title: Combretastatin-A4 prodrug induces mitotic catastrophe in chronic lymphocytic leukemia cell line independent of caspase activation and poly(ADP-ribose) polymerase cleavage.
abstract: 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.
authors.affiliation: Division of Hematology and Oncology, Department of Internal Medicine, Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan 48201, USA.
authors.forename: Sanaa M
authors.initials: SM
authors.lastname: Nabha
authors.forename: Ramzi M
authors.initials: RM
authors.lastname: Mohammad
authors.forename: Mahmoud H
authors.initials: MH
authors.lastname: Dandashi
authors.forename: Brigitte
authors.initials: B
authors.lastname: Coupaye-Gerard
authors.forename: Amro
authors.initials: A
authors.lastname: Aboukameel
authors.forename: George R
authors.initials: GR
authors.lastname: Pettit
authors.forename: Ayad M
authors.initials: AM
authors.lastname: Al-Katib
mesh_terms: D048948:14-3-3 Proteins; D000972:Antineoplastic Agents, Phytogenic / Q000494:pharmacology; D014408:Biomarkers, Tumor; D015153:Blotting, Western; D016203:CDC2 Protein Kinase / Q000378:metabolism; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D002470:Cell Survival; D000071877:Checkpoint Kinase 1; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005092:Exonucleases / Q000378:metabolism; D005095:Exoribonucleases; D005434:Flow Cytometry; D016195:G2 Phase; D005819:Genetic Markers; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000188:drug therapy*; D008938:Mitosis; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D010766:Phosphorylation; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D011355:Prodrugs / Q000494:pharmacology*; D011494:Protein Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D051736:Proto-Oncogene Proteins c-mdm2; D013267:Stilbenes / Q000494:pharmacology*; D013997:Time Factors; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D051028:bcl-2-Associated X Protein
chemical_list: D048948:14-3-3 Proteins; D000972:Antineoplastic Agents, Phytogenic; C494826:BAX protein, human; D014408:Biomarkers, Tumor; C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004791:Enzyme Inhibitors; D005819:Genetic Markers; D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D011355:Prodrugs; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D013267:Stilbenes; D016159:Tumor Suppressor Protein p53; D051028:bcl-2-Associated X Protein; C497206:MDM2 protein, human; D051736:Proto-Oncogene Proteins c-mdm2; D011065:Poly(ADP-ribose) Polymerases; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D016203:CDC2 Protein Kinase; D005092:Exonucleases; D005095:Exoribonucleases; C413442:SFN protein, human; C505407:CASP3 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases; C058728:fosbretabulin