Pyranocoumarins Isolated fromPeucedanum praeruptorumas Differentiation Inducers in Human Leukemic HL-60 Cells

Differentiation therapy for myeloid leukemia offers great potential as a supplement to the current treatment modalities. In the present report, we investigated if the pyranocoumarins, (±)-4′- O -acetyl-3′- O -angeloyl- cis -khellactone (or angular pyranocoumarin, APC) isolated from the medicinal plant Peucedanum praeruptorum Dunn, could induce human acute myeloid leukemic HL-60 cells to differentiate and elucidated the molecular mechanism(s) involved. The ability of HL-60 cells to reduce nitroblue tetrazolium (NBT) was significantly increased after APC treatment for 72 h. In these differentiating HL-60 cells, cell surface differentiation markers CD11b (for myeloid cells) and CD14 (for monocytic cells) were detected in 90.3 % and 70.1 % of the cells, respectively. The differentiation inducing effect of APC was time- and dose-dependent. Treatment with 20 μg/mL APC for 72 h inhibited cell growth by 90 % and cell cycle analysis revealed an increase in the proportion of G1 phase cells. In these growth-inhibited cells the expression of the cyclin-dependent kinase inhibitor p27 kip1 , but not p21 WAF1 , was up-regulated as shown by Western blotting. Differentiation inducing signal pathways were investigated and it was shown that phospho-MEK and phospho-ERK were elevated shortly after the addition of APC. Pre-incubation of the cells with MEK1 inhibitor PD98059 blocked this APC-induced differentiation. Our results suggest that APC are potent inducers of HL-60 cell differentiation along both the myelocytic and monocytic lineages and are potential agents for differentiation-treatment of leukemia. Abbreviations APC:(±)- cis -3′- O -angeloyl-4′- O -acetyl-khellactone MAPK:mitogen-activated protein kinase ERK:extracellular signal-regulated kinase MEK:MAPK/ERK kinase NBT:nitroblue tetrazolium RA:all- trans -retinoic acid TPA:12- O -tetradecanoylphorbol 13-acetate PE: R -phycoerythrin FITC:fluorescein isothiocyanateide HPLC:high-performance liquid chromatography NMR:nuclear magnetic resonance