fosbretabulin and Leukemia--Lymphocytic--Chronic--B-Cell

Microtubule targeting agents, such as vinblastine, are usually thought to arrest cells in mitosis and subsequently induce apoptosis. However, they can also cause rapid induction of apoptosis in a cell-cycle phase independent manner. BNC105 is a novel vascular and microtubule disrupting drug that also induces apoptosis rapidly but with markedly increased potency compared to vinca alkaloids and combretastatin A4. BNC105 binds to the colchicine-binding site on tubulin resulting in activation of c-Jun N-terminal kinase (JNK), phosphorylation of ATF2, and induction of ATF3 and Noxa leading to acute apoptosis in chronic lymphocytic leukemia (CLL) cells. Apoptosis induced by BNC105 is dependent upon both JNK activation and Noxa induction. Normal leukocytes and one CLL sample also exhibited JNK activation but not Noxa induction and were resistant to BNC105. This study emphasizes the importance of Noxa and JNK for induction of apoptosis in CLL cells by microtubule targeting drugs, and highlights the potential of BNC105 as a potent therapeutic to treat haematopoietic malignancies.

Topics: Anisoles; Apoptosis; Benzofurans; Cell Line, Tumor; Humans; JNK Mitogen-Activated Protein Kinases; Leukemia, Lymphocytic, Chronic, B-Cell; MAP Kinase Signaling System; Microtubules; Stilbenes; Transfection; Tubulin Modulators; Vinblastine

We have previously reported that combretastatin-A4 prodrug (CA4P), anantitubulin/antiangiogenic agent isolated from the South African willow tree Combretum caffrum, induced cell death primarily through mitotic catastrophe in a panel of human B-lymphoid tumors. In this study, we investigated the molecular aspects of the mitotic catastrophe and whether or not it shares the same pathways of apoptosis. For this we studied the effect of CA4P on selected markers of apoptosis [caspases 9 and 3, poly(ADP-ribose) polymerase (PARP), bcl-2, and bax] and G2-M protein regulators (p53, MDM2, 14-3-3sigma, GADD45, cdc2, cdc25, chk1, wee1, p21, and cyclin B1). The chronic lymphocytic leukemia cell line WSU-CLL was used for this purpose. Western blot analysis showed that 24 h of CA4P (5 nM) exposure induces caspase 9 activation and PARP cleavage. However, the addition of Z-Val-Ala-Asp-fluoromethylketone (a general caspase inhibitor) or Z-Leu-Glu(OMe)-His-Asp(OMe)-CH2F (a caspase 9 inhibitor) before CA4P treatment did not block cell death. No change in bcl-2 or bax protein expression was observed. Exposure of WSU-CLL cells to 4 and 5 nM CA4P was associated with overproduction of total p53 and no dramatic change in MDM2, 14-3-3sigma, GADD45, the cyclin-dependent kinase cdc2, its inhibitory phosphorylation, the cdc2-inhibitory kinase (wee1), chk1, or cdc25 hyperphosphorylation. The overaccumulation of p21 and cyclin B1 protein was obvious at 24 h. Furthermore, CA4P treatment showed an increase in the expression of a marker of mitosis (mitotic protein monoclonal-2 antibody) and an overaccumulation of the cyclin B in the nucleus. Our findings suggest that CA4P induces mitotic catastrophe and arrest of WSU-CLL cells mostly in the M phase independent of p53 and independent of chk1 and cdc2 phosphorylation pathways. Apoptosis is a secondary mechanism of death in a small proportion of cells through activation of caspase 9 and PARP cleavage. The two mechanisms of cell death, i.e., mitotic catastrophe and apoptosis, are independent of each other in our model.

Topics: 14-3-3 Proteins; Antineoplastic Agents, Phytogenic; bcl-2-Associated X Protein; Biomarkers, Tumor; Blotting, Western; Caspase 3; Caspase 9; Caspases; CDC2 Protein Kinase; Cell Nucleus; Cell Survival; Checkpoint Kinase 1; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Enzyme Activation; Enzyme Inhibitors; Exonucleases; Exoribonucleases; Flow Cytometry; G2 Phase; Genetic Markers; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Mitosis; Neoplasm Proteins; Nuclear Proteins; Phosphorylation; Poly(ADP-ribose) Polymerases; Prodrugs; Protein Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Proto-Oncogene Proteins c-mdm2; Stilbenes; Time Factors; Tumor Cells, Cultured; Tumor Suppressor Protein p53