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Glyfoline exhibits cytotoxic activityin vitroand 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 usingS-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.