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


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Duechler
Markus
M
Ludwig Boltzmann Institute for Cytokine Research, Department of Hematology, Clinic of Internal Medicine I, University of Vienna, Austria.
Linke
Anna
A
Cebula
Barbara
B
Shehata
Medhat
M
Schwarzmeier
Josef D
JD
Robak
Tadeusz
T
Smolewski
Piotr
P
D017304:Annexin A5 / Q000032:analysis; D000970:Antineoplastic Agents / Q000633:toxicity; D017209:Apoptosis / Q000187:drug effects; D001897:Boronic Acids / Q000633:toxicity*; D000069286:Bortezomib; D002470:Cell Survival / Q000187:drug effects*; D017338:Cladribine / Q000633:toxicity; D004305:Dose-Response Relationship, Drug; D006801:Humans; D016130:Immunophenotyping; D015451:Leukemia, Lymphocytic, Chronic, B-Cell; D061988:Proteasome Inhibitors; D011719:Pyrazines / Q000633:toxicity*; D014407:Tumor Cells, Cultured
D017304:Annexin A5; D000970:Antineoplastic Agents; D001897:Boronic Acids; D061988:Proteasome Inhibitors; D011719:Pyrazines; D017338:Cladribine; D000069286:Bortezomib