Cell lines
Human cervical adenocarcinoma cell line (HeLa) and a highly metastatic breast adenocarcinoma cell line (MDA-MB-231) were chosen to demonstrate the effects of medium consisting of varying metabolic states. The HeLa cell line was purchased through Sterilab Services (Pty) Ltd, Johannesburg, South Africa, from the American Tissue Culture Collection (ATCC), Maryland, United States of America. The HeLa cell line is the oldest and most distributed immortalized cell line that presents with aggressive growth and doubles on average every 24 h [8]. The primary source of energy in HeLa cells is glutamine rather than glucose, thus demonstrating that oxidative phosphorylation is preferential to generate ATP [9]. Furthermore, HeLa cells are capable of adapting their mitochondrial network, structure and function depending on substrate basis to generate energy exclusively from oxidative phosphorylation by remodelling its mitochondria [10].
The MDA-MB-231 cell line was provided by Microsep (Pty) Ltd Johannesburg, South Africa. MDA-MB-231 is a triple negative tumorigenic breast cell line. This indicates that MDA-MB-231 cells do not express receptors for steroid hormones (estrogen and progesterone), type II receptor tyrosine kinase (RTK) Her-2, but do possess upregulation of basal cytokeratins and epidermal growth factor response [11–13]. Further, the MDA-MB-231 cell line is highly metastatic and presents with elevated glycolytic activity under normoxic conditions [14]. MDA-MB-231 cells mainly use glycolysis rather than mitochondrial respiration to produce energy required for cell functioning and proliferation [15]. Furthermore, MDA-MB-231 cells contain mitochondria presenting with deoxyribonucleic acid (DNA) mutations resulting in decreased oxidative metabolism [16].
General reagents
Actinomycin D, glucose, l-glutamine and sodium pyruvate free-Dulbecco’s minimum essential medium eagle (DMEM), as well as high glucose (25.52 mM, 4500 mg/l), l-glutamine (4 mM) and sodium pyruvate (1 mM, 110 mg/l) containing DMEM, bicarbonate, l-glutamine, glucose, trypsin, crystal violet, NaCl, KCl, KH2PO4 and Na2HPO4, acridine orange and 2,7-dichlorofluorescein diacetate (DCF-DA) were supplied by Sigma Chemical Co. (St. Louis, United States of America). Heat-inactivated fetal calf serum (FCS), sterile cell culture flasks and plates were obtained through Sterilab Services (Kempton Park, Johannesburg, South Africa). Penicillin, streptomycin and fungizone were purchased from Highveld Biological Ltd (Pty). (Sandringham, Gauteng, South Africa).
General cell culture procedures
Cells were grown and maintained in 25 cm2 tissue culture flasks in a humidified atmosphere at 37 °C, 5 % CO2 in a Forma Scientific water-jacketed incubator (Ohio, United States of America). Cells were cultured in DMEM with 25.52 mM glucose, 4 mM l-glutamine, and 1 mM sodium pyruvate supplemented with 10 % heat-inactivated fetal calf serum (56 °C, 30 min), 100 U/ml penicillin G, 100 μg/ml streptomycin and fungizone (250 μg/l). The media according to metabolic state were prepared 24 h before exposure and autoclaved to ensure sterility.
Cells were exposed to varying metabolic conditions as described below:
Control: DMEM with 25.52 mM glucose, 4 mM l-glutamine, and 1 mM sodium pyruvate supplemented with 10 % heat-inactivated FCS (56 °C, 30 min), 100 U/ml penicillin G, 100 μg/ml streptomycin and fungizone (250 μg/l).
Experimental Condition 1: DMEM with 6 mM glucose, 1 mM l-glutamine, and 0 mM sodium pyruvate supplemented with 10 % heat-inactivated FCS (56 °C, 30 min), 100 U/ml penicillin G, 100 μg/ml streptomycin and fungizone (250 μg/l).
Experimental condition 2: DMEM with 3 mM glucose, 0.5 mM l-glutamine, and 0 mM sodium pyruvate supplemented with 10 % heat-inactivated FCS (56 °C, 30 min), 100 U/ml penicillin G, 100 μg/ml streptomycin and fungizone (250 μg/l).
Experimental Condition 3: DMEM with 0 mM glucose, 0 mM l-glutamine, and 0 mM sodium pyruvate supplemented with 10 % heat-inactivated FCS (56 °C, 30 min), 100 U/ml penicillin G, 100 μg/ml streptomycin and fungizone (250 μg/l).
Positive control: Growth medium containing 0.1 μg/ml actinomycin D was used as a positive control to induce cell death via apoptosis.
General experimental procedures for short term exposures and recovery experiments:
Short term exposure: Cells were seeded at 500 000 cells per 25 cm2 flask or 5 000 cells/well into Nunc F96 microwell plates (AEC-Amersham Soc (Ltd), Kyalami, South Africa). After 24 h, cells were exposed to varying metabolic conditions for 2 h, 4 h and 6 h. Thereafter, all experimental methods were conducted as described below.
