Pyranocoumarins isolated from Peucedanum praeruptorum as 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 pyranocournarins, (+/-)-4'-O-acetyl-3'- O-angeloyl-cis-khellactone (or angular pyranocoumarin, APC) isolated from the medicinal plant Peticedanum 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 mug/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.