Bortezomib - A Potential Therapeutic Approach in B Chronic Lymphocytic Leukaemia.

Abstract

B-cell lymphocytic leukaemia (B-CLL) is an indolent non-Hodgkin’s lymphoma and the most frequent leukaemia. Even though the capacity of B-CLL leukemic cells to proliferate has been underestimated until recently, the accumulation of tumor cells mostly results from a defect in the apoptotic program. The ubiquitin/proteasome system is important in the turnover of regulatory proteins and as regulator of cell proliferation and apoptosis. This critical link between the apoptotic machinery and the ubiquitin/proteasome system led to an increased interest in designing inhibitory agents that target these pathways. A promising new drug, bortezomib (BZ), a dipeptidyl boronic acid proteasome inhibitor, was approved for treatment of relapsed multiple myeloma. Extensive preclinical data are being developed to study the potential application of this new drug in other cancers. Our preliminary results show that BZ induces a marked decrease in LLC-B cell viability by increasing the percentage of apoptosis. The aim of this study is to evaluate the potential therapeutic value of BZ in B-CLL patients studying the cytotoxicity mechanisms induced by this proteasome inhibitor. For this purpose, mononuclear cells isolated from 22 patients with B-CLL (14 without and 8 with conventional therapy) were cultured in absence and presence of bortezomib (BZ) (ranging concentration from 0.01 μM to 10 μM), as single agent, or plus 75 or 150 μM fludarabine (FDN), during 24 hours. Directly conjugated monoclonal antibodies to CD5 and CD19 was used to identify LLC-B cells. Cell death was evaluated by annexin V incorporation and detected by flow cytometry. The expression of the proteins involved in apoptosis regulation, namely the proapoptotic proteins, Bax and p53, and the antiapoptotic protein Bcl-2 was determined by flow cytometry using monoclonal antibodies. Our results show that BZ induces a marked decrease in B-CLL cell viability by increasing the percentage of apoptosis in a dose dependent manner (20 to 80% apoptosis). However, in concentrations higher than 1 mM a saturable effect is observed. On the other hand, a lesser sensitivity to BZ was observed in normal mononuclear cells (≤ 30% apoptosis). BZ apoptotic effect seems to be independent of previous therapy. We observed higher levels of apoptosis in B-CLL cells treated with BZ compared with cells not treated or in response to treatment with FDN, which may relate with the increase in Bax expression (average difference of 37.3 ± 18%). However, we observe identical apoptosis levels in B-CLL cells, as opposed to others (Duechler, M. et al., 2005), and an increase in apoptotic T cells levels in the presence of treatment with BZ plus FDN compared with those observed in cells treated with BZ alone. On the other hand, when compared with the results obtained with FDN alone (in 150 μM), an increase in apoptosis levels was detected (average difference of 69.5 ± 28% increase in apoptosis, for B-CLL cells and 33.6 ± 11.6%, for T cells). Our results support that bortezomib induces apoptosis as a single agent in a Bax-dependent way. The apoptotic effect show some selectivity for the transformed cells (B-CLL). These results suggests that this proteasome inhibitor may be useful as a therapeutic approach in B-CLL patients.