E7389, a macrocyclic
ketone analog of the marine
natural product halichondrin B, currently is undergoing clinical trials for
cancer. This fully synthetic agent exerts its highly potent in vitro and in vivo anticancer effects via
tubulin-based
antimitotic mechanisms, which are similar or identical to those of parental
halichondrin B. In an attempt to understand the impressive potency of E7389 in animal models of human
cancer, its ability to induce apoptosis following prolonged mitotic blockage was evaluated. Treatment of U937 human
histiocytic lymphoma cells with E7389 led to time-dependent collection of cells in the G2-M phase of the cell cycle, beginning as early
as 2 h and becoming maximal by 12 h. Increased numbers of hypodiploid events were seen beginning at 12 h, suggesting initiation of apoptosis after prolonged E7389-induced mitotic blockage. The identity of hypodiploid events as apoptotic cells under these conditions was confirmed by two additional morphologic criteria: green to orange/yellow shifts on
acridine orange/
ethidium bromide staining, and cell surface
annexin V binding as assessed by flow cytometry. Several biochemical correlates of apoptosis also were seen following E7389 treatment, including phosphorylation of the antiapoptotic
protein Bcl-2,
cytochrome c release from mitochondria, proteolytic activation of
caspase-3 and -9, and cleavage of the
caspase-3 substrate
poly(ADP-ribose) polymerase (PARP). In LNCaP human
prostate cancer cells, treatment with E7389 also led to generation of hypodiploid cells, activation of
caspase-3 and -9, and appearance of cleaved PARP, indicating that E7389 can activate cellular apoptosis pathways under anchorage-independent and -dependent cell culture conditions. These results show that prolonged mitotic blockage by E7389 can lead to apoptotic cell death of human
cancer cells in vitro and can provide a mechanistic basis for the significant in vivo anticancer efficacy of E7389.