Induction of apoptosis by 3-amino-6-(3-aminopropyl)-5,6-dihydro-5,11-dioxo-11H-indeno[1,2-c]isoquinoline via modulation of MAPKs (p38 and c-Jun N-terminal kinase) and c-Myc in HL-60 human leukemia cells

Abstract

Recently, we reported that 3-amino-6-(3-aminopropyl)-5,6-dihydro-5,11-dioxo-11H-indeno[1,2-c]isoquinoline (AM6-36), sharing structural similarity with naturally occurring isoquinolines, induced activities mediated by retinoid X receptor (RXR) response element accompanied by antiproliferative effects on breast cancer cells. To further characterize the biologic potential of AM6-36, we currently report studies conducted with HL-60 human leukemia cells. AM6-36 significantly inhibited cellular proliferation in a dose- and time-dependent manner with an IC(50) value of 86 nM. When evaluated at low test concentrations (≤0.25 μM), AM6-36 induced arrest in the G2/M phase of the cell cycle. At higher concentrations (1 and 2 μM), the response shifted to apoptosis, which was consistent with the effect of AM6-36 on other apoptotic signatures including an increase of apoptotic annexin V(+) 7-AAD(-) cells, loss of mitochondrial membrane potential, induction of poly(ADP-ribose) polymerase cleavage, and activation of several caspases. These apoptotic effects are potentially due to up-regulation of p38 MAPK and JNK phosphorylation and down-regulation of c-Myc oncogene expression. Taken together, AM6-36 might serve as an effective anticancer agent by inducing G2/M cell cycle arrest and apoptosis through the activation of MAPKs and inhibition of c-Myc.

Figure 1

Effect of AM6-36 on the proliferation of HL-60 cells. Human acute leukemia cells (HL-60) were seeded at a density of 2×10⁴ cells per well in 96-well cell culture plates. After 24 h of incubation, cells were treated with AM6-36 in the concentration range from 0.0625-1 µM and the incubation was continued for 24, 48, 72, and 96 h. Each bar presents a fold-change (mean ± SD) of OD at 540 nm in each group divided by the OD at 540 nm of the time zero control group (n = 6 per group). *P value less than 0.05 is considered statistically significant from control group (white bars) at each incubation time.

Figure 2

Effect of AM6-36 on the cell cycle distribution of HL-60 cells. Cells were treated with various concentrations of AM6-36 for 24 h and stained with NIM-DAPI. The cell cycle distribution of each group was evaluated by DNA content using flow cytometry. Representative histograms of DNA content are shown in (A) and % population in each phase of cell cycle is demonstrated as mean ± SD from three independent experiments (B). *P value less than 0.05 is considered statistically significant from control group (white bars) at each phase.

Figure 3

Induction of apoptotic markers by AM6-36 in HL-60 cells. (A) Effect of AM6-36 on Annexin V-FITC/7-AAD double staining: HL-60 cells were treated with AM6-36 for 24 h. The whole cells were collected, and then further incubated with Annexin V-FITC and 7-AAD. After incubation, cells were analyzed by flow cytometry within 30 min. Data are presented as biparametric dot plots showing fluorescence intensity of Annexin-V FITC (FL-1) versus 7-AAD (FL-3). Dots in the each quadrant represent the cell population containing the information on the status of cell viability: Viable cells in the lower left quadrant (Annexin V⁻ 7-AAD⁻), the early and late apoptotic cells in the lower and upper right quadrant (Annexin V⁺ 7-AAD⁻, and Annexin V⁺ 7-AAD⁺), and necrotic cells in the upper left quadrant (Annexin V⁻ 7-AAD⁺). (B) Effect of AM6-36 on the mitochondrial membrane potential: HL-60 cells were treated with AM6-36 (1 µM) for 24 h and incubated with 100 nM of DiOC6(3) for 30 min at 37 °C. The data are demonstrated as histograms of intensity of fluorescence in FL-1 using a flow cytometer. (C) Effect of AM6-36 on the cleavage of PARP: HL-60 cells were treated with the indicated concentrations of AM6-36 for 24 h, and protein expression was assessed by Western blot analysis.

Figure 4

Effects of AM6-36 on the expression of caspases. (A) HL-60 cells were treated for 24 h with various concentrations of AM6-36 and protein expression was measured by Western blot analysis. (B) HL-60 cells were treated with serially diluted AM6-36 for 24 h and then incubated with the substrates of caspase-3/-7, caspase-8, or caspase-9. The fold induction in comparison with vehicle (DMSO)-treated control is demonstrated as a bar graph. *P value less than 0.05 is considered statistically significant from control group (white bars) at each time and caspase.

Figure 5

Effect of AM6-36 on the expression of MAPKs and c-Myc. HL-60 cells were treated for the indicated times with 1 µM AM6-36, and protein expression was measured by Western blot analysis.