In vitro sensitivity of B-cell chronic lymphocytic leukemia to cladribine and its combinations with mafosfamide and/or mitoxantrone.

We examined in vitro sensitivity of B-CLL cells exposed to cladribine, mafosfamide, mitoxantrone and combinations ofcladribine with mafosfamide and/or mitoxantrone. The results revealed that each applied treatment of leukemic cells, besides having a cytotoxic effect, affected the events associated with apoptosis. All drugs used alone, and cladribine combinations with mafosfamide and/or mitoxantrone induced DNA fragmentation and the changes in expression/proteolysis level of caspase-3, caspase-9 precursors, PARP-1, lamin B, Bax and Bcl-2; however, each to a different degree. The exposure of leukemic cells to both cladribine combinations induced stronger effects. Moreover, the data showed that the expression of regulatory antiapoptotic protein Bcl-2 generally decreased in drug-treated B-CLL cells, whereas proapoptotic polypeptide Bax increased, resulting in enhancement of Bax-Bcl-2 ratios in comparison with untreated cells. Drug-treatment of the studied cells induced the translocation of Bax protein from the cytosol to the cellular pellet, containing mitochondria, where this polypeptide indicated the capacity for oligomerization. These observations suggest that the examined drugs are able to induce apoptosis of B-CLL cells via the mitochondria pathway.

Determination of the in vivo effects of cladribine alone and its combination with cyclophosphamide or cyclophosphamide and mitoxantrone on Bax and Bcl-2 protein expression in B-CLL cells.

The present study investigated a correlation between expression of Bcl-2 family members, Bax and Bcl-2 (the Bax/Bcl-2 ratio values) in B-CLL cells in vivo and the response of these cells to chemotherapy. Western blot technique combined with videodensitometry was used for Bax and Bcl-2 determination in homogenate, nuclear and postnuclear fractions of mononuclear cells isolated from peripheral blood of B-CLL patients treated with cladribine alone (C), and in combination with cyclophosphamide (CC) or mitoxantrone and cyclophosphamide (CMC). The Bax/Bcl-2 ratio values were changed in B-CLL cells originated from blood samples of patients treated by the three therapy protocols, and was the most elevated in the case of CMC treatment. High degree of B-CLL cell apoptosis induction with cladribine combined with mitoxantrone and cyclophosphamide was confirmed by DNA fragmentation and an appearance of apoptotic morphology among leukemic cells from the blood of patients treated with this form of combined therapy.