Cell and mammosphere Culture
Two breast cancer cell lines, MCF-7 and MDA-MB-231, were obtained from the American Type Culture Collection (Rockville, MD, USA). All human breast cancer cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, CA, USA) and 1% penicillin/streptomycin (HyClone, Thermo Fisher Scientific). Cancer cells (3.5x104 or 0.5x104) were incubated in an ultralow attachment 6-well plate in MammoCult culture media (STEMCELL Technologies, Vancouver, BC, Canada). Breast cancer cells were cultured in an incubator containing 5% CO2 under 37°C. The formed mammospheres were counted by using the NICE program . Ability of mammosphere formation was estimated by determining the mammosphere formation efficiency (MFE) (%) as in a previous study .
Antibodies and small interfering RNAs (siRNAs)
Antibody-specific against YAP, pYAP, and GR proteins were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-ubiquitin, anti-β-actin, and anti-Lamin b antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-CD44 FITC and anti-CD24 PE antibodies were purchased from Abcam (Cambridge, MA, USA). The GR- and YAP-specific siRNAs were obtained from BIONEER (Daejeon, Korea).
Cell proliferation assay was used as in a previous study . Breast cancer was incubated in a 96-well plate in the presence of ciclesonide for 1 day. Cell proliferation was tested using a CellTiter 96® Aqueous One Solution cell kit (Promega, Madison, WI, USA), and the optical density of 490 nm was analyzed using a 96-well plate reader (SpectraMax, San Jose, CA, USA).
Colony formation and migration assays
MDA-MB-231 cancer cells (1,000 cells/well) were incubated with ciclesonide for 7 days in DMEM containing 10% FBS and 1% penicillin/streptomycin. The colonies were incubated and counted. For the migration assay, breast cancer cells were incubated in a 24-well plate, and a scratched line in a cell monolayer was made using a pipette tip .
Annexin V/PI staining and cell apoptosis
For the Annexin V/PI assay, breast cancer cells were incubated with ciclesonide (20 μM) in 6-well plates. Apoptotic cells were analyzed by FITC-Annexin V/PI staining according to the manufacturer’s protocol (BD, San Jose, CA, USA). The stained cells were detected by flow cytometry. For Hoechst 33258 staining, cancer cells were cultured with 20 μM ciclesonide for 24 h, and were stained with Hoechst 33258 (10 mg/ml) solution for 30 min at 37°C. The stained cells were detected under a fluorescence microscope (Lionheart FX, BioTek, Winooski, VT, USA).
Flow cytometric analysis and aldehyde dehydrogenase (ALDH1) activity
Flow cytometric analysis was used in a previous study . Cancer cells (1x106) were incubated with anti-CD44-FITC and anti-CD24-PE antibodies (Abcam, Cambridge, MA, USA) for 20 min. The cells were centrifuged and washed three times with 1x FACS buffer and analyzed using an Accuri C6 (BD, San Jose, CA, USA). ALDH activity was assayed using an ALDEFUORTM assay kit (STEMCELL Technologies) used in a previous study . Cancer cells were incubated in ALDH assay buffer at 37°C for 30 min. ALDH-positive population was examined by using flow cytometer.
Gene expression analysis
Total RNA from cancer cells and mammosphere were extracted and purified, and real-time reverse transcription-quantitative PCR (RT-qPCR) was tested using a one-step RT-qPCR kit (Enzynomics, Daejeon, Korea) used as in a previous study . The specific primers are shown in online resource, supplementary Table S1.
Breast cancer cells and mammospheres were lysed with RIPA buffer containing protease inhibitors and total proteins were quantified with BCA protein quantitation kit. The protein samples were electrophoresed on 12% SDS gel. The gel with separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes (EMD Millipore, Burlington, MA, USA). Membranes were incubated first in Odyssey blocking buffer for 1 h and then overnight with primary antibodies. The anti-GR, anti-YAP, anti-pYAP, anti-lamin B, and anti-β-actin antibodies were used. After washing and drying, membranes were incubated with IRDye 680RD- and 800W-conjugated secondary antibodies, and images were acquired using an ODYSSEY CLx (LI-COR, Lincoln, NE, USA).
Caspase-3/7 assay was used in a previous study . Cancer cells were cultured with ciclesonide. Caspase-3/7 activity was measured using a Caspase-Glo 3/7 kit (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Then, 100 μl of caspase reagent was added to 96-well microplates and incubated, and the activity was assayed in a GloMax® luminometer (Promega, Madison, WI, USA).
SiRNA of GR and YAP
To examine the inhibitory function of GR and YAP on mammosphere formation, breast cancer cells were transfected with siRNAs targeting human GR and YAP (Bioneer, Daejeon, South Korea). The GR and YAP siRNAs (NM_181651.1) and a scrambled siRNA were purchased from Bioneer (Daejeon Cor., South Korea). For siRNA transfection, cancer cells were cultured and transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The protein levels of GR and YAP were determined via immunoblot analysis.
Mammospheres were washed with 1x PBS and resuspended in lysis buffer. The lysates were incubated with an anti-ubiquitin antibody for 16 h at 4°C. Protein A/G agarose (Thermo Scientific, IL, USA) was added to the mixtures. The mixtures were centrifuged, and the precipitates were washed with lysis buffer 5 times, run on SDS-PAGE gels and subjected to immunoblotting.
Cancer cells were fixed with 3.8 % paraformaldehyde for 40 min, permeabilized with 0.5% Triton X-100 for 15 min, blocked with 3% bovine serum albumin (BSA) for 1 hour and labeled with an anti-GR primary antibody followed by an Alexa Fluor 488-conjugated anti-mouse secondary antibody. We used nonspecific signal conditions to confirm the specificity of the primary antibodies for immunofluorescence. The nuclei were stained with DAPI, and stained GR was visualized with a fluorescence microscope (Lionheart, BioTek, VT, USA).
In vivo mice experiments
Twelve female nude mice were injected with three million MDA-MB-231 cells and after one week, injected with/without ciclesonide (10 mg/kg). Tumor volumes were examined for 45 days using the following formula: (width2 x length)/2. The mouse experiments were used as in a previous study . Animal experimentation was performed in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Jeju National University (JNU-IACUC; Approval Number 2017-003). Female nude mice (5 weeks old) were obtained from OrientBio (Seongnamsi, South Korea) and maintained in mouse facility for 1 week.
Our all data were processed using GraphPad Prism 7.0 software (GraphPad Prism Inc., San Diego, CA, USA). Our all data values are presented as the means ± standard deviations (SDs). Our data were analyzed with one-way ANOVA. P-values of less than 0.05 or 0.01 were considered to indicate significance.