Acteoside inhibits human promyelocytic HL-60 leukemia cell proliferation via inducing cell cycle arrest at G ₀ /G ₁ phase and differentiation into monocyte

Abstract

We investigated the in vitro effects of acteoside on the proliferation, cell cycle regulation and differentiation of HL-60 human promyelocytic leukemia cells. Acteoside inhibited the proliferation of HL-60 cells in a concentration- and time-dependent manner with an IC ₅₀ , ∼30 μM. DNA flow cytometric analysis indicated that acteoside blocked cell cycle progression at the G ₁ phase in HL-60 human promyelocytic leukemia cells. Among the G ₁ phase cell cycle-related proteins, the levels of cyclin-dependent protein kinase (CDK)2, CDK6, cyclin D1, cyclin D2, cyclin D3 and cyclin E were reduced by acteoside, whereas the steady-state level of CDK4 was unaffected. The protein and mRNA levels of CDK inhibitors (cyclin-dependent kinase inhibitors), such as p21 ^CIP1/WAF1 and p27 ^KIP1 , were gradually increased after acteoside treatment in a time-dependent manner. In addition, acteoside markedly enhanced the binding of p21 ^CIP1/WAF1 and p27 ^KIP1 to CDK4 and CDK6, resulting in the reduction of CDK2, CDK4 and CDK6 activities. Moreover, the hypophosphorylated form of retinoblastoma increased, leading to the enhanced binding of protein retinoblastoma (pRb) and E2F1. Our results further suggest that acteoside is a potent inducer of differentiation of HL-60 cells based on biochemical activities and the expression level of CD14 cell surface antigen. In conclusion, the onset of acteoside-induced G ₁ arrest of HL-60 cells prior to the differentiation appears to be tightly linked to up-regulation of the p21 ^CIP1/WAF1 and p27 ^KIP1 levels and decreases in the CDK2, CDK4 and CDK6 activities. These findings, for the first time, reveal the mechanism underlying the anti-proliferative effect of acteoside on human promyelocytic HL-60 cells.