Briefly, cells were cultured in 75 cm 2 cell-culture flask (Sigma-Aldrich, Milan, Italy) in high glucose (4.5 g/L) Dulbecco's modified Eagle medium (DMEM, Sigma-Aldrich) with 10 % fetal bovine serum (FBS, Sigma-Aldrich), 1% L-glutamine (Sigma-Aldrich), and 1% penicillin-streptomycin solution (Sigma-Aldrich). 5 x 10 5 cells/well were seeded into 96-well plates and incubated overnight. Then, cells were preincubated for 30 min with the selected compounds (10 µM) and then treated for 24 h with colchicine (Sigma-Aldrich, 10 µM) or vincristine (Sigma-Aldrich, 10 µM), two cytotoxic drugs that are known P-gp substrates. Zosuquidar (Sigma-Aldrich) (10 µM) was used as a reference drug. Cell viability was then quantified using the Cell Counting Kit-8 (CCK-8, Sigma-Aldrich) and absorbance at 450 nm was measured with a Victor Nivo multimode plate reader (PerkinElmer, Waltham, MA). The increase of cytotoxicity of these drugs when co-administered with another compound could be used as a measure of the inhibition of P-gp mediated efflux . Experiments were performed in triplicate.