The MCF-12A cell line is a non-tumorigenic spontaneously immortalized adherent human breast epithelial cell line and forms domes in confluent cultures. The MCF-12A cells were a gift from Professor Parker (Department of Medical Biochemistry, University of Cape Town, South Africa).
All required reagents of cell culture analytical grade were purchased from Sigma (St. Louis, United States of America) unless otherwise specified. Heat-inactivated fetal calf serum (FCS), sterile cell culture flasks and plates were purchased from Sterilab Services (Kempton Park, Johannesburg, South Africa). Penicillin, streptomycin and fungizone were obtained from Highveld Biological Pty (Ltd) (Sandringham, South Africa). The Annexin V fluorescein isothiocyanate (FITC) kit, MitoCapture TM Mitochondrial apoptosis detection kit and a rabbit polyclonal anti-LC3B conjugated to DyLight 488 were purchased from BIOCOM biotech (Pty) Ltd. (Clubview, South Africa). 2,7-Dichlorofluorescein diacetate and hydroethidine was acquired from Sigma (St. Louis, United States of America). Since 2MEBM is not commercially available, it was synthesized by Professor Vleggaar from the Department of Chemistry (University of Pretoria, Pretoria, South Africa).
Cells were grown in sterile 25 cm2 tissue culture flasks at a humidified atmosphere at 37°C and 5% CO2. MCF-12A cells were cultured in medium consisting of a 1:1 mixture of Dulbecco’s minimum essential medium eagle (DMEM) and Ham’s-F12 medium, 20 ng/ml epidermal growth factor, 100 ng/ml cholera toxin, 10 μg/ml insulin and 500 ng/ml hydrocortisone, supplemented with 10% heat-inactivated fetal calf serum (56°C, 30 min), 100U/ml penicillin G, 100 μg/ml streptomycin and fungizone (250 μg/l).
A stock solution of 2x10-3 M 2MEBM dissolved in dimethyl sulphoxide (DMSO) was prepared and diluted with medium to the desired concentrations prior to exposure of the cells. Media of all control experiments were supplemented with an equal volume of DMSO (vehicle) with the DMSO content of the final dilutions never exceeding 0.05% (v/v). Experiments entailed seeding 500 000 exponentially growing MCF-12A cells per 25 cm2 cell culture flasks containing a final volume of 5 ml of maintenance medium. A 24 h incubation period at 37°C was allowed for cell adherence, the medium was discarded and cells were exposed to 0.4 μM 2MEBM and incubated for 48 h at 37°C. These conditions were selected since previous studies in our laboratory have demonstrated successful antiproliferative activity in tumorigenic cell lines [5, 6].
Cell cycle progression
Flow cytometry was utilized to investigate the DNA content to determine cell cycle distribution, G2/M block and the presence of a sub-G1 apoptotic peak. After 48 h of exposure to 0.4 μM 2MEBM, cells were trypsinized and resuspended in 1 ml growth medium. 1x106 cells were centrifuged for 5 min at 300xg. The pellet resuspended twice in ice-cold phosphate buffer solution (PBS). The supernatant was discarded and the cells were resuspended in 200 μl of ice-cold PBS containing 0.1% FCS. Ice-cold 70% ethanol (4 ml) was added in a drop wise manner and cells were stored at 4°C for 24 h. After 24 h, cells were pelleted by centrifugation for 5 min. The supernatant was removed and cells were resuspended in 1 ml of PBS containing propidium iodide (40 μg/ml) and incubated at 37°C, 5% CO2 for 45 min. Subsequently, cells were analysed by means of FACS FC500 System flow cytometer (Beckman Coulter South Africa (Pty) Ltd) equipped with an air-cooled argon laser excited at 488 nm. Data from at least 10 000 cells were captured with CXP software [Beckman Coulter South Africa (Pty) Ltd, Johannesburg, Gauteng, South Africa] and analyzed with Cyflogic (CyFlo Ltd., Turku, Finland).
