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.