Abstract
Bortezomib (Velcade™) is a reversible proteasome inhibitor that is approved for the treatment of multiple myeloma (MM). Despite its demonstrated clinical success, some patients are deprived of treatment due to primary refractoriness or development of resistance during therapy. To investigate the role of the duration of proteasome inhibition in the anti-tumor response of bortezomib, we established clonal isolates of HT-29 adenocarcinoma cells adapted to continuous exposure of bortezomib. These cells were ∼30-fold resistant to bortezomib. Two novel and distinct mutations in the β5 subunit, Cys63Phe, located distal to the binding site in a helix critical for drug binding, and Arg24Cys, found in the propeptide region were found in all resistant clones. The latter mutation is a natural variant found to be elevated in frequency in patients with MM. Proteasome activity and levels of both the constitutive and immunoproteasome were increased in resistant cells, which correlated to an increase in subunit gene expression. These changes correlated with a more rapid recovery of proteasome activity following brief exposure to bortezomib. Increased recovery rate was not due to increased proteasome turnover as similar findings were seen in cells co-treated with cycloheximide. When we exposed resistant cells to the irreversible proteasome inhibitor carfilzomib we noted a slower rate of recovery of proteasome activity as compared to bortezomib in both parental and resistant cells. Importantly, carfilzomib maintained its cytotoxic potential in the bortezomib resistant cell lines. Therefore, resistance to bortezomib, can be overcome with irreversible inhibitors, suggesting prolonged proteasome inhibition induces a more potent anti-tumor response.
Highlights
The proteasome is a multicatalytic proteolytic structure that is responsible for the degradation of intracellular proteins [1]
The introduction of bortezomib to the armamentarium of myeloma therapy has resulted significant clinical success but primary refractoriness and treatment-emergent drug resistance has deprived a subset of patients of effective therapy [7,8]
In order to better understand the mechanism of drug resistance, we developed multiple lines of solid tumor HT-29 cells adapted to continuous bortezomib pressure
Summary
The proteasome is a multicatalytic proteolytic structure that is responsible for the degradation of intracellular proteins [1]. Three distinct catalytic activities comprise the proteasome: chymotrypsinlike (CT-L), caspase-like (C-L), and trypsin-like (T-L). These activities are encoded in the broadly expressed constitutive (c20S) form of the proteasome by b5, b1, and b2, respectively. Another form of the proteasome that is primarily expressed in cells of hematopoietic origin and cells exposed to inflammatory cytokines, known as the immunoproteasome (i20S), has the three catalytic activities represented by LMP7, LMP2, and MECL1. Three signaling modules of the unfolded protein response are activated: i)
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