In vitro cytotoxic effect of proteasome inhibitor bortezomib in combination with purine nucleoside analogues on chronic lymphocytic leukaemia cells

Abstract

The anti-tumour in vitro activity of proteasome inhibitor bortezomib (PS-341, VELCADE) in combination with purine nucleoside analogues, cladribine (2-CdA) and fludarabine (FA) was tested in lymphocytes derived from 26 patients with B-cell chronic lymphocytic leukaemia (B-CLL). Cell viability was assessed by propidium iodide staining, and apoptosis by annexin-V and caspase activation flow cytometry assays. Additionally, expression of the apoptosis-regulating proteins Bax, Bak, Bid, Bcl-w, Bcl-2, XIAP and Mcl-1 was evaluated in B-CLL lymphocytes. Bortezomib alone induced significant, dose-dependent cytotoxicity starting from the low concentration 2.5 nm, inducing apoptosis of B-CLL cells. Combination of this agent with 2-CdA or FA resulted in an increase of cytotoxicity when compared with that mediated by single drugs. The observed increase was especially evident when 5 nm of bortezomib were combined with suboptimal doses of 2-CdA or FA. The combination index (CI) was 0.87 for bortezomib + 2-CdA and 0.82 for bortezomib + FA, indicating an evident additive effect of these combinations. Moreover, B-CLL cells were more sensitive to proteasome inhibitor used alone or combined with 2-CdA or FA comparing to CD3+ lymphocytes. Corresponding to enhanced apoptosis, the expression levels of several apoptosis-regulating proteins were altered. The most pronounced changes were down-regulation of XIAP and up-regulation of Bid proteins by the combination of bortezomib with either 2-CdA or FA. This study suggest that the in vitro cytotoxic effect through proteasome inhibition by bortezomib can be increased substantially with low doses of the purine nucleoside analogues, 2-CdA and FA, and that this effect on B-CLL cell is selectively higher than on normal, CD3-positive lymphocytes.