Anti-proliferative and Pro-apoptotic Effects from Sequenced Combinations of Andrographolide and Cisplatin on Ovarian Cancer Cell Lines

Materials and Methods

Materials. Cisplatin was prepared based on the Dhara method (8). Andrographolide was purchased from Sigma-Aldrich, Sydney, Australia. Foetal calf serum (FCS), RPMI 1640, 200 mM L-glutamine, and 5.6% sodium bicarbonate were obtained from Trace Biosciences Pty Ltd, Sydney, Australia. Other chemicals were purchased from Sigma, Sydney Australia unless stated otherwise. The ovarian cancer cell lines were generous gifts from Ms. Mei Zhang, Royal Prince Alfred Hospital, Sydney, Australia. Stock solutions of platinum compounds (1 mM) were prepared in (DMF-mQ) water mixture and those of plant compounds (1 mM) were prepared in ethanol.

Cell culture. Human ovarian cancer cell lines A2780 (cisplatin-sensitive, parental cell line), and A2780^cisR (cisplatin-resistant) were seeded in 25-cm² tissue culture flasks in carbon dioxide incubator at 37°C in a humidified atmosphere consisting of 5% CO₂ in air. The cells were maintained in logarithmic growth phase in a complete medium consisting of RPMI 1640, 10% heat-inactivated FCS, 20 mM Hepes, 0.112% bicarbonate, and 2 mM glutamine (9). Each cell line was seeded at a density of 4,000 to 5,500 cells/well in the 96-well plates in 10% FCS/RPMI 1640 culture medium and left overnight in the carbon dioxide incubator for treatment.

Single-drug treatments. For single-drug treatments, stock solutions were subjected to serial dilutions to give final concentrations ranging from 0.005 to 10 μM. The dilutions were made using 10% RMPI 1640 medium without serum as the vehicle added to equal volumes of cell culture in triplicate wells, then cells were left to incubate in the carbon dioxide incubator for 72 h. Treatments were carried out to determine (IC₅₀) values.

Combination studies. In combination studies, cells were treated with solutions of compounds alone and in combination at three different concentrations, generally at constant ratios of their IC₅₀ values. One hundred microliters of cells were seeded in each well of a 96-well plate prior to treatment with single and combinations of compounds. Cisplatin was combined with andrographolide according to the following five sequences of administration: a) simultaneous addition (0/0 h), b) Cisplatin administered first followed by Andro 4 h later (0/4 h), c) andrographolide administered first followed by cisplatin 4 h later (4/0 h), d) cisplatin administered first followed by andrographolide 24 h later (0/24h) and e) andrographolide administered first followed by cisplatin 24 h later (24/0 h). Non-treated cells served as control. The plates containing cells and compounds were left to incubate under normal growth conditions for a period of 72 h from the time of addition of the first compound. The inhibition of the cell growth was determined using the (MTT) reduction assay (10). At the end of 72 h of incubation, MTT (50 μl per well of 1 mg/ml MTT solution) was added to each plate. Four hours after the addition of MTT, the yellow formazan crystals produced from the reduction of MTT was dissolved in 150 μl of DMSO and the absorbance of the resulting solution was read using Bio-Rad Model 3550 microplate Reader (Agilent) set at 570 nm. The absorbance values of the treated and untreated (control) cells were used to calculate growth inhibition due to single-drug treatment and that due to drug combinations. In the case of treatment with single drugs, IC₅₀ values (i.e. concentrations of compounds needed to cause 50% cell kill) were obtained from the dose response curves whereas in the case of drug combinations, combination index (CI) values were obtained to provide a quantitative measure for combined drug action in terms of synergism, additiveness or antagonism. Each experiment was repeated at least three times.

Combination analysis. CIs used as measures of combined drug action were calculated using the program CalcuSyn (11, 12). The (CI) for binary combination of drugs is defined by the following equation: D₁ and D₂ represent the mean doses of compounds 1 and 2 respectively, in combination required to cause x% inhibition, whereas D_1x and D_2x represent the doses of compounds 1 and 2 required to cause x% inhibition when present alone. D_x can be readily calculated from the following form of the median-effect equation: where D_x denotes the dose of drug, D_m is the median-effect dose, f_a is the fraction of cells affected (killed) by the dose, f_u is the fraction of cells remaining unaffected so that f_u=1-f_a and m is the exponent defining the shape of the dose-effect curve.

CI values of <1, =1 and >1 indicate synergism, additivity and antagonism in combined drug action, respectively.

DNA fragmentation assay (apoptosis assay). DNA fragmentation, as a measure of apoptotic cell death following drug treatment, was assayed using Deadend™ Colorimetric Apoptosis Detection System (Promega, Madison, USA). In brief, 1×10⁵ cells were plated into four sets of chamber slides and incubated in an atmosphere of 5% CO₂ for 24 h. Following this, cells were treated with andrographolide and cisplatin alone on separate slides. For studies on combination between cisplatin and andrographolide, cisplatin was administered first followed by andrographolide 4 h later. Cells treated with DNase 1 served as the positive control. The apoptosis assay was carried out according to the manufacturer's protocol. The apoptotic cells (that produced yellow fluorescence) and non-apoptotic cells (that produced red colour) were examined under a fluorescence microscope (13). The experiments were repeated at least three times. The percentage of apoptotic cells was determined as follows. All experiments were performed at least in triplicate and the data are presented as the mean±S.D.