Doxorubicin d1515

Cell viability of osteosarcoma cells treated with chemotherapy and decitabine was assessed using MTT assay according to previously report [15] (link). Osteosarcoma cells were seeded in 96-well tissue culture plates (5 × 10³ cells/100 μl freshly prepared culture media/well) and incubated at 37 °C with 5% CO₂ overnight. Osteosarcoma cells were treated with decitabine (Selleckchem, Houston, TX, USA) at concentrations of 0, 0.01, 0.1, 1, 10, and 100 μM for 72 h. The cells were further treated with clinically achievable concentrations of decitabine in combination with doxorubicin (D1515; Sigma, St. Louis, MO, USA) or cisplatin (P4394; Sigma, St. Louis, MO, USA) for 72 h. After a 72-hour incubation, the culture medium was removed, and 100 μl of fresh medium containing 5 mg/ml of MTT solution was added to each well which were then incubated for 2 h at 37 °C. The MTT-formazan crystal had been dissolved in 100 μl of dimethyl sulfoxide with vigorous mixing. Finally, a spectrophotometer was used to measure absorbance at 550 nm. IC50 values were calculated by performing nonlinear regression analysis using GraphPad Prism version 8.0 (GraphPad Software, Inc., La Jolla, CA, USA).
To evaluate the synergism of the combination drug treatment, we used a reference model named zero interaction potency (ZIP) [18] (link). Synergy score values were calculated using SynergyFinder software [19] (link).