Betulinic acid enhances 1α,25-dihydroxyvitamin D3-induced differentiation in human HL-60 promyelocytic leukemia cells

Betulinic acid (BA) is a pentacyclic triterpene found in a number of medicinal plants and has been shown to cause apoptosis in a number of cell lines. We report here that BA may also have an effect on HL-60 cell differentiation. BA was cytotoxic to HL-60 cells with an IC50 of 5.7 muM after a 72-h treatment. Flow cytometry analysis showed that after exposure to 1-12 muM of BA for 72 h, approximately 10% of viable cells were in the sub-G(1), presumably apoptotic, phase. At the same time differentiation was induced in approximately 10% (at 1 muM BA) to a maximum of 20% (at 6 muM BA) of cells as judged by the NBT-reduction test and the expression of membrane markers CD11b and CD14. On the other hand, at 1 and 5 nM, 1alpha,25-dihydroxyvitamin D-3 (DHD3) induced differentiation in approximately 10 and 70% of cells, respectively. At 1 nM DHD3, the addition of 1 muM BA increased differentiated cells from 10 to 43% and with 3 muM BA the increase was to 80%. BA also enhanced the effects of DHD3 in the expansion of the G(1) cell population with a concomitant decrease of S phase cells. The effects of DHD3 and BA on CD11b and CD14 expression were inhibited by PD98059, a MEK inhibitor. Our results suggest that BA may enhance the effect of DHD3 in inducing mitogen-activated protein kinase kinase/extracellular signal-regulated protein kinase-mediated HL-60 cell differentiation. (C) 2004 Lippincott Williams Wilkins.