In vitro antileukemic activity of novel adenosine derivatives bearing boron cluster modification.

A series of adenosine derivatives bearing a boron cluster were synthesized and evaluated for their cytotoxicity against primary peripheral mononuclear cells from the blood of 17 patients with leukemias (16 CLL and 1 very rare PLL), as well as from 5 healthy donors used as a control. Among the tested agents, two, i.e., compounds 1 and 2, displayed high in vitro cytotoxicity and proapoptotic potential on leukemic cells, with only scarce activity being seen against control cells. Biological tests related to apoptosis revealed the activation of the main execution apoptotic enzyme, procaspase-3, in CLL and PLL cells exposed to compounds 1 and 2. Moreover, the above compounds indicated high activity in the proteolysis of the apoptotic markers PARP-1 and lamin B1, fragmentation of DNA, and the induction of some changes in the expression of the Mcl-1, protein apoptosis regulator in comparison with control cells.

In vivo and ex vivo responses of CLL cells to purine analogs combined with alkylating agent.

BACKGROUND: The heterogeneity of chronic lymphocytic leukemia (CLL) is thought to be due to differences in the expression of factors that regulate apoptosis and cell cycle, giving rise to diverse apoptotic disturbances and tumor properties. Therefore, the primary goal in CLL treatment is to overcome resistance to apoptosis and efficiently trigger this process in leukemic cells. METHODS: Mononuclear cells were obtained from the blood of CLL patients by Histopaque-1077 sedimentation. CLL cell samples from the blood of drug treated patients, (cladribine or fludarabine with cyclophosphamide; CC or FC), as well as the cell samples of untreated patients exposed to the used drug combinations (CM, FM) or mafosfamide alone for 48 h were fractionated into nuclear and cytoplasmic fractions or were lysed. DNA fragmentation was evaluated by agarose electrophoresis and also cytometrically as sub-G1 population. The expression of apoptosis related proteins and H1.2 histone translocation were evaluated in lysates and nuclear and cytoplasmic fractions, respectively with appropriate antibodies. RESULTS: Cladribine (C) and fludarabine (F) combined with cyclophosphamide/mafosfamide in vivo, as well as ex vivo trigger apoptosis in CLL cells. These drug combinations (CC; FC/CM; FM) induce leukemic cell apoptosis confirmed by DNA fragmentation, sub-G1 cell number, down-regulation of anti-apoptotic proteins (Mcl-1, Bcl-2), and H1.2 histone translocation in comparison with appropriate control cells, however, to a different degree. CONCLUSIONS: The kinetics and rate of drug-induced apoptosis in leukemic cells under ex vivo experiments differ between patients, mirroring the differences noticed during in vivo treatment. Individual model cell samples indicate comparable susceptibility to the used drug combinations under in vivo and ex vivo conditions.

Promising anti-leukemic activity of atorvastatin.

There is a current need for novel therapeutic strategies for the treatment of chronic lymphocytic leukemia (CLL), a still incurable hematological cancer involving mainly deregulated apoptosis. The purpose of the present study was to determine ex vivo the effect of the synthetic statin, atorvastatin, a known cholesterol-lowering drug, on peripheral blood mononuclear cells obtained from CLL patients. Using flow cytometry, we investigated the viability and induction of apoptosis in leukemic cells exposed to statin by the Vybrant apoptosis assay kit #4, compared with untreated control cells. We also examined the expression levels of apoptosis-regulatory proteins (Mcl-1, Bcl-2 and Bax), as well as products of the expression/proteolysis of lamin B, poly(ADP-ribose) polymerase­1 (PARP­1) and p27Kip1 by western blot analysis. Moreover, the number of sub-G1 cells and DNA fragmentation in atorvastatin-treated leukemic cells were examined by flow cytometry and agarose gel electrophoresis, respectively. The obtained results indicated that CLL cells ex vivo were extremely sensitive to atorvastatin. The cytotoxic effect of this statin was caused by the induction of apoptosis in the leukemic cells. The induction of apoptosis in the drug-treated model cells was confirmed by the reduction or proteolysis of apoptotic markers, such as PARP-1, lamin B and p27Kip1, the increase in the number of sub-G1 cells and DNA ladder formation. During atorvastatin-triggered apoptosis, changes in the expression levels of mitochondrial outer membrane permeability regulatory proteins of the Bcl-2 family were also observed. Ex vivo promising data indicate the strong cytotoxic and pro-apoptotic potential of atorvastatin against leukemic cells, but not normal cells. The obtained data suggest that atorvastatin be considered as a therapeutic option for the treatment of CLL.

Can ex vivo evaluation (testing) predict the sensitivity of CLL cells to therapy with purine analogs in conjunction with an alkylating agent? A comparison of in vivo and ex vivo responses to treatment.

