4.2. Cell Viability Assays and Drug Combination Studies

Cells were cultured in 96-well plates and, after 24 h of culturing, they were treated with escalating concentrations of Bosutinib or Alpelisib for 24 h. Cell viability was evaluated with the Colorimetric Assay XTT Cell Proliferation Kit II (Roche, Basel, Switzerland). Background absorbance (medium only) was subtracted, and the data (average of six replicates of each drug concentration) were normalized as percentages of vehicle control. Each experiment was performed at least three times, and each concentration point was replicated six times within each experiment. The concentrations of Bosutinib corresponding to its 25, 50 and 75 inhibitory concentrations (IC) (IC₂₅, IC₅₀ and IC₇₅) were calculated with Graph Pad Prism5 software and are shown in Table 2. These values are defined as the concentration of drug causing decreases of 25%, 50% and 75% in cell viability, as measured by XTT, respectively, and they were used for the rest of the experiments in this manuscript (two-drug combination studies, cell cycle analysis and Western blotting).

For the combined viability assays, we followed the method described by Chou and Talalay for two-drug combination studies [29] that calculates a “Combination Index” (CI) to quantitatively depict synergism (CI < 1), additive effects (CI = 1) and antagonism (CI > 1). Briefly, two-fold serial dilutions of the IC₅₀ were performed for each drug alone and their mixture to create 4–5 concentrations. The combination of the two drugs was performed at a constant ratio. For this purpose, the IC₅₀ concentration for Alpelisib for some of the HNSCC cell lines had to be established (Table 2). The CI was calculated using CompuSyn software (Available online: http://www.combosyn.com/).