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Mechanisms of action of the anti-VEGF monoclonal antibody bevacizumab on chronic lymphocytic leukemia cells.

INTRODUCTION
Chronic lymphocytic leukemia (CLL) remains incurable; therefore searching for new therapeutic strategies in this disease is necessary. An important mechanism of tumor development is neoangiogenesis. A potent antiangiogenic factor, bevacizumab (Avastin, AVA), has been poorly explored in CLL so far. In the current study we assessed cytotoxic activity of AVA alone or in combinations with drugs routinely used in this disease.


MATERIALS AND METHODS
Cells isolated from 60 CLL patients were treated with AVA alone or in combination with anti-CD20 monoclonal antibody (MoAb), rituximab (RIT), anti-CD52 MoAb, alemtuzumab (ALT), 2-CdA (2-chlorodeoxyadenosine), FA (fludarabine), MAF (mafosfamide) or RAPA (rapamycin). Cytotoxicity was assessed by propidium iodide staining. Apoptosis was evaluated using annexin-V and TUNEL assays. Additionally, a drop of mitochondrial potential (DYm) as well as expression of apoptosis-regulating proteins Bax, Bak, Bid, Bad, Bcl-2, Mcl-2, XIAP, FLIP, Akt and Bcl-2-A1 were determined by flow cytometry.


RESULTS
At the dose of 40 μg/ml, after 48 hours of incubation, AVA induced significant cytotoxicity against CLL cells. The drug triggered apoptosis, with activation of caspase-3 and -9, but not caspase-8, along with a drop of DYm. Incubation with AVA induced significant overexpression of proapoptotic Bak and Bad as well as downregulation of antiapoptotic Mcl-2 and Akt proteins. Combination of AVA with RIT, ALT or RAPA significantly increased cytotoxicity when compared with the effects of single drugs.


DISCUSSION
In conclusion, this is the first report showing proapoptotic activity of AVA against CLL cells. Combination of AVA with RIT, ALT or RAPA may be a promising therapeutic strategy, which requires confirmation in further studies.