2.2. CCK-8 Assay and Analysis of Drug Synergy

Cell viability was measured using a Cell Counting Kit-8 assay (#CK04, CCK-8, Dojindo Molecular Technologies, Inc., Rockville, MD, USA). In light of previous studies indicating that cancer cell treatment with 5~20 mM metformin or 15~25 μM everolimus effectively reduced cell proliferation [40,41,42], CaSki and C33A cells (2 × 10⁴/well) were seeded into 96-well plates and then treated with different concentrations (10 and 20 mM in sterilized water) of metformin (#D150959, Sigma-Aldrich, St. Louis, MO, USA) with or without (10 and 20 μM in dimethyl sulfoxide, Sigma-Aldrich) everolimus (#S1120, Selleck Chemicals, Houston, TX, USA) for 48 h. Control cells were treated with 0.1% DMSO in the culture medium. Subsequently, CCK-8 (10 μL) was added to each well, and cells were incubated for 1 h at 37 °C. Absorbance at 450 nm was measured using a microplate reader (FLUOstar Galaxy, BMG Labtech, Ortenberg, Germany). The synergistic effect of the drugs was analyzed using the Chou–Talalay method and CalcuSyn software (Biosoft, Cambridge, UK) [43]. A combination index (CI) value of <1 indicated synergy, >1 indicated antagonism, and 1 indicated an additive effect of the two agents. In further experiments, cells were pretreated with various inhibitors, such as z-VAD-fmk (#1009-20C, 10 μM, BioVision, Inc., Milpitas, CA, USA) and BIRB796 (#S1574, 5 μM, Selleck Chemicals), prior to metformin and everolimus treatment.