Platycodin d induces g₂/m cell-cycle arrest, leading to endoreduplication, inhibition of proliferation and apoptosis in leukemia cells

B3

Platycodin D (PD), a major constituent of triterpene saponins in Platycodon grandiflorum, has also become an interesting candidate for cancer chemotherapy however, little is known about apoptotic mechanisms on cancer cells. We herein investigated the mechanisms that are related to PD-induced anti-proliferation and cell death in human leukemia cells (U937, THP-1 and K562 cells). Cell growth was assessed with proliferation assays, cell counting, flow cytometry, phase contrast microscopy and Western blot assay. Microtubule (MT) formation was measured with immunofluorescent staining and in vitro tubulin polymerization assay. Apoptotic effect was analyzed by assessing increase in annexin V-staining and caspase-3 activity. Treatment of synchronized leukemia cells with varying concentrations of PD resulted in significant G₂/M cell cycle arrest and endoreduplication (END) via downregulation of Cdc2/cyclin B1 and upregulation of wee1 expression, and elevated the Cdk2 protein via downregulation of p21 within 48 h. We also researched PD’s induction of polyploidy through the MT polymerization. Immunofluorescent microscopy and Western blot analysis revealed that PD significantly caused MT polymerization in leukemia cells. We also found that very highconcentrations of PD (> 200 µM) were required to directly induce MT polymerization in vitro. Finally, PD exposure induced apoptosis and cell death significantly increased PARP and lamin A cleavage, and caspase-3 activity. We conclude that theprimary means by which PD induces END not only arrests cells in mitosis by suppressing spindle MT dynamics, but also promotes mitotic cell apoptosis in human leukemia cells.