Reagents
Luteolin (2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4H-1-benzopyran-4-one) (LU) was purchased from Tocris (Minneapolis, MN, USA) and dissolved in sterile filtered dimethyl sulfoxide (DMSO; Sigma-Aldrich; St. Louis, MO, USA). Medroxyprogesterone acetate (MPA), progesterone, norethindrone, norgestrel, and RU-486 were purchased from Sigma-Aldrich. Pierce bicinchoninic acid protein reagents were obtained from Fisher Scientific (Pittsburgh, PA, USA). 17-β estradiol (E2; 1.7 mg), MPA (10 mg), and placebo 60-day release pellets were obtained from Innovative Research of America (Sarasota, FL, USA).
Cell lines and culture
Hormone-responsive BT-474 and T47-D human breast cancer cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA) and maintained at 37 °C in phenol red-free DMEM/F12 medium (Invitrogen, Waltham, MA, USA) supplemented with 10 % fetal bovine serum (FBS; Sigma-Aldrich) in a humidified atmosphere of 5 % CO2. For all in vitro experiments, cells were maintained in DMEM/F12 supplemented with 5 % dextran-coated charcoal (DCC)-stripped FBS for 24 h prior to treatment. Subsequently, cells were washed and further incubated in fresh 5 % DCC-stripped FBS-DMEM/F12.
Cell viability assay
Viable cells were quantitated using sulforhodamine B (SRB) assays (Skehan et al. 1990). In brief, breast cancer cells in 100 µl DMEM/F12/10 % FBS medium were seeded into each well of a 96-well plate and incubated at 37 °C overnight in 5 % CO2. Cells were treated (in six replicates) with either LU or DMSO (controls) in DMEM/5 % FBS for periods up to 48 h, then subjected to SRB assays.
Apoptosis assay
Apoptosis was evaluated by staining with Annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI) as described previously (Liang et al. 2006). T47-D cells were grown to 50–60 % confluence in DMEM/F12/10 % FBS, at which point the media was switched to 5 % DCC-stripped FBS-DMEM/F12. After 24 h, cells were treated with LU ± MPA for an additional 16 h. Treated cells were harvested using 0.05 % trypsin–EDTA, stained, and subjected for fluorescence-activated cell sorting (FACS) analysis per the manufacturer’s protocol (BioVision Inc, Milpitas, CA, USA).
VEGF enzyme-linked immunosorbent assay (ELISA)
The Quantikine human VEGF ELISA kit was purchased from R&D Systems, Inc. (Minneapolis, MN, USA). Supernatant from treated cells was collected and VEGF concentrations measured according to the manufacturer’s protocol. Experiments were performed in triplicate, and each sample was analyzed in duplicate on a microplate reader. Inter- and intra-assay coefficients of variance given by the manufacturer for cell culture supernatant assays are 5–8.5 and 3.5–6.5 %, respectively.
Bicinchoninic acid protein assay
Cells were harvested and pellets resuspended in 300 µl lysis buffer (50 mM Tris/HCl, pH 8, 150 mM NaCl, and 1 % Nonidet P-40). Protein concentration was determined by measuring absorbance at 562 nm on a microplate reader, using bovine serum albumin (Thermo Fisher Scientific; Waltham, MA, USA) as standard. Experiments were performed in triplicate, and samples were analyzed in duplicate.
Reverse transcription-polymerase chain reaction (RT-PCR)
RNA from progestin-treated cells was purified and RT-PCR conducted as described previously (Mafuvadze et al. 2010). The primers used were:
VEGF
$${\text{F}} \quad 5^{\prime} {\text{-}} {\text{CTGCTGTCTTGGGTGCATTGG}}$$
$${\text{R}} \quad 5^{\prime} {\text{-}} {\text{CACCGCCTCGGCTTGTCACAT}}$$
Glyceraldehyde phosphate dehydrogenase (GAPDH)
$${\text{F}} \quad 5^{\prime} {\text{-}} {\text{ATGAGA AGTATGACAGCC}}$$
$${\text{R}} \quad 5^{\prime} {\text{-}} {\text{TGAGTCCTTCCACGATACC}}$$
FACS analyses
For all FACS analysis, treated breast cancer cells were harvested using Accutase (BD Biosciences; San Jose, CA, USA) in place of trypsin–EDTA. Cells (1 × 106) were then suspended in 100 µl staining buffer and placed in microcentrifuge tubes.
