Materials
Unless otherwise specified, all reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).
Cell lines
Most studies were performed on a bladder carcinoma cell line, HT1376, obtained from the American Type Culture Collection (Rockville, MD, USA). Cells were maintained as a monolayer at 37°C in a humidified atmosphere of 5% CO2 in air and subcultured weekly. Culture medium was composed of DMEM/HAM F12 (1:1) supplemented with foetal calf serum (10%), glutamine (2 mM), non-essential aminoacids (1%) (Mascia Brunelli s.p.a., Milan, Italy), and insulin (10 mg/mL). Cells were used in the exponential growth phase in all the experiments.
Some experiments were performed on U937 myelomonocytic cell line, obtained from the same source. Cells were grown as liquid cultures at 37°C in a humidified atmosphere of 5% CO2 in air and subcultured every three days. Culture medium was RPMI 1640 (Biowhittakers, Verviers, Belgium) supplemented with foetal calf serum (10%), glutamine (2 mM), penicillin (100 IU/L), and streptomycin (0.1 g/L).
Cell treatments
DOC was obtained from Aventis Pharma S.A. (Paris, France). The drug was stored at a concentration of 10 mg/mL (12.6 mM) in 13% w/w ethanol at 4°C and diluted in medium before use. The final concentration of ethanol in the medium never exceeded 0.01%, and therefore had no effect on cell growth and viability. Control cells received the same amount of solvent.
After performing an initial evaluation of drug cytotoxicity by treating the cell cultures with DOC at concentrations ranging from 0.03 to 3 μg/mL, the highest dose was used in all subsequent experiments.
Chemosensitivity assay
The Sulforhodamine B (SRB) assay was used according to the method by Skehan et al. [37]. Briefly, cells were collected by trypsinisation, counted and plated at a density of 10,000 cells/well in 96-well flat-bottomed microtitre plates (100 μl of cell suspension/well). In the chemosensitivity assay, DOC was tested at scalar concentrations ranging from 0.03 μg/mL to 3 μg/mL (3.7 μM) for 24 hours or for 1 hour followed by 23-hour culture in drug-free medium. Experiments were run in octuplet, and each experiment was repeated three times. The optical density (OD) of treated cells was determined at a wavelength of 540 nm by means of a fluorescence plate reader. Growth inhibition and cytocidal effect of the drug were calculated according to the formula reported by Monks et al. [38]: [(ODtreated - ODzero)/(ODcontrol - ODzero)] × 100%, when ODtreated is > to ODzero. If ODtreated is above ODzero, treatment induced a cytostatic effect, whereas if ODtreated is below ODzero, cell killing has occurred. The ODzero depicts the cell number at the moment of drug addition, the ODcontrol reflects the cell number in untreated wells and the ODtreated reflects the cell number in treated wells on the day of the assay. In this way, if a drug does not affect cell growth or viability, the test outcome will be 100. Conversely, if it induces a cytostatic effect, the result will be between 0 and 100 (upper part of the diagram), whereas if it induces a cytocidal effect, the result will be between 0 and -100 (lower part of the diagram), indicating that the number of cells at the end of the observation time is lower than the initial one.
Induction of apoptosis
Induction of apoptosis was tested at a concentration of 3 μg/mL (3.7 μM) DOC for 1 hour followed by a 6-, 24-, 48- 72- and 96-hour culture in drug-free medium. Data are the average of two to three experiments, with errors under 10%.
Cyclosporin A treatment
Cyclosporin A (CsA) is the most widely used blocker of calcium efflux from the mitochondria. For this preliminary experiment we used a concentration of 1 μM [39]. CsA was added to HT 1376 cells 30 min before DOC treatment. After a 1-hour incubation, both drugs were washed out. Evaluation of JC-1, AnnV and PI staining was carried out immediately after the end of drug exposure and after a 16- and 24-hour culture in drug-free medium. Control (untreated) and DOC-treated cultures were run in parallel and evaluated at the same times as CsA+DOC-treated cultures.
Flow cytometry
For cytofluorimetric evaluations, the medium was removed at the indicated wash-out times and cells were detached from the flasks by trypsin treatment, washed twice with PBS and stained according to the different methods specified below. A FACS Vantage flow cytometer (Becton Dickinson, San Diego, CA, USA), equipped with an argon laser (488 nm) was used. Data acquisition and analysis were performed using CELLQuest Pro software (Becton Dickinson, San Diego, CA, USA).
Cell cycle distribution
Cell cycle distribution and percentage of cells within the sub-G1 peak (i.e. with fragmented DNA) were estimated by PI staining. Cells (1 × 106/mL) were fixed in 70% ethanol, washed twice in PBS, then incubated overnight at 4°C in the dark in a PBS solution containing propidium iodide (10 μg/mL), RNAse (10 Kunits/mL) and NP40 (0.01%). Data were elaborated using Modfit software (DNA Modelling System, Verity Software House, Inc., Topsham, ME, USA) and expressed as fractions of cells in the different cycle phases. Samples were run in triplicate, and each experiment was repeated three times. Diagrams of growing cultures display the characteristic x-axis distribution according to the DNA content, the first peak corresponding to the diploid (2c) peak, i.e. to cells in the G0/1 phase, and the second peak to cells with 4c DNA content, i.e. to cells in G2/M phase. Cells with an intermediate DNA content are in the S phase. When DNA is fragmented, as in apoptotic cells, the affinity with the intercalating PI dye is decreased and a so-called hypodiploid peak (or area) becomes apparent to the left of the G0/1 peak.
