Antiproliferative effect of aaptamine on human chronic myeloid leukemia K562 cells

Abstract

We previously isolated aaptamine, a benzonaphthyridine alkaloid, from marine sponge Aaptos suberitoids. In this study, we investigated the anti-proliferative effect of aaptamine on chronic myeloid leukemia (CML) K562 cells. Aaptamine inhibited growth of K562 with a GI50 as 10 μM, and arrested cell cycle at G2/M phase. Western blot analysis indicated that aaptamine induced p21 expression in K562 cells. Moreover, p21 promoter was activated by aaptamine treatment in p21 transfected K562 cells. Since K562 is p53 negative, aaptamine was demonstrated to be a p53-independent p21 inducer in CML cells.

Keywords: G2/M arrest; K562 cells; aaptamine; p21.

Figure 1

Chemical structure of aaptamine.

Figure 2

Effect of aaptamine on growth of K562 cells. K562 cells were incubated with various concentrations of aaptamine in 96-well plate at 37 °C. Two days later, WST-8 solution was added and the cells were further incubated for 3 h followed by measurement of the absorbance (450 nm) with microplate spectrophotometer. The number of remaining cells after treatment was calculated using the following formula: Cell number (% control) = 100 × (absorbance of a given sample − absorbance of Blank well)/(absorbance of Control well − absorbance of Blank well), where the Blank well contained medium only and the Control well contained cells without aaptamine.

Figure 3

Effect of aaptamine on the cell cycle of K562 cells. K562 cells was incubated with various concentrations of aaptamine for 48 h. The collected cells were dyed with DNA-Prep Reagents Kit and analyzed by flow cytometer (λex = 493 nm, λem = 630 nm). The analysis was quantified by ModFit software.

Figure 4

Aaptamine induced p21 expression in K562 cells. The cells were incubated with 100 μM of aaptamine for the indicated times. Cell lysates were prepared and applied to 4–20% SDS-Polyacrylamide gel electrophoresis (SDS PAGE). After being transferred to polyvinylidene difluoride (PVDF) membrane, the blots were exposed to anti-Cip1/WAF-1/p21 or anti-β-actin, and then to anti-mouse horseradish peroxidase (HRP) conjugated IgG antibody.

Figure 5

Activation of p21 promoter in K562 cells. K562 cells were transfected with the human p21 promoter-luciferase fusion plasmid (pWWP), followed by treatment with various concentrations of aaptamine for 24 h, and then analyzed by luciferase assay. Fold increase of luciferase activity by aaptamine was calculated in comparison with the luciferase activity of the untreated cells. The result represents three independent experiments.