Glyfoline exhibits cytotoxic activity in vitro and antitumor activity in mice bearing murine or human solid
tumors, but the underlying mechanisms are unknown. In our study, we found that
glyfoline inhibited cell growth and induced accumulation of mitotic cells in human
cancer cell lines.
Glyfoline induced the appearance of spindle
abnormalities, chromosome mis-segregation, multipolar cell division and multiple nuclei, all of which are indicative of mitotic catastrophe. However,
glyfoline did not bind to
DNA and did not inhibit or stabilize
tubulin polymerization, but slightly increased the resistance of mitotic spindles to
nocodazole-induced disassembly. In addition, microtubule aster formation was significantly enhanced in the extract prepared from
glyfoline-arrested mitotic cells compared to that from synchronized mitotic cells. When Eg5, a mitotic
kinesin that plays an essential role in establishing mitotic spindle bipolarity, was inhibited using S-trityl-
cysteine in
glyfoline-treated cells, formation of spindle multipolarity, multipolar cell division, and multinuclei was significantly reduced. After
glyfoline-mediated arrest of cells at mitosis, considerable
poly(ADP-ribose) polymerase degradation was induced and the number of
annexin V-positive cells significantly increased, indicating that
glyfoline ultimately induces apoptosis.
Small interfering RNA-mediated silencing of the spindle checkpoint
proteins BUBR1 and MAD2 markedly reduced induction of mitotic cell accumulation, but did not affect
glyfoline-induced mitotic catastrophe and apoptosis. Thus,
glyfoline induces mitotic catastrophe probably by enhancing microtubule aster formation and subsequent apoptosis in
cancer cells independently of spindle checkpoint function.