Cantharidin-induced Mitotic Arrest is Associated with the Formation of Aberrant Mitotic Spindles and Lagging Chromosomes Resulting in part from the Suppression of PP2Aα

摘要

Cantharidin, a natural vesicant, inhibits the activity of several PPP-family phosphatases, displays antitumor activity and induces apoptosis in many types of tumor cells. However, the molecular mechanisms underlying the antitumor activity of cantharidin are not clear. Here, dose-response studies confirm a strong correlation between the suppression of phosphatase activity and cell death. Flow cytometry analysis indicates that prior to apoptosis, cantharidin delays cell cycle progression following DNA replication with no apparent effect on G1/S- or S/G2-phase progression. In contrast, studies with double thymidine-synchronized populations of cells indicate that cantharidin can rapidly arrest growth when added during G2- or early M-phase. Immunostaining indicates that cell cycle arrest occurs prior to the completion of mitosis and is associated with the appearance of aberrant mitotic spindles. Live-cell imaging with time-lapse microscopy demonstrates that cantharidin disrupts the metaphase alignment of chromosomes, and produces a prolonged mitotic arrest, with the onset of apoptosis occurring prior to the onset of anaphase. To explore the contribution of individual phosphatases, antisense-oligonucleotides and siRNA were developed to suppress the expression of cantharidin sensitive phosphatases. The suppression of PP2Aα, but not PP2Aβ, is sufficient to induce metaphase-arrest, during which time lagging chromosomes are observed moving between the spindle poles and the metaphase plate. Immunostaining revealed slightly abnormal, yet predominately bipolar, mitotic spindles. Nonetheless, after a 10–15 hour delay the cells enter anaphase, suggesting an additional cantharidin sensitive phosphatase is involved in the progression from metaphase into anaphase or to prevent the onset of apoptosis in cells arrested during mitosis.