Cytotoxic and apoptosis-inducing effects of bendamustine used alone and in combination with rituximab on chronic lymphocytic leukemia cells in vitro

Aim: The aim of our study was to compare the cytotoxic effects of bendamustine (BENDA) and rituximab (RIT) used either alone or in combination and to evaluate the influence of the above mentioned drugs on apoptosis measured as changes in mitochondrial transmembrane potential (Delta psi(m)), expression of caspases and selected apoptosis-regulating proteins in freshly isolated peripheral blood mononuclear cells of chronic lymphocytic leukemia (CLL) patients. Material/Methods: Cytotoxic effect of tested drugs, as well as induction of apoptosis, drop in Delta psi(m) and expression of selected proteins involved in regulation of apoptosis were assessed in 48 hour cultures containing autologous serum (AS) using flow cytometry. BENDA was used at the concentration of 40 mu g/ml and RIT at the concentration of 10 mu g/ml. Control cultures were incubated without drugs. Results: BENDA used either alone or in combination with RIT strongly induced apoptosis as well as enhanced expression of selected apoptotic proteins, especially those involved in the intrinsic apoptotic pathway: P53, PUMA and BAX, which cause mitochondrial transmembrane potential changes leading to activation of caspase-9 and -3. Conclusions: Our results indicate that both BENDA and RIT participate in the induction of apoptosis of CLL lymphocytes in vitro in the presence of AS in the culture medium. The drug-induced apoptosis occurs mainly via intrinsic pathway and activation of P53 and PUMA proteins, however the extrinsic pathway is likely to be involved as well. We also found that the combination of these drugs induces the expression of P53, caspase-8 and -9 more potently than either of them used separately.