Apoptosis induction via microtubule disassembly by an antitumour compound, pironetin.
We reported previously that pironetin and its derivatives were potent inhibitors of cell cycle progression at the M-phase and showed antitumour activity against a murine tumour cell line, P388 leukaemia, transplanted in mice. In this paper, we investigated the mechanism of action of pironetins in antitumour activity and cell cycle arrest at the M-phase. As reported previously for murine leukaemia P388 cells, pironetin showed antitumour activity in 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 activity of pironetin is thought to be due to the induction of apoptosis. Pironetin 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 apoptosis caused by the phosphorylation of Bcl-2, and that pironetin targets the microtubules. Pironetin and demethylpironetin exhibited reversible disruption of the cellular microtubule network in normal rat fibroblast 3Y1 cells. However, epoxypironetin, which contains epoxide instead of the double bond of pironetin, showed only weak activity. Since the concentrations that inhibit cell cycle progression at the M-phase were the same as those for disruption of the microtubule network, it was suggested that the mitotic arrest induced by pironetin was the result of the loss of the mitotic spindle. These compounds also inhibited the microtubule-associated protein-induced and glutamate-induced tubulin assembly in vitro. Pironetin inhibited the binding of [3H]vinblastine, but not that of [3H]colchicine, to tubulin, and the Kd values revealed that the affinity of pironetin for tubulin is stronger than that of vinblastine. These results suggest that pironetins are novel antitumour agents which inhibit microtubule assembly.
Kondoh
M
M
Antibiotics Laboratory, The Institute of Physical and Chemical Research (RIKEN), Hirosawa 2-1, Wako-shi, Saitama, 351-0198, Japan.
Usui
T
T
Nishikiori
T
T
Mayumi
T
T
Osada
H
H
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D018922:HL-60 Cells; D006801:Humans; D007941:Leukemia P388 / Q000378:metabolism / Q000473:pathology; D007951:Leukemia, Myeloid / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008870:Microtubules / Q000187:drug effects*; D010766:Phosphorylation; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D011753:Pyrones / Q000494:pharmacology*; D051381:Rats
D000970:Antineoplastic Agents; D019253:Proto-Oncogene Proteins c-bcl-2; D011753:Pyrones; C088446:pironetin