Modulation of Cisplatin Cytotoxicity due to its Combination with Bortezomib and the Nature of its Administration

Materials and Methods

Materials. CS and YH12 were prepared according to previously described methods (9, 14). Bort was purchased from LC Laboratories Woburn, MA, USA. Foetal calf serum (FCS), RPMI-1640, 200 mM L-glutamine, and 5.6% sodium bicarbonate were obtained from Trace Biosciences Pty Ltd., Australia. Other chemicals were mostly purchased from Sigma-Aldrich, Sydney, Australia. A2780, A2780^cisR and A2780^ZD0473R ovarian cancer cell lines were gifts from Ms. Mei Zhang, Royal Prince Alfred Hospital, Sydney, Australia. Stock solutions of platinum compounds (1 mM) were prepared in 1:1 DMF-mQ water mixture and that of Bort (1 mM) were made in ethanol.

Cell culture. Human ovarian cancer cell lines A2780, its cisplatin-resistant form A2780^cisR and its ZD0473-resistant form A2780^ZD0473R were seeded in 25 cm² tissue culture flasks in an incubator at 37°C in a humidified atmosphere consisting of 5% CO₂ and 95% air. The cells were maintained in logarithmic growth phase in complete medium consisting of RPMI-1640, 10% heat inactivated FCS, 20 mM Hepes, 0.11% bicarbonate, and 2 mM glutamine without antibiotics (15). Each cell line was seeded at a density of 4,000 and 6,000 cells/well in a flat-bottomed 96-well culture plate in 10% FCS/RPMI-1640 culture medium. The plate was then incubated for 24 h at 37°C in a humidified atmosphere to allow cells to attach.

Single-drug treatment. For single-drug treatment, each of the stock solutions was subjected to serial dilutions to give final concentrations ranging from 0.0008 to 20 μM. The dilutions were performed using 10% RMPI-1640 medium without serum as the vehicle and were added to equal volumes of cell culture in triplicate wells, then cells were left to incubate for 72 h. These treatments were carried out to determine (IC₅₀) values i.e. drug concentrations required for 50% cell kill.

Combination analysis. Dose-response curves and combination indices (CIs) were used as measures of synergism and generated/calculated using the program CalcuSyn (16-18). The CI for binary combinations of drugs was calculated according to the equation: where D₁ and D₂ represent 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 respectively required to cause x% inhibition when present alone. D_x can be readily calculated from the following form of median effect equation: where D_x denotes the dose of drug, Dm is the median-effect dose, f_a is the fraction of cells affected 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. Recently, Huq defined a new term called the ‘enhancement factor’ (EF) giving a direct relationship between EF and synergism (10).

Platinum cellular accumulation and platinum–DNA binding studies. As the action of platinum-containing drugs is associated with their binding with DNA, cellular accumulation of platinum and platinum–DNA binding levels in A2780 and A2780^cisR cell lines were determined for combination of CS-Bort applying to the sequences 0/0 h and 0/2 h. Furthermore, the platinum content in cells treated with CS in bolus and CS in two aliquots with a time gap (2 h and 4 h) were also determined. Combinations of CS with Bort (at IC₅₀ values) and the addition of CS in two aliquots (at 50 mM final concentration) were added to culture plates containing exponentially growing A2780 and A2780^cisR cells in 10 ml 10% FCS/RPMI-1640 culture medium (cell density=1×10⁶ cells/ml). The cells containing the drugs were incubated for 24 h at the end of which cell monolayers were trypsinized and cell suspensions (10 ml) were transferred to centrifuge tubes and spun at 3,500 rpm for 2 min at 4°C. The cells were washed twice with ice-cold phosphate-buffered saline (PBS) and the pellets were stored at −20°C until assayed. At least three independent experiments were performed.

Cellular accumulation. Following drug incubation, the cell pellets were suspended in 0.5 ml 1% Triton-X, held on ice then sonicated. Total intracellular platinum contents were determined by graphite furnace atomic absorption spectrophotometry.

Drug–DNA binding. Following drug incubation, high molecular weight DNA was isolated from the cell pellet using H440050 JETQUICK Blood DNA Spin Kit/50 from Austral Scientific Pty Ltd., Sydney, Australia, and the modified protocol of Bowtell (19). The cell pellets, resuspended in PBS to a final volume of 200 μl and mixed with 10 μl of RNase A, were incubated for 4 min at 37°C. About 25 μl Proteinase K and 200 μl Buffer K1 (containing guanidine hydrochloride and a detergent) were added to the mixture followed by incubation for 10 min at 70°C. Then 200 μl of absolute ethanol was added and mixed thoroughly to prevent any precipitation of nucleic acids. The samples were centrifuged for 1 min at 10,600 rpm through the silica membrane using JETQUICK micro-spin column. The columns containing the samples were washed with 500 μl of buffer KX (containing high-salt buffer to remove residual contaminations) and centrifuged for 1 min at 10,600 rpm. These were again washed with 500 μl buffer K2 (containing low-salt buffer to change the high-salt conditions on the silica membrane to low-salt) and centrifuged for 1 min at 10,600 rpm. To further clear the silica membrane from residual liquid, the sample columns were centrifuged again for 2 min at full speed (13,000 rpm). The column receivers were changed and the purified DNA in the column was eluted from the membrane with 200 μl of 10 mM Tris–HCl buffer (pH 8.5). DNA content was determined by UV spectrophotometry (260 nm) and platinum levels were determined by graphite furnace AAS.