Incompatibility of chemical protein synthesis inhibitors with accurate measurement of extended protein degradation rates

Protein synthesis inhibitors are commonly used for measuring protein degradation rates, but may cause cytotoxicity via direct or indirect mechanisms. This study aimed to identify concentrations providing optimal inhibition in the absence of overt cytotoxicity. Actinomycin D, cycloheximide, emetine, and puromycin were assessed individually, and in two‐, three‐, and four‐drug combinations for protein synthesis inhibition (IC (50)) and cytotoxicity (CC (50)) over 72 h. Experiments were conducted in HepG2 cells and primary rat hepatocytes (PRH). IC (50) for actinomycin D, cycloheximide, emetine, and puromycin were 39 ± 7.4, 6600 ± 2500, 2200 ± 1400, and 1600 ± 1200 nmol/L; with corresponding CC (50) values of 6.2 ± 7.3, 570 ± 510, 81 ± 9, and 1300 ± 64 nmol/L, respectively, in HepG2 cells. The IC (50) were 1.7 ± 1.8, 290 ± 90, 620 ± 920, and 2000 ± 2000 nmol/L, with corresponding CC (50) values of 0.98 ± 1.8, 680 ± 1300, 180 ± 700, and 1600 ± 1000 (SD) nmol/L, respectively, in PRH. CC (50) were also lower than the IC (50) for all drug combinations in HepG2 cells. These data indicate that using pharmacological interference is inappropriate for measuring protein degradation over a protracted period, because inhibitory effects cannot be extricated from cytotoxicity.