Mitochondrial membrane potential
Mitochondrial integrity was investigated using the MitoCapture TM mitochondrial kit. The mitochondrial (intrinsic) apoptosis pathway is activated by stress signals that results in the permeabilization of the mitochondrial outer membrane. This will be demonstrated by the MitoCapture TM mitochondrial kit that supplies quantitive apoptosis data . After 48 h of exposure to 0.4 μM 2MEBM, cells were trypsinized and centrifuged at 13 000 x g. Cells were resuspended in 1 ml MitoCapture solution (1 μl MitoCapture: 1 ml pre-warmed incubation buffer provided by the MitoCaptureTM mitochondrial kit), incubated at a humidified atmosphere (37°C, 5% CO2) for 20 min and subsequently centrifuged at 500 x g. After the supernatant was discarded, cells were resuspended in 1 ml prewarmed incubation buffer (37°C). Afterwards, cells were analyzed immediately using fluorescence activated cell sorting (FACS) FC500 System flow cytometer (Beckman Coulter South Africa (Pty) Ltd). Apoptotic cells were detected in the FITC channel (usually FL1) showing diffused green fluorescence. Data from at least 10 000 cells were analysed using CXP software [Beckman Coulter South Africa (Pty) Ltd, Johannesburg, Gauteng, South Africa] and analyzed with Cyflogic (CyFlo Ltd., Turku, Finland).
Flow cytometry utilizing the rabbit polyclonal anti-LC3B conjugated to DyLight 488 demonstrated possible autophagy induction. LC3-I is converted to LC3-II by a series of reactions . Enhancement of LC3-I conversion to LC3-II results in the upregulation of autophagy. Thus, detection of LC3-II is a useful indication for the presence of autophagy . After 48 h of exposure to 0.4 μM 2MEBM cells were trypsinised and centrifuged. Cells were washed with cold PBS, pelleted and fixed with 3 ml 0.01% formaldehyde in PBS for 10 min at 4°C. Cells were centrifuged and resuspended in 200 μl PBS, followed by 1 ml ice-cold methanol (−20°C) for 15 min at 4°C. Afterwards cells were washed twice with cold PBS. Cells were stained with 0.5 ml of the antibody cocktail (0.05% Triton X-100, 1% bovine serum albumin (BSA), 40 μg/ml propidium iodide and 0.5 μg/ml conjugated rabbit polyclonal anti-LC3B antibody) prepared in PBS for 2 h at 4°C. Cells were washed trice with PBS/0.05% Triton/1% BSA and analyzed by means of flow cytometry. Data from at least 10 000 cells were analyzed employing Cyflogic version 1.2.1 software (Pertu Therho, Turko, Finland).
Reactive oxygen species generation
Hydrogen peroxide (H2O2) generation was assessed using 2,7-dichlorofluorescein diacetate (DCFDA), a non-fluorescent probe, which, upon oxidation by ROS and peroxides is converted to the highly fluorescent derivative 2,7-dichlorofluorescein (DCF). Superoxide generation was assessed using hydroethidine (HE) that is oxidized by superoxide, to a red fluorescent compound. After 0.4 μM 2MEBM exposure for 48 h exposure, cells were trypsinized and 1x106 cells were resuspended in 1 ml PBS and washed with PBS. Cells were incubated with 20 μM DCFDA for 25 min and 10 μM HE for 15 min at 37°C. DCF (FL1) and HE fluorescent product fluorescence (FL2) were measured with a FACS FC500 System flow cytometer equipped with an air-cooled argon laser excited at 488 nm. The information generated from at least 10 000 cells were analyzed by means of Cyflogic version 1.2.1 software (Pertu Therho, Turko, Finland).
Measurement of FITC-, HE- and DCF-derived fluorescence was expressed as a ratio of the value measured for the 2MEBM-treated cells compared to vehicle-treated exposed cells (mean relative fluorescence). Flow cytometry analysis involved data from at least 10 000 events that was repeated thrice where after a representative figure was chosen for each experiment. Flow cytometry data were analyzed by means of Cyflogic version 1.2.1 software (Pertu Therho, Turko, Finland).