Inhibitory effects of local anesthetics on the proteasome and their biological actions

Abstract

Local anesthetics (LAs) inhibit endoplasmic reticulum-associated protein degradation, however the mechanisms remain elusive. Here, we show that the clinically used LAs pilsicainide and lidocaine bind directly to the 20S proteasome and inhibit its activity. Molecular dynamic calculation indicated that these LAs were bound to the β5 subunit of the 20S proteasome, and not to the other active subunits, β1 and β2. Consistently, pilsicainide inhibited only chymotrypsin-like activity, whereas it did not inhibit the caspase-like and trypsin-like activities. In addition, we confirmed that the aromatic ring of these LAs was critical for inhibiting the proteasome. These LAs stabilized p53 and suppressed proliferation of p53-positive but not of p53-negative cancer cells.

Figure 1

Effects of LAs on 20S proteasome activity and binding to the 20S proteasome. (a) Effects of LAs and other related chemical agents (■: pilsicainide; ▼: lidocaine; ∆: MG132; ♦: bortezomib; □: verapamil; ○: E-4031; ◊: TTX; ▲: 4-F; ●: M-3) on 20S proteasome activity in vitro. Each point represents the mean ± SEM of more than 7 determinations. 20S proteasome activities were expressed relative to their values in the absence of drugs (100%). The calculated IC₅₀ values were 4.1 ± 2.4 nM, 1.1 ± 1.8 nM, 3.5 ± 1.5 μM, 23 ± 0.2 μM, 20 ± 2.5 μM, 14 ± 6.3 μM for MG132, bortezomib, pilsicainide, lidocaine, 4F, and mexiletine, respectively. (b) Inhibitory action of pilsicainide, 2-F, 3-F, 4-F, 2,4-F, 2,6-F and M-3 at 10 μM (left) and 100 μM (right) on the chymotrypsin-like activity of the proteasome. (c) Inhibition of binding of [³H]-pilsicainide to the 20S proteasome by cold pilsicainide, 4-F, M-3 and lidocaine in vitro. Each point represents the mean ± SEM of more than 7 determinations. The binding fraction of [³H]-pilsicainide to the 20S proteasome was expressed relative to the values in the absence of cold pilsicainide, 4-F, M-3 or lidocaine (100%). K _d values for pilsicainide, lidocaine, and 4-F are1.0 ± 2.8 μM, 21.2 ± 3.5 μM, and 4-F, respectively, whereas no binding capacity of M-3 was observed.

Figure 2

Predicted binding mode of LAs to the proteasome. (a) Docking simulations of pilsicainide against two parts of the β-subunit ring. (b) The binding mode of pilsicainide in the β5 subunit. The approximate position of S1 site pocket is designated. CPK model of pilsicainide and the surrounding protein matrix surface (left panel). Subunits β4 and β5 are colored yellow and orange, respectively. Amino acid residues surrounding the pilsicainide molecule and the nucleophilic active site residue of Thr1 are represented by the stick model and named (right panel). Green broken lines denote putative hydrogen bonds between the ligand and the protein. (c) Docking simulations of pilsicainide and its derivative 4-F, and lidocaine against β4-β5-β6 subunits.

Figure 3

Pilsicainide and lidocaine increase transfected and endogenous p53. (a), (b) Dose-dependent effects of MG132 and LAs on ubiquitinated p53-FLAG protein. Cell lysates were analyzed by Western blot analysis (a). Quantitative analysis of p53-FLAG stability using densitometry is shown in (b). Each point was expressed relative to the values in the absence of drugs (100%). (c), (d) Dose-dependent effects of LAs on endogenous p53 expression in MKN45 cells. MKN45 cells were cultured for 24 h in the presence of pilsicainide, lidocaine, 4-F, M-3 or MG132. Cell lysates were analyzed by Western blot analysis (c). Quantitative analysis of the protein level of endogenous p53 was conducted using densitometry (d).

Figure 4

Effects of LAs on cancer cells. (a) Effects of LAs on the proliferation of MKN45 cells. MKN45 cells were cultured for 24 h in the presence of MG132, pilsicainide, lidocaine, 4-F or M-3. Proliferation of MKN43 was estimated by MTT assays. (b) Effects of LAs on the proliferation of MKN45 and Kato III cells cultured for 24 h in the presence of MG132 (10 nM), pilsicainide (1 μM) and lidocaine (20 μM). (c) In the in vivo experiments, lidocaine significantly decreased the proliferation of MKN45 cells dispersed among mouse mesenchymal cells to a similar extent as MG132.