Drug interaction assay

Cells (6 × 10³ cells/well) were seeded in 96-well plates and incubated at 37°C for 24 h. They were then exposed to different concentrations of resveratrol and cisplatin for an additional 24 h. The treatments included resveratrol (24 μg/mL [105.15 μM] and 12 μg/mL [52.57 μM]), cisplatin (9 μg/mL [30.00 μM] and 4.5 μg/mL [15.00 μM]), 24 μg/mL (105.15 μM) resveratrol + 9 μg/mL (10.95 μM) cisplatin and 12 μg/mL (52.57 μM) resveratrol + 4.5 μg/mL cisplatin (15.00 μM). A total of 20 μL of MTT solution (5 mg/mL [12.7 μM]) was added to each well and incubated for 4 h at 37°C. Next, 150 μL of DMSO was added to each well to dissolve the formazan. Absorbance was recorded at 490 nm using a microtiter plate reader (BioTek, USA). Each experiment was repeated three times. The interaction between two drugs was evaluated using the coefficient of drug interaction (CDI), which is based on the Pharmacological Additivity model (42, 43), Statistical Independence model (44), and Pharmacological Independence model, respectively. CDI was calculated using the formula: CDI = P (a + b)/(Pa × Pb), where P (a + b) represents the cell viability after treatment with both drugs A and B, while Pa and Pb represent cell viability after treatment with a single compound alone. CDI < 1 represents the synergy of A and B, CDI = 1 represents the additivity of A and B, and CDI > 1 represents the antagonism of A and B. According to the Statistical Independence model, if the probabilities of death are statistically independent, then P (a + b) = 1 − (1 − Pa) × (1 − Pb), which means there is no interaction between the two drugs. If P(a + b) > 1 − (1 − Pa) × (1 − Pb), it indicates a synergistic effect between these two drugs. Conversely, if P(a + b) < 1 − (1 − Pa) × (1 − Pb), these two drugs exhibit antagonism.