Apoptosis induction via microtubule disassembly by an antitumour compound,pironetin

We reported previously that pironetin and its derivatives were potent inhib itors of cell cycle progression at the M-phase and showed antitumour activi ty against a murine tumour cell line, P388 leukaemia, transplanted in mice. In this paper, we investigated the mechanism of action of pironetins in an titumour activity and cell cycle arrest at the M-phase. As reported previou sly for murine leukaemia P388 cells, pironetin showed antitumour activity i n a dose-dependent manner in the human leukaemia cell line HL-60. Since DNA fragmentation was observed in both P388 and HL-60 cells, the antitumour ac tivity of pironetin is thought to be due to the induction of apoptosis. Pir onetin also induced the rapid phosphorylation of Bcl-2 before formation of the DNA ladder in HL-60 cells, as seen with several tubulin binders. These results suggest that the antitumour activity of pironetin is due to apoptos is caused by the phosphorylation of Bcl-2, and that pironetin targets the m icrotubules. Pironetin and demethylpironetin exhibited reversible disruptio n of the cellular microtubule network in normal rat fibroblast 3Y1 cells. H owever, epoxypironetin, which contains epoxide instead of the double bond o f pironetin, showed only weak activity. Since the concentrations that inhib it cell cycle progression at the M-phase were the same as those for disrupt ion of the microtubule network, it was suggested that the mitotic arrest in duced by pironetin was the result of the loss of the mitotic spindle. These compounds also inhibited the microtubule-associated protein-induced and gl utamate-induced tubulin assembly in vitro. Pironetin inhibited the binding of [H-3]vinblastine, but not that of[H-3]colchicine, to tubulin, and the K- 4, values revealed that the affinity of pironetin for tubulin is stronger t han that of vinblastine. These results suggest that pironetins are novel an titumour agents which inhibit microtubule assembly.