Evaluation of apoptosis induced in vitro by cladribine (2-CdA) combined with anthracyclines in lymphocytes from patients with B-cell chronic lymphocytic leukemia.

The aim of the study was to evaluate the effect of three anthracyclines [doxorubicin (DOX), mitoxantrone (MIT), and idarubicin (IDA)] on the rate of apoptosis triggered by 2-chlorodeoxyadenosine (2-CdA) in peripheral blood mononuclear cells isolated from 52 untreated patients with B-cell chronic lymphocytic leukemia (B-CLL). The cells were cultured up to 48 h in the presence of drugs alone and in the following combinations: 2-CdA+DOX, 2-CdA+MIT, and 2-CdA+IDA. Apoptosis was assessed after 24 h and 48 h of incubation using annexin V/propidium iodide assay by flow cytometry. The apoptotic index (AI) was defined as a percentage of annexin V-positive B-CLL cells. Additionally, in some patients other hallmarks of apoptosis (activation of caspases, DNA fragmentation) were assessed in parallel for confirmation of apoptotic mode of induced cell death. All of the cytostatics induced apoptosis of B-CLL cells at a rate significantly higher than the index of spontaneous apoptosis occurring during 24 h and 48 h of cell culture. 2-CdA in combination with DOX significantly increased the percentage of annexin V-positive cells, particularly after 48 h of incubation, as compared with DOX used in monotherapy (median AI for 2-CdA+DOX=37.9%, median AI for DOX =13.8%, P=0.0011, and median AI for 2-CdA=22.1%, P=0.013). Combination of 2-CdA with MIT induced a similar effect, also more distinct after 48 h (median AI for 2-CdA+MIT=41.05%, median AI for MIT=16.3%, p=0.0012, and median AI for 2-CdA=22.1%, p=0.017). For both combinations median AI were similar to the sum of median AI for each drug when used alone. IDA in a concentration ten times lower (0.1 micro g/ml) than used before in acute leukemia cells produced high cytotoxic effects, masking the additive effect of combination with 2-CdA. Only at a dose of 25 ng/ml of IDA, significant differences in AI after 24 h and 48 h were detected between samples treated with 2-CdA+IDA (median 27.5% and 65.0%, respectively) and those incubated with IDA alone (median 10.5% and 33.4%; P=0.0004 and 0.0274, respectively). Similarly, there were significant differences between AI of cells treated with 2-CdA+IDA and 2-CdA alone (median 9.5% at 24 h and 23.5% at 48 h; P=0.0013 and 0.0207, respectively). In conclusion; these data indicate an additive cytotoxic effect on B-CLL cells of DOX, MIT, and IDA applied in vitro with 2-CdA; all of them induced apoptosis with similar efficacy. We suggest that further preclinical and clinical studies concerning combined use of 2-CdA with anthracyclines are desirable. High sensitivity of B-CLL cells to IDA suggests the possibility of lowering its dose in patients, especially when combined with 2-CdA.