Cell culture experiments
Human breast epithelial adenocarcinoma cell lines (MCF7, MDA-MB-231, MDA-MB-436), and a human ovarian adenocarcinoma cell line (OVCAR-5) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Human breast ductal carcinoma cell line; HCC1937 and HCC1937 reconstituted with wild type BRCA1 (HCC1937/wt BRCA1) were kind gifts from Dr. Grant Mc Arthur, Peter MacCallum Cancer Centre, VIC, Australia. All the cell lines were maintained in culture medium containing 10 % Fetal Bovine Serum (FBS) (PAN-Biotech GmBH, Aidenbach, Germany), 0.1 g/L Streptomycin (Sigma Aldrich, St. Louis, MO, USA) and 100U/L Penicillin in a humidified incubator at 37 °C in 5 % (v/v) CO2. MCF7, MDA-MB-231 and OVCAR-5 were maintained in Dulbecco’s Modified Eagles’ Medium (Gibco, Carlsbad, CA, USA), MDA-MB-436 in RPMI 1640 (PAN-Biotech GmBH, Aidenbach, Germany) and HCC1937 and HCC1937/wt BRCA1 in RPMI 1640 with insulin (5 μg/ml). Passage numbers post thawing of the cell lines were noted and experiments were carried out within a maximal passage number (<30).
Cell viability assay
MTT assay to assess the effects of 48 h treatment with PB and CP on HCC1937 and HCC1937/wt BRCA1 was performed as described elsewhere . 3–(4,5-Dimethyl thiazol-2-yl) 2,5-diphenyl tetrazolium bromide (MTT) was purchased from USB, Cleveland, OH, USA, PB and CP were from Sigma Aldrich, St. Louis, MO, USA. CP solutions were prepared in phosphate buffered saline (PBS) immediately before the experiment. PB was dissolved in Dimethyl Sulfoxide (DMSO) and the vehicle control (DMSO) did not show any effect on cell viability. All results are expressed as the percentage cell viability over control ± S.D. of quadruplicate determinations from three independent experiments.
Side population analysis
Cells were stained with 5 μg/ml of Hoechst 33,342 (Sigma Aldrich, St. Louis, MO, USA) for 90 min in a 37 °C water bath. Cells stained with 5 μg/ml Hoechst in the presence of 50 μM Verapamil Hydrochloride (calcium channel blocker that prevents Hoechst efflux from cells) (Sigma Aldrich, St. Louis, MO, USA) was used as negative control. The cells were then stained with propidium iodide (PI) (2 μg/106 cells) for dead cell exclusion and sorted using BD FACSAriaII flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA). Hoechst 33,342 stained cells were analyzed using 350 nm excitation with blue (635 nm) and red (488 nm) emission. BD FACSDiva Software was used for analysis.
Surface marker profiling
Cells were stained with Mouse anti-human CD44-APC (C26) and CD24-FITC (ML5) antibodies (BD Biosciences (San Jose, CA., USA)) for 30 min on ice. Cells stained with CD44-APC and CD24-FITC separately (single color) were taken as the controls. The 633 nm and 488 nm lasers were used for excitation and 530/30 nm BP and 660/20 BP detectors were used for CD24-FITC and CD44-APC respectively. The CD44+/24–/low (putative stem cell) population was identified and analyzed using flow cytometry as mentioned above.
The ALDEFLUOR assay (The ALDEFLUOR Assay kit, Stem Cell Technologies (Durham, NC, USA)) was used to identify and isolate the ALDH+ cells from the various cell lines. Briefly, cells in growing conditions or treated with PB or CP for 48 h were suspended in ALDEFLUOR assay buffer containing ALDH substrate (BODIPY-aminoacetaldehyde or BAAA, 1 μmol/l per 1 × 106 cells) and incubated for 40 min at 37 °C in a water bath to assess the ALDH enzymatic activity. As negative control, an aliquot was treated with 50 mmol/l diethylaminobenzaldehyde (DEAB), a specific ALDH inhibitor for each sample of cells. The ALDH1 positive (ALDH1+) and ALDH1 negative (ALDH1-) populations were sorted out by flow cytometry using the 488 nm laser and the 530/30 nm BP detector as mentioned above.
In vitro propagation of BCSCs: mammosphere culture
Cells were seeded for primary mammosphere formation at 2000 cells/ml in ultralow attachment plates (Corning Inc., Corning, NY, USA) in MEBM (Mammary Epithelial Basal Medium Bullet kit from Lonza (Basel, Switzerland)) containing insulin (5 μg/ml) hydrocortisone (1 μg/ml), EGF (10 ng/ml), bFGF (20 ng/ml) (BD Biosciences, San Jose, CA), 2 % B27 serum free supplement (Life Technologies, Carlsbad, CA, USA) and heparin (4 μg/ml) (Sigma Aldrich, St. Louis, MO, USA). Medium was supplemented every 4 days. Mammospheres (>50 μm) were counted and photographed on day 7. The cells were treated with sub-cytotoxic concentrations of PB (single dose) and cultured for 7 days to study the sphere forming efficiency and ABCG2 expression. During the treatment, growth medium without PB was supplemnted every 4 days.
