Fig. 7.

Luteolin suppresses MPA-induced stem cell-like properties of T47-D breast cancer cells. a, b Luteolin suppresses MPA-induced breast cancer cell CD44 expression. T47-D cells were incubated at 37 °C for 24 h with 10 nM MPA, 10 or 25 µM luteolin (LU), 1 µM RU-486 (RU), or 10 nM MPA + 10 µM LU, 25 µM, or 1 µM RU-486. Following treatment, cells were harvested and labeled with CD44-APC and CD24-PE antibodies and analyzed by FACS. a Upper panel Displays flow cytometry data for control (DMSO only), 10 nM MPA, 10 nM MPA + 25 µM LU, and 10 nM MPA + 1 µM RU-486. b Lower panel Quantitative data from three different FACS analysis experiments. Results represent mean ± SEM (n = 3). *Significantly different from control. **Significantly different from MPA (P < 0.05, ANOVA). b Luteolin inhibits MPA-induced ALDH^bright induction. T47-D cells were treated with 10 nM MPA ± 25 µM LU or 25 µM LU alone at 37 °C for 24 h. Following treatment, cells were harvested and ALDH activity measured by FACS analysis using the ALDEFLUOR assay. Upper panel Representative flow cytometry data; ALDH^bright cells are shown in red. The ALDH^bright gate was set using negative controls DEAB+. Viable cells were gated using PI; once the gates were set, they were applied throughout the analyses. Lower panel Quantitative data from four different ALDEFLUOR assay experiments. Results represent mean ± SEM (n = 4). *Significantly different from control (DMSO only). **Significantly different from control and MPA (P < 0.05, ANOVA followed by a Newman–Keuls multiple comparison test). c Luteolin suppresses MPA-induced breast cancer cell mammosphere formation. T47-D cells were treated at 37 °C for 48 h with 10 nM MPA, 10 nM MPA + 25 µM LU, or 25 µM LU alone. Following treatment, cells from each group were seeded into six-well plates (5000 cells/well) and treated for seven more days in Complete MammoCult medium. Cells were re-treated with the agent(s) of interest in 1 ml culture medium on days 2, 4, and 6. Upper panel Representative light microscopic images of T47-D mammospheres formed after 7 days. Scale bar 60 μm. Lower panel Results represent mean number of mammospheres ± SEM (n = 3). Number of mammospheres ≥60 μm was quantitated from 6 to 9 images per well, three wells per group. *Significantly different from control (DMSO only) (P < 0.05); **Significantly different from MPA (P < 0.05, ANOVA on ranks followed by Dunn’s method of multiple comparisons)