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Apoptosis induction via microtubule disassembly by an antitumour compound, pironetin
M Kondoh1, T Usui, T Nishikiori
1Antibiotics Laboratory, The Institute of Physical and Chemical Research (RIKEN), Hirosawa 2-1, Wako-shi, Saitama, 351-0198, Japan.
Abstract:
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.
Insights
Pironetins show anticancer effects by inducing apoptosis and targeting microtubules, leading to cell cycle arrest. These novel agents inhibit microtubule assembly, offering potential as new antitumour drugs.
Area of Science:
- Pharmacology
- Cell Biology
- Biochemistry
Background:
- Pironetin and its derivatives were previously identified as inhibitors of M-phase cell cycle progression and demonstrated antitumour activity.
- The precise mechanism underlying pironetin's antitumour effects and M-phase cell cycle arrest required further investigation.
Purpose of the Study:
- To elucidate the mechanism of action of pironetins in mediating antitumour activity and M-phase cell cycle arrest.
- To investigate the effects of pironetin on apoptosis, Bcl-2 phosphorylation, and microtubule dynamics.
Main Methods:
- Dose-dependent antitumour activity assessment in human leukaemia (HL-60) and murine leukaemia (P388) cell lines.
- Analysis of DNA fragmentation and Bcl-2 phosphorylation to evaluate apoptosis induction.
- Microtubule disruption assays in rat fibroblast (3Y1) cells and in vitro tubulin assembly inhibition studies.
- Binding assays to investigate the interaction of pironetin with tubulin.
Main Results:
- Pironetin demonstrated dose-dependent antitumour activity in HL-60 cells, similar to its effects on P388 cells.
- DNA fragmentation and Bcl-2 phosphorylation were observed, suggesting apoptosis induction as a key mechanism.
- Pironetin and demethylpironetin disrupted the microtubule network, leading to M-phase arrest, while epoxypironetin showed weaker activity.
- Pironetin inhibited tubulin assembly in vitro and bound to tubulin with higher affinity than vinblastine, but not colchicine.
Conclusions:
- Pironetins exert antitumour effects by inducing apoptosis, likely through Bcl-2 phosphorylation and subsequent DNA fragmentation.
- Pironetins act as novel antitumour agents by targeting microtubules, inhibiting their assembly, and disrupting the mitotic spindle, leading to M-phase arrest.