Disorazole C₁: A potent antimitotic agent that induces premature senescence

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Disorazole C₁ is a potent, cytotoxic natural product, with IC₅₀ values of < 20 nM in a variety of cancer cell lines. The addition of disorazole C₁ disrupted microtubules in cells as early as 24 h after treatment, causing multinucleation and disorganized microtubules at interphase and misaligned chromosomes at the metaphase plate during mitosis. A549 cells that survived a 7 day treatment of disorazole C₁ displayed an enlarged, flattened morphology similar to senescent cells treated with doxorubicin. These surviving cells displayed the characteristic β-galactosidase staining at pH 6.0 seen in senescent cells and changes in cell cycle markers indicative of premature or hypermitogenic senescence. A549 cells treated with the IC₅₀ concentration of disorazole C₁ (2 nM) for 7 days had increased protein levels of cyclin D, p53 and cyclin-dependent kinase inhibitor p21 as detected by Western blot. This resulted in decreased phosphorylation of Rb (Ser780) and a decrease in total Rb protein levels. A dramatic decrease in BrdU incorporation was also observed in disorazole C₁ treated cells, further implicating the induction of senescence. Finally, DC3F/VCRd-5L cells, which are >2000-fold resistant to vincristine as compared to DC3F cells, were only 5-fold resistant to disorazole C₁. These results provide the first evidence of a microtubule disrupter that induces premature senescence.