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

Abstract

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 (IC₅₀ ) and cytotoxicity (CC₅₀ ) over 72 h. Experiments were conducted in HepG2 cells and primary rat hepatocytes (PRH). IC₅₀ for actinomycin D, cycloheximide, emetine, and puromycin were 39 ± 7.4, 6600 ± 2500, 2200 ± 1400, and 1600 ± 1200 nmol/L; with corresponding CC₅₀ values of 6.2 ± 7.3, 570 ± 510, 81 ± 9, and 1300 ± 64 nmol/L, respectively, in HepG2 cells. The IC₅₀ were 1.7 ± 1.8, 290 ± 90, 620 ± 920, and 2000 ± 2000 nmol/L, with corresponding CC₅₀ values of 0.98 ± 1.8, 680 ± 1300, 180 ± 700, and 1600 ± 1000 (SD) nmol/L, respectively, in PRH. CC₅₀ were also lower than the IC₅₀ 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.

Keywords: cytotoxicity; protein degradation rate; protein synthesis inhibitor.

Figure 1

IC ₅₀ and CC ₅₀ of the four individual protein synthesis inhibitors in HepG2 and primary rat hepatocytes (PRH). (A–D) Cell viability was measured by standard MTT assay and expressed as viability as a percentage of untreated control. (E–H) Protein synthesis inhibition across different concentrations of inhibitors was measured by [³H]‐Leucine incorporation assay and shown as percentage of inhibition of control. Dotted line shows PRH and solid line for HepG2 cells. Dose–response curves were produced by Prism software and IC ₅₀ and CC ₅₀ values were calculated from linear regression models. Data are shown as mean ± SD from n = 3 independent experiments.

Figure 2

Linear regression analysis of IC ₅₀ and CC ₅₀ between HepG2 and PRH cell types. (A) shows linear regression between CC ₅₀ values of the four protein synthesis inhibitor drugs for the different cell types. (B) shows linear regression between IC ₅₀ values with cycloheximide omitted but shown in inset graph, of the two cell types. (C) shows IC ₅₀:CC ₅₀ ratio of HepG2 and PRH cell types omitting cycloheximide. Cycloheximide is included in the inset graph.

Figure 3

Isobolograms generated based on CC ₅₀ and IC ₅₀ values showing the interaction between protein synthesis inhibitor pairs. Six combinations of inhibitor pairs are shown in (A–F) The dotted line corresponds to the predicted curve if drug pairs showed an additive effect. The black line corresponds to drug pair interactions for protein synthesis inhibition. The grey line shows drug pair interactions for cytotoxicity. FIC_A and FIC_B correspond to the fractional inhibitory concentrations of the first and second drugs in each drug pair listed. Cytotoxicity analysis was not performed for cycloheximide–emetine, cycloheximide–puromycin, and emetine–puromycin drug pairs (A–C) as these showed strong antagonism for protein synthesis inhibition. N = 4 independent experiments were carried out in HepG2 cells.

Figure 4

The level of [³H]Leucine incorporation for three‐ and four‐inhibitor combinations at subcytotoxic concentrations (CC ₁₀). Leucine incorporation assays were carried out in HepG2 cells and the percentage of incorporation compared to control was calculated. Combination APCE corresponds to actinomycin D, puromycin, cycloheximide, and emetine; ACE to actinomycin D, cycloheximide, and emetine; APE to actinomycin D, puromycin, and emetine; APC to actinomycin D, puromycin, and cycloheximide; and PCE to puromycin, cycloheximide, and emetine. Data are shown as mean ± S.D from n = 3 independent experiments.

Figure 5

Measuring cytotoxicity for the three‐ and four‐inhibitor combinations at subcytotoxic concentrations. Three‐ and four‐inhibitor combinations were prepared at CC ₁₀ concentrations. A range of cytotoxicity assays including standard MTT, GSH, ATP, and trypan blue exclusion assays were conducted on HepG2 cells. APCE corresponds to actinomycin D, puromycin, cycloheximide, and emetine; ACE to actinomycin D, cycloheximide, and emetine; APE to actinomycin D, puromycin, and emetine; APC to actinomycin D, puromycin, and cycloheximide; and PCE to puromycin, cycloheximide; and emetine. Data are shown as mean ± SD from n = 3 independent experiments.