Proteasome inhibition correlates with intracellular bortezomib concentrations but not with antiproliferative effects after bolus treatment in myeloma cell lines

Abstract

Although bortezomib is successfully used against multiple myeloma, the pharmacodynamics of proteasome inhibition and its association with efficacy or resistance is poorly understood. Using ultra performance liquid chromatography coupled to tandem mass spectrometry, site-specific luminogenic substrate assays and 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazoliumbromide (MTT) assays, effects of bortezomib on cellular drug concentrations, chymotrypsin- , caspase- , and trypsin-like activities, and cytotoxic efficacy were evaluated in eight myeloma cell lines directly after 1 h of exposure and additionally after a 23-h washout phase. Bortezomib accumulated in myeloma cells by up to 100-fold and concentration-dependently inhibited the proteasomal activities with the chymotrypsin-like activity being the most sensitive. Whereas intracellular concentrations correlated with the inhibition of the chymotrypsin- and the caspase-like activities of the proteasome, the cytotoxic efficacy of bortezomib did not correlate with either intracellular concentrations or proteasomal inhibition. However, the ratio of concentrations measured directly after the exposure and after the washout phase (indicating drug disposition) correlated with efficacy, suggesting that the cell's ability to dispose bortezomib at least in part influences bortezomib's cytotoxicity. In conclusion, this data argues against a direct association of intracellular concentration or proteasomal inhibition with cytotoxic efficacy but advocates for an important role of cellular drug disposition. Moreover, this study underlines the pleiotropic mode of action of bortezomib going beyond proteasome inhibition.

The Influence of Metabolism on Drug Response in Cancer

“…Bortezomib resistance is associated with mutations in the proteasomal bortezomib-binding pocket and upregulation of the proteasomal machinery, both of which lower the efficacy of the drug ( 149 – 154 ). However, intracellular concentrations of bortezomib seem to correlate with proteasome inhibition, but not cytotoxicity ( 155 ). This suggests that adaptive resistance mechanisms are involved, which allow cells to proliferate even when proteasome function is impaired.…”

Approaching sites of action of drugs in clinical pharmacology: New analytical options and their challenges

“…This may also inform on the optimal uptake modus at the cellular and molecular sites of action. As an example, when multiple myeloma cells were exposed to the reversible proteasome inhibitor bortezomib, both extracellular concentration (exposure) and subsequent intracellular drug concentration were correlated with proteasome activity, 52 while for the irreversible proteasome inhibitor carfilzomib, uptake kinetics (fast accumulation due to steep concentration gradient) rather than steady‐state concentrations modulated proteasome inhibition and cytotoxicity 53 Abundance of the molecular target and number of available specific binding sites.…”

Bosentan enhances in vitro bortezomib's anti‐proliferative potency against multiple myeloma by mechanisms going beyond endothelin receptor blockade

“…However, the inhibitory potency of bortezomib was not significantly altered when combined with bosentan. Inhibition of the chymotrypsin‐like activity is commonly accompanied by a compensatory increase of the trypsin‐like‐site activity (Shabaneh et al , ; Dettmer et al , ) (Fig A, B). As this physiological compensation was not constantly observed in all cell lines exposed to bortezomib plus bosentan, we suspected additional bosentan cytotoxicity, damaging the cell (and thus proteasome functional integrity) unspecifically.…”