Phycoerythrin (PE)-conjugated mouse anti-human CD24 (20 µl) and allophycocyanin (APC)-conjugated mouse anti-human CD44 (20 µl) antibodies (both from BD Biosciences) were added to each sample, along with the necessary FACS dye controls, and the samples incubated on ice for 45 min. Cells were washed twice in staining buffer, resuspended in 500 µl staining buffer and 1 μl of 250 μg/ml PI, and subjected to FACS analysis.
Aldehyde dehydrogenase (ALDH) activity was assessed using the ALDEFLUOR kit (STEMCELL Technologies Inc.; Vancouver, BC, Canada), according to the manufacturer’s protocol. All samples were processed within 15 min of the final wash. Cells were visualized using a Beckman Coulter CyAn ADP FACS machine running Summit 5.2 software and results analyzed as previously described (Ginestier et al. 2007).
Mammosphere-formation assay
T47-D cells were grown in 10 % FBS DMEM/F12 medium, then cultured in 5 % DCC-stripped FBS-DMEM/F12 medium for 24 h. Cells were then treated for 48 h with indicated agent(s) of interest. Cells from each group were subsequently seeded into six-well plates (5000 cells/well) in Complete MammoCult medium (STEMCELL Technologies Inc.) and treatment continued for six more days. Culture medium (1 ml) was refreshed on days 2, 4, and 6 to ensure drug availability. Light microscopy (10×) pictures of mammospheres formed were captured after 7 days using an EVOS light microscope. Mammospheres ≥60 µm in diameter (determined by size exclusion) were counted.
Human breast cancer cell xenograft studies
Xenograft experiments were performed as described previously (Liang et al. 2007). All facilities were approved by the American Association for Accreditation of Laboratory Animal Care in accordance with current federal regulations and standards. In brief, an E2 60-day release pellet (1.7 mg) was implanted in each nude mouse. Two days later, T47-D cells were suspended in DMEM/F12 medium and injected subcutaneously (1 × 107 cells per 150 µl) into each flank of nude mouse (n = 2–4 animals/group). Mice were then implanted with a 60-day release MPA (10 mg) or placebo pellet 10 days after breast cancer cell injection. When tumors reached about 60 mm3, intraperitoneal treatment with LU (20 mg/kg/day) or vehicle commenced. LU was administered daily for 2 days (loading dose), then every other day until day 79.
Immunohistochemical analyses
Xenograft tumor-bearing mice were sacrificed at day 79 and tumors collected and processed for immunohistochemical analysis as previously described (Liang et al. 2007). Tumors were collected from both flanks of each mouse and at least three tumors per group were collected for analysis. One section from each individual tumor was subjected to immunohistochemical staining using anti-VEGF (1:100 dilution; Santa Cruz Biotechnology; Dallas, TX, USA), anti-PR, or anti-CD31 polyclonal antibodies, both at 1:50 dilution (DAKO; Carpinteria, CA, USA).
Four random fields were captured from every stained section to minimize errors due to differences in cellularity. Regions of staining within tumors were recorded. Fovea Pro 3.0 software (Reindeer Graphics; Ashville, NC, USA) was used to quantitate the percent area of VEGF staining, while the percent of PR-positively stained cells was calculated using the color threshold in Image J (NIH). This facilitated precise discrimination between positive/negative cells and background. For quantitating blood vessels, five CD31-labeled 10x sections were taken from each tumor to minimize intra-tumoral variation (Liang et al. 2007). The total number of vessels was counted in each section and then averaged per corresponding tumor.
Immunohistochemical staining data were reported as mean ± standard error of the mean (SEM) per treatment group, with each group having an n ≥ 3 tumors analyzed.
Statistical analysis
Statistical significance was tested using one-way analysis of variance (ANOVA) followed by a Newman–Keuls multiple comparison test to determine the difference in mean between groups. A two-way repeated measures ANOVA was used for animal weights. If normality failed, the data were tested using a nonparametric one-way ANOVA on ranks (Kruskal–Wallis), followed by Newman–Keuls comparison test. Data were reported as mean ± SEM. For all comparisons, P ≤ 0.05 was regarded as statistically significant. Analyses were performed using SigmaPlot 12.5 software.