JC-1
The cationic fluorochrome JC-1 (BD Biosciences Pharmingen, San Diego, CA, USA) was used to evaluate the decrease in transmembrane mitochondrial potential (Δψm) that accompanies the efflux of pro-apoptotic molecules fromthe mitochondrion. JC-1 forms aggregates that fluoresce red in the presence of high mitochondrial membrane potential, as in normal cells. When mitochondrial membrane potential decreases, as occurs when mitochondrial pores open during the apoptotic process [8, 9], JC-1 becomes monomeric and fluoresces green.
After DOC treatments, cells were harvested, washed once in PBS and then immediately incubated in JC-1 working solution containing 1 μM JC-1 monomer at a concentration of 1 × 106 cells/mL for 15 min in a humidified atmosphere at 37°C in the dark, according to the manufacturers' instructions. Cells were then washed twice, resuspended in Assay Buffer and analysed by flow cytometry. For each sample, 15,000 events were recorded.
Propidium iodide staining
The DNA-intercalating dye, propidium iodide, does not permeate viable cells. Therefore, when added to unpermeabilised cells, it is possible to assess the amount of necrosis in a cell population. When used in combination with a marker of apoptosis, it permits detection of so-called "secondary necrosis" or "late apoptosis", i.e. the phenomenon whereby apoptotic cells, over time, lose their membrane integrity. Thus, cells that are stained by PI alone have undergone bona fide necrotic cell death, whereas PI+ cells that are also stained with a marker of apoptosis can be classified as apoptotic. PI was added to a final concentration of 5 μg/mL immediately before cytometric evaluation (longer incubation times may cause aspecific staining).
Lectin staining
MAA lectin binds to 2,3-linked syalil residues on cell surface. Cells (5 × 105) were stained for 15 min in the dark at room temperature with 1.5 μg FITC-conjugated MAA lectin [40] dissolved in 100 μL PBS. Cells were thenwashed twice in PBS and fixed in a 0.74% solution of formaldehyde in PBS. Immediately before cytometric analysis, propidium iodide was added to a final concentration of 5 μg/mL to distinguish between early (PI-) and necrotic or late (PI+) apoptotic cells. For each sample, 15,000 events were recorded.
Annexin V assay
Annexin V binds to phosphatidylserine, a lipid component of the plasma membrane that is flopped from the inner to the outer cell surface during apoptosis and is believed to play a role as an "eat-me" signal [41].
After DOC treatments, cells were harvested, washed once in PBS and incubated with 5 μl/mL Annexin V-FITC in binding buffer (Bender MedSystems, Vienna, Austria) for 10 min at room temperature in the dark. Cells were then washed in PBS and suspended in binding buffer. Immediately before flow cytometry analysis, propidium iodide was added to a final concentration of 5 μg/mL to distinguish between early (PI-) and necrotic or late (PI+) apoptotic cells. For each sample, 15,000 events were recorded.
TUNEL assay
In the TUNEL assay, 3' OH termini, resulting from DNA cleavage, are labelled with FITC-conjugated nucleotides in a reaction utilsing exogenous terminal deoxynucleotide transferase (TdT).
After DOC treatments, cells were harvested, fixed in 1% paraformaldehyde in PBS on ice for 15 min, suspended in ice cold ethanol (70%) and stored overnight at -20°C. Cells were then washed twice in PBS and incubated with 50 μl of solution containing TdT and FITC-conjugated dUTP (Roche Diagnostic GmbH, Mannheim, Germany) in a humidified atmosphere for 60 min at 37°C in the dark. Samples were then washed in PBS containing Triton X-100 (0.1%), counterstained with 3 μg/mL propidium iodide and RNase (10 Kunits/mL) for 30 min at 4°C in the dark, and finally analysed by flow cytometry. For each sample, 10,000 events were recorded.
Cell count and viability assay
HT1376 cells were seeded in duplicate at a concentration of 2 × 105 cells/ml in 35-mm well dishes. At the indicated times, cells were detached by trypsin treatment, washed and resuspended in PBS. An aliquot of the cell suspension was combined with an equal volume of 0.4% Trypan Blue and incubated for 8–10 min. at 37°C. Cells were counted in a counting chamber (Kova Glasstic Slide Hemocytometer, Hycor Biomedical Inc., Garden Groove, USA). Both total cell count and the percentage of viable and non viable cells were recorded. Cells are considered to be viable when they exclude the dye.
DNA gel electrophoresis
In the course of the apoptotic process, DNA is cleaved in a distinctive way at internucleosomal sites by a specific caspase-activated endonuclease [42], thus yielding fragments in multiples of 200 bp, which appear as a characteristic "ladder" when DNA is separated by gel electrophoresis.
Fragmented DNA was isolated from samples of 2 × 106 cells as described by Zhivotovsky [42]. Electrophoresis was carried out in 1.5% agarose gel.
Morphological investigation
After DOC exposure, samples were immediately stained with the cationic fluorochrome JC-1 as described above and with the cell-permeable dye 4',6-DAPI 10 μL/mL (Molecular Probes, Leiden, The Netherlands). Cells were fixed in ethanol (70%) and then examined under a fluorescence photomicroscope (Zeiss, Axioscope 40) to visualise the mitochondrial membrane depolarisation and concurrently the chromatin condensation and/or fragmentation typical of apoptotic cells.