Cell cycle arrest and suppression of the Akt/mammalian target of rapamycin signaling mediated by yuanhuadine, a daphnane diterpenoid, isolated from Daphne genkwa in human lung cancer cells

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Natural products have been played major roles in drug discovery programs including development of anticancer agents. In our continuous efforts to identify and characterize antitumor agents from natural products we recently isolated daphnane-type diterpenoids from the flower of Daphne genkwa (Thymelaceae) by a bioassay-guided fractionation with a potential anti-proliferative activity against human lung cancer cells. In this study, the anti-proliferative activity and the mechanisms of actions were elucidated with yuanhuadine, one of the most potent isolates from the plant extracts, in cultured human non-small lung cancer A549 cells. Yuanhuadine showed a potent growth inhibition and a relatively strong selectivity against lung cancer cells compared to a panel of several cancer cell lines and normal lung cells. In A549 cells, yuanhuadine exhibited the growth inhibition of the cells in a time- and dose-dependent manner with an IC₅₀ value of 14 nM at 72 h incubation. Flow cytometric analysis revealed that yuanhuadine (20 nM) induced cell cycle arrest at either G₀/G₁ or G₂/M phase depending on incubation time. The cell cycle arrest was well correlated with the expression of checkpoint proteins including the upregulation of cyclin-dependent kinase inhibitor p21, and down-regulation of cyclin D1, cyclin A, cyclin E, cyclin B1, cyclin dependent kinases, c-myc and Rb phosphorylation. In addition, yuanhuadine inhibited the phosphorylation of Akt, the mammalian target of rapamycin (mTOR) and its downstream effector molecules, p70 S6 kinase 1 and eukaryotic initiation factor 4E binding protein 1, indicating that the induction of cell cycle arrest by yuanhuadine might be in part mediated by the suppression of Akt/mTOR signaling pathway. These data suggest that yuanhuadine might have the potential to serve as a selective anti-tumor agent against human lung cancer cells by virtue of arresting the cell cycle and modulation of signal transduction in an Akt/mTOR pathway.