3 D culture of spheroids
In order to recapitulate the mammosphere data in a system that involves the extracellular matrix components as would be seen in vivo, cells were seeded at 10,000 cells/ml in RPMI with insulin (5 μg/ml /ml), hydrocortisone (1 μg/ml), EGF (10 ng/ml) and 5 % Growth Factor Reduced Matrigel (GFRM) to 8-well chamber slides (BD Falcon, San Jose, CA, USA) pre-coated with 50 μl GFRM (BD Biosciences, San Jose, CA, USA). 0.5 μM PB was added to the cells. Cells were viewed on Day 5, Day 7, Day 10 and Day 15 using the Olympus IX71 microscope and photographed.
Day 7 mammospheres were washed with Phosphate Buffered Saline (PBS) and fixed in 4 % paraformaldehyde. Following permeabilization with 0.25 % Triton × 100, and blocking with 1 % Bovine Serum Albumin (BSA), spheres were incubated overnight at 4 °C in the primary antibody. After PBS wash and incubation with fluorochrome tagged secondary antibody for 2 h, spheres were mounted in Prolong Gold Antifade with DAPI (Life Technologies, Carlsbad, CA, USA) and viewed using the Olympus IX71 microscope. Immunofluorescence analysis was performed with Rabbit anti-human Snail + Slug, rabbit anti-human β catenin (E247), rabbit anti-human α-SMA (Smooth Muscle Actin), and goat anti-human Oct 4 purchased from Abcam (Cambridge, MA, USA), goat anti-human Vimentin (C20), rabbit anti-human BRCA1, mouse anti-human ABCG2 (6D17) from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
ROS production in mammospheres was assessed from the levels of bright green colored 2′,7′-dichlorofluorescein (DCF), produced by the oxidation of DCF-DA (2′,7′-Dichlorodihydrofluorescein diacetate) dye by ROS induced in mammospheres after a 10 min treatment with 1 μM PB followed by exposure to 500 nM DCF-DA (Sigma Aldrich, St. Louis, MO, USA) (stock dissolved in Dimethyl formamide and further diluted in PBS) for 20 min in dark at 37 °C. The green fluorescence was detected by the Leica DMI 6000B microscope at 495/529 nm.
A single gel electrophoresis method for comet assay with modifications as described by Olive et al., 2006 was done. Briefly, cells treated with PB for 4 h were lysed using neutral lysis buffer overnight at 37 °C and seeded on to low melting agarose gel in a frosted slide. Then electrophoresis was done in Tris Borate EDTA buffer for 30 min at 50 V. The slides were then stained with propidium iodide and observed under an Olympus 1X71 fluorescent microscope for comets. The comets formed were scored using Casplab software.
Cell lysates were analyzed for expression of ABCG2, β-catenin and β-actin. Whole cell lysates were prepared in RIPA buffer supplemented with protease inhibitor cocktail. Supernatant was collected by centrifugation at 13,000 rpm for 25 min at 4 °C. Samples were then subjected to sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membranes and immunoblotted. Protein bands were observed by Enhanced Chemiluminescence detection (Amersham, Piscataway, NJ, USA) of the specifically bound antibody. The quantitation was done by densitometric analysis by “Quantity One” software.
Six-week old female Severe Combined Immunodeficient (SCID) mice with tumors left untreated for 28 days were randomized into two groups (ten animals in a group). Group 1 was treated with 25 % polyethylene glycol (PEG) alone. Group 2 was treated with PB in 25 % PEG at doses of 1 mg/kg/day intraperitoneally. Tumors were measured twice a week, and primary tumor volumes were calculated using the formula V = 1/2a x b2, where ‘a’ is the longest tumor axis, and ‘b’ is the shortest tumor axis.
Immunohistochemical analysis (IHC)
After 3 weeks of treatment, all OVCAR-5 tumor-bearing mice were sacrificed by asphyxiation with CO2; tumors were removed, measured, and prepared for IHC. Tumors were removed and fixed in neutral buffered 10 % formalin at room temperature for 24 h. Subsequently, the samples were embedded in paraffin and sections were taken. Sections were deparaffinized and then subjected to Oct 4 (Cell Signaling Technology, Beverly, MA), Vimentin (Clone V9, Chemicon International, Temcula, CA) and N-cadherin (Clone 13A9, Santa Cruz Biotechnology, Santa Cruz, CA) according to the manufacturer’s instructions (DAB 150, (Millipore, Billerica, MA). The secondary anti-mouse antibody was used before adding chromogen substrate. Stable diaminobenzidine was used as a chromogen substrate, and the sections were counterstained with a hematoxylin solution. Photographs of the entire cross-section were digitized using an Olympus camera (DP70).
The independent-sample paired two tailed student t-test was used to test the probability of significant differences between different experimental groups. FACS results were expressed as mean ± S.D from at least three independent experiments. Statistical significance was defined as (*) p ≤ 0.05 and (**) p ≤ 0.005. Error bars were given on the basis of calculated S.D values. All experiments were repeated at least thrice.