Non-covalent immunoproteasome inhibitors induce cell cycle arrest in multiple myeloma MM.1R cells

Abstract

Proteasome inhibition is a promising strategy for the treatment of multiple myeloma; unfortunately, this disease is often associated with an increasing chemoresistance. One novel approach may be to target the immunoproteasome, a proteasomal isoform mainly present in cells of hematopoietic origin. We investigated the activity of a panel of amides against immunoproteasome core particles as potential agents for the treatment of multiple myeloma (MM). Amide 6 showed an ideal profile since it was able to inhibit both the chymotrypsin-like activities of the immunoproteasome with K_i values of 4.90 µM and 4.39 µM for β1i and β5i, respectively, coupled with an EC₅₀ =17.8 µM against MM.1R cells. Compound 6 inhibited also ubiquitinated protein degradation and was able to act on different phases of MM cell cycle reducing the levels of cyclin A/CDK1, cyclin B/CDK1 and cyclin D/CDK4/6 complexes, which turns in cell cycle arrest.

Keywords: Immunoproteasome; cyclins; multiple myeloma.

Figure 1.

Structure of peptidomimetic amides 1–6.

Figure 2.

Effects of several concentrations of compounds 5 and 6 on vitality of MM.1R cells. Each point is obtained from the mean of 5 separate experiments.

Figure 3.

Effects of compounds 5–6, in panels A and B respectively, on ubiquitin accumulation in MM.1R cells. The upper blots depicts ubiquitin (10 KDa) and the lower β-actin (45 KDa). Each bar is obtained from the mean of 3 separate experiments. *p < .005 versus ctrl.

Figure 4.

Figures depict western blots results relatively to cyclin A and CDK1, both normalized for β-actin (lower bands); panels A and B show results for compound 5 and 6, respectively. Each bar is obtained from the mean of 3 separate experiments. *p < .005 versus ctrl; #p < .05 versus ctrl.

Figure 5.

Figures depict western blots results relatively to cyclin B and CDK1, both normalized for β-actin (lower bands); panels A and B show results for compound 5 and 6, respectively. Each bar is obtained from the mean of 3 separate experiments.

Figure 6.

Figures depict western blots results relatively to cyclin D1 and CDK4/6, both normalized for β-actin (lower bands); panels A and B show results for compound 5 and 6, respectively. Each bar is obtained from the mean of 3 separate experiments.

Figure 7.

Figures depict western blots results relatively to NF-kB and phospho-NF-kB, both normalized for β-actin (lower bands); panels A and B show results for compound 5 and 6, respectively. Each bar is obtained from the mean of 3 separate experiments.

Figure 8.

Figures depict western blots results relatively to PARP and Caspase-3, both normalized for β-actin (lower bands); panels A and B show results for compound 5 and 6, respectively. Each bar is obtained from the mean of 3 separate experiments.