were monitored for 10 days, and colonies were stained with crystal violet and counted manually. Data are expressed as mean±s.e.m. of three independent experiments. (C) A mammosphere assay was performed using 4 × 10³ cells (ALDH⁻ and ALDH⁺ cells) on ultra-low attachment plates. Cells were treated with IC₅₀ dose of Pso and allowed to grow for 2 weeks followed by counting of mammospheres. Data are expressed as mean±s.e.m. of two independent experiments. (D) To assess apoptosis induced by Pso, ALDH⁻ cells, ALDH⁺ cells, and commercial BCSCs were treated with Pso for 24 h. Cells were stained with FITC-Annexin-V and propidium iodide and analyzed by flow cytometery. Data are expressed as mean±s.e.m. of three independent experiments. Student's t-test was used to calculate statistical significance. *P<0.05, **P<0.005, and ***P<0.0001. The full colour version of this figure is available at British Journal of Cancer online.

Figure 4

Psoralidin inhibits NOTCH1 signaling and EMT in ALDH⁻ and ALDH⁺ cells. (A) ALDH⁻ and ALDH⁺ cells were treated with vehicle or the IC₅₀ dose of Pso. Total cell lysates were prepared, and equal amounts of protein were subjected to western blot analysis for NOTCH1, HES1, and actin proteins. (B) Total protein lysates were utilized for western blot analysis to determine the expression of the EMT markers E-cadherin, β-catenin, and vimentin. Actin was used as a loading control. (C) E-cadherin, and (D) β-catenin expression and localization were visualized by confocal microscopy in vehicle- and Pso-treated ALDH⁻ and ALDH⁺ cells.

Figure 5

Psoralidin inhibits migration and invasion in BCCs and BCSCs. (A) ALDH⁻ cells, ALDH⁺ cells, and commercial BCSCs were plated in six-well plates and grown until confluent. A uniform wound was created in the center of the monolayer. The wound gap was photographed in a Biostation CT programmed to take pictures every 2 h. The distance between the edges of wound was measured in μM using NIS-Element software, and statistical analysis was performed. Data are expressed as mean±s.e.m. of two independent experiments. (B) A transwell invasion assay was performed with ALDH⁻ cells, ALDH⁺ cells, and commercial BCSCs using Boyden chambers. The cells were treated with vehicle or the IC₅₀ dose of Pso and allowed to migrate towards the lower chamber. The invasive cells were stained with crystal violet and counted. Data are expressed as mean±s.e.m. of three independent experiments. Student's t-test was used to calculate statistical significance. *P<0.05, **P<0.005, and ***P<0.0001.

Figure 6

Psoralidin inhibits pro-survival genes and activates the pro-apoptotic cascade in ALDH⁻ and ALDH⁺ cells. (A) ALDH⁻ and ALDH⁺ cells were treated with vehicle or an IC₅₀ dose of Pso. Total cell lysates were prepared and equal amounts of proteins were subjected to western blot analysis to examine the expression pattern of p65, BAX, and BCL-2. (B) Total cell lysates were subjected to western blot analysis for caspase-3, caspase-9, and PARP. Actin was used as a loading control. (C) ALDH⁻ and ALDH⁺ cells were plated in six-well plates. Cells were transiently transfected with scrambled (Scr) or NOTCH1 siRNA (siNOTCH1) for 36 h. Cells were harvested, and cell proliferation was measured. (D) Whole-cell lysates were prepared and utilized for western blot analysis for NOTCH1, HRT-1, vimentin, β-catenin, and Slug. Actin was used as a loading control. Data are expressed as mean±s.e.m. of two independent experiments. Student's t-test was used to calculate statistical significance. **P<0.005 and ***P<0.0001.