Cytotoxic and apoptosis-inducing efects of bendamustine used alone and in combination with rituximab on chronic lymphocytic leukemia cells in vitro* Cytotoksyczny i proapoptotyczny wplyw bendamustyny zastosowanej pojedynczo lub w skojarzeniu z rytuksymabem na komórki przewlek lej bialaczki limfocytowej in vitro Ewelina Ziolkowska 1, , , , , Dariusz Wolowiec 2, , , Barbara Cebula-Obrzut 3, , Jerzy Z. Blonski 1, , Piotr Smolewski 3, , Tadeusz Robak 1, , , , Anna Korycka-Wolowiec 1, , , , , 1 Department of Hematology, Medical University of Lodz, Poland 2 Department of Hematology, Medical University of Wroclaw, Poland 3 Department of Experimental Hematology, Medical University of Lodz, Poland Summary The aim of our study was to compare the cytotoxic efects of bendamustine (BENDA) and rituximab (RIT) used either alone or in combination and to evaluate the infuence of the above mentioned drugs on apoptosis measured as changes in mitochondrial transmembrane potential (Δψ m ), expression of caspases and selected apoptosis-regulating proteins in freshly isolated peripheral blood mononuclear cells of chronic lymphocytic leukemia (CLL) patients. Cytotoxic efect of tested drugs, as well as induction of apoptosis, drop in Δψ m and expression of selected proteins involved in regulation of apoptosis were assessed in 48 hour cultures containing autologous serum (AS) using fow cytometry. BENDA was used at the concentration of 40 µg/ml and RIT at the concentration of 10 µg/ml. Control cultures were incubated without drugs. 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. 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. chronic lymphocytic leukemia • bendamustine • rituximab • cytotoxicity • mitochondrial potential changes • apoptosis-regulatory proteins Received: 2014.06.26 Accepted: 2014.09.04 Published: 2014.12.07 Postepy Hig Med Dosw (online), 2014; 68 * Te study was supported from Mundipharma Research Ltd grant, statutory means of the Department of Hematology Medical University of Lodz (No. 503/1-093-01/503-01) and the Foundation of Diagnostics and Terapy, Warsaw. www.phmd.pl Original Article 1433 Postepy Hig Med Dosw (online), 2014; 68: 1433-1443 e-ISSN 1732-2693 Aim: Material/Methods: Keywords: Conclusions: Results: Authors’ Contribution: Study Design Data Collection Statistical Analysis Data Interpretation Manuscript Preparation Literature Search Funds Collection