Recovery experiment: Cells were seeded at 60 000 cells per 25 cm2 flask or 850 cells/well into Nunc F96 microwell plates (AEC-Amersham Soc (Ltd), Kyalami, South Africa). After 24 h, cells were exposed to the varying metabolic conditions for 2 h, 4 h and 6 h. Subsequently, cells were washed with PBS and medium was replaced with DMEM containing 6 mM glucose and 1 mM l-glutamine. Medium was subsequently replaced every 2 days. Subsequently, all experimental methods were conducted as described below.
Polarization-optical transmitted light differential interference contrast
Polarization-optical transmitted light differential interference contrast (PlasDIC) is a contrast method used to view morphology. PlasDIC displays the required phase profile which is relative to the product of the section thickness and the refractive index difference between the environment and the average refractive index of quartz. PlasDIC has high-quality DIC imaging of individual cells, cell clusters and thick individual cells in plastic cell-culture vessels [16, 17]. PlasDIC was conducted according to Visagie, et al. [17].
Cell cycle progression and apoptosis induction
Flow cytometry was employed to measure the DNA content of cells after exposure to the various metabolic conditions and to monitor the effect on cell cycle progression [18]. The latter was accomplished by means of ethanol fixation and propidium iodide staining which was conducted according to Mqoco, et al. [16]. Cell cycle Propidium iodide fluorescence was measured with fluorescence activated cell sorting (FACS) FC500 System flow cytometer (Beckman Coulter South Africa (Pty) Ltd). Data from at least 10 000–30 000 events were analyzed with CXP software (Beckman Coulter South Africa (Pty) Ltd. (Pretoria, Gauteng, South Africa). Cell cycle distributions was calculated with Cyflogic 1.2.1 released 2008/11/19 (Perttu Terho & Cyflo Ltd) by assigning relative DNA content per cell to sub-G1, G1, S and G2M fractions.
Mitochondrial membrane potential
A reduction in mitochondrial membrane potential is an early indicator of apoptosis induction [19]. Changes in the mitochondrial membrane potential were investigated using the mitocapture antibody BIOCOM biotech Pty (Ltd) (Clubview, South Africa. Mitocapture is a cationic dye that accumulates in the mitochondria of healthy cells. However, the Mitocapture is unable to accumulate in the mitochondria of apoptotic cells due to the altered mitochondrial membrane potential and therefor the Mitocapture remains in the cytoplasm in its monomer form (green) [20]. After above-mentioned general experimental procedures were followed, diluted Mitocapture solution (mixed according to suppliers’ instructions) was pipetted to all samples. Samples were then incubated for 60 min in a humidified atmosphere (37 °C, 5 % CO2). Samples were then incubated the fluorescence was measured at excitation wavelength of 485 nm and emission wavelength of 520 nm using fluorometrics (Department of Pharmacology, University of Pretoria).
Hydrogen peroxide generation
Hydrogen peroxide generation was measured using 2, 7-dichlorofluorescein diacetate (DCFDA). DCFDA, a non-fluorescent probe, which upon oxidation by ROS and peroxides is converted to the highly fluorescent derivative DCF [21]. After above-mentioned general experimental procedures were followed, DCF-DA (200 μl; 10 μM) was pipetted to all the samples and were subsequently incubated for 60 min in a humidified atmosphere (37 °C, 5 % CO2. Fluorescence was measured at excitation wavelength of 485 nm and emission wavelength of 520 nm using fluorometrics (Department of Pharmacology, University of Pretoria).
Acridine orange staining
Acridine orange is a lysosomotropic fluorescent compound that moves freely across cell membranes when uncharged [22]. However, acridine orange accumulates in its protonated form in acidic compartments and thus serves as a tracer for acidic vesicular organelles including autophagic vacuoles and lysosomes [22]. After above-mentioned general experimental procedures were followed, PBS containing acridine orange (200 μl; 5 mg/ml) was added to all the samples and samples were incubated for 60 min in a humidified atmosphere (37 °C, 5 % CO2). Fluorescence was measured at excitation wavelength of 485 nm and emission wavelength of 520 nm using fluorometrics (Department of Pharmacology, University of Pretoria).
Statistics
At least three independent experiments were conducted for all techniques. Each independent fluorometrical experiment had a sample size of 3. Averages of each experiment were represented in bar charts, with T-bars referring to standard deviations. P-values < 0.05 were regarded as statistically significant and were indicated by an asterisk (*). Cell cycle progression data from at least 10 000–30 000 events were analyzed using CXP software (Beckman Coulter South Africa (Pty) Ltd. (Pretoria, Gauteng, South Africa)). Cell cycle distributions was calculated with Cyflogic 1.2.1 released 2008/11/19 (Perttu Terho & Cyflo Ltd).