Malfunctions in the regulation of apoptosis cause the accumulation of malignant, long-lived B CD19+/CD5+ cells in chronic lymphocytic leukemia (CLL). The primary goal in CLL therapy is to overcome resistance to apoptosis and efficiently trigger programmed cell death in leukemic cells. This study demonstrated that the in vivo responses of malignant cells from CLL patients after administration of purine analogs (cladribine/fludarabine) with cyclophosphamide vary significantly. For comparative purposes, the sensitivity of leukemic cells obtained from the same CLL patients to conventional purine analogs and the selective CDK inhibitor R-roscovitine (ROSC) was determined, with and without the addition of an alkylating agent, prior to the onset of in vivo therapy. The kinetics and rate of spontaneous and drug-induced apoptosis of CLL cells under ex vivo conditions differed significantly between patients, mirroring the variability observed during in vivo treatment. Interestingly, individual patients' leukemic cells were comparably sensitive to the drugs under both conditions. Of the drugs examined, ROSC exerted the highest therapeutic efficacy under ex vivo conditions. Our results indicate that ex vivo testing might be useful for identifying the most potent first-line therapeutic regimen for specific CLL patients and possibly for the design of therapies tailored for individual CLL patients.

R-roscovitine (Seliciclib) affects CLL cells more strongly than combinations of fludarabine or cladribine with cyclophosphamide: Inhibition of CDK7 sensitizes leukemic cells to caspase-dependent apoptosis.

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of malignant, apoptosis-resistant B CD19(+)/CD5(+) cells. Populations of CLL cells are heterogeneous and consist primarily of quiescent cells with a minor subset of dividing cells. In this study the efficacy of a first-line in vivo therapy was compared with treatment by R-roscovitine (ROSC) alone or by purine analogues (cladribine and fludarabine) combined with maphosphamide for 14 CLL patients under ex vivo conditions. ROSC induced the highest reduction in numbers of living B-cells, coinciding with an increased rate of apoptosis. After 24 h the percentage of apoptotic cells in ROSC-treated cultures was markedly higher than in untreated controls. ROSC also induced strong activation of the apoptosome and effector caspases in CLL cells. During progression of apoptosis the plasma membrane became permeable, resulting in the release of activated caspases into the culture medium. Leukemic cells were more sensitive to ROSC than normal mononuclear cells. Treatment with ROSC did not affect the activating phosphorylation of CDK2 or CDK1. However, ROSC decreased phosphorylation of survivin, CDK7, and RNA-Pol II, resulting in inhibition of transcription elongation and subsequent down-regulation of levels of anti-apoptotic factors, thereby facilitating apoptosis. Unlike ROSC, two other purine analogues barely affected the cellular levels of anti-apoptotic proteins and more weakly activated effector caspases. In addition, the efficacies of in vivo and ex vivo therapies were found to be correlated. Marked between-patient differences in expression patterns of apoptosis-regulating factors in CLL cells were observed, explaining the variations in patients' sensitivity to therapy.

Roscovitine triggers apoptosis in B-cell chronic lymphocytic leukemia cells with similar efficiency as combinations of conventional purine analogs with cyclophosphamide.

B-cell chronic lymphocytic leukemia (CLL) is characterized by an accumulation in peripheral blood of many long-lived lymphocytes that do not die because of the deregulation of apoptosis. Most CLL cells are quiescent, and therefore the leukemic lymphocytes are resistant to conventional chemotherapy. The aim of this study was to evaluate in vitro the chemosensitivity of CLL cells to cladribine or fludarabine used alone or in combinations with mafosfamide (Mf; the active form of cyclophosphamide) as well as to roscovitine, a potent inhibitor of cyclin-dependent kinases with proapoptotic potential. The results of flow cytometry revealed that tested agents differentially reduced the viability of leukemic cells. Interestingly, roscovitine exerts a similar cytotoxic effect as the combinations of the used purine analogs with Mf, but with other kinetics. Roscovitine kills leukemic cells after a much shorter exposure time. Immunoblotting analysis showed that the reduction of the number of living cells coincides with marked changes of the balance between pro- and antiapoptotic factors. The latter were markedly reduced. The activation of proapoptotic proteins became evident especially after exposure of cells to roscovitine alone or to combinations of purine analogs and Mf. Furthermore, exposure of CLL cells to tested drugs degraded p27(KIP1) protein. Our findings demonstrate that roscovitine alone significantly reduces the number of viable CLL cells by inducing them to undergo apoptosis, and it acts earlier than clinically applied combinations of purine analogs with Mf/cyclophosphamide. These results confirm the high efficacy of roscovitine against CLL cells.