Effects of LG268 on Cell Proliferation and Apoptosis of NB4 Cells

Abstract

Aims: To investigate the effect of LG100268 (LG268) on cell proliferation and apoptosis in NB4 cells.

Methods: NB4 cells were treated with LG268 for 24 h or 48 h. The effect of LG268 on cell proliferation was assessed by the CCK-8 assay and colony-forming assay. Apoptosis and cell cycle were evaluated by flow cytometry. The protein expression levels of Survivin, PARP, c-Myc, cyclin D1, ERK, p-ERK, p38 MAPK, and p- p38 MAPK were detected by western blot.

Results: We found that LG268 inhibited the proliferation of NB4 cells in a dose-dependent manner. Flow cytometry analysis showed that LG268 accelerated apoptosis in NB4 cells in a time- dependent manner and that LG268 treatment led to cell cycle arrest at G0/G1 phase. Moreover, LG268 significantly decreased the protein levels of Survivin, c-Myc, and cyclinD1. Cleaved PARP was observed in the LG268 treatment group but not in the control group. In addition, LG268 increased the phosphorylation level of p38 MAPK and decreased the phosphorylation level of ERK.

Conclusions: LG268 inhibited cell proliferation and promoted cell apoptosis in NB4 cells.

Keywords: ERK; LG268; NB4 cells; apoptosis; p38 MAPK; proliferation.

Figure 1

LG268 inhibited the proliferation of NB4 cells. (A) Cytotoxicity was detected by CCK-8 assay. Data are expressed as means ± SD. (B) The in vitro colony-forming ability of NB4 cells was examined in the absence or presence of 2 μM LG268. *P < 0.05.

Figure 2

LG268 induced G0/G1 arrest in NB4 cells. NB4 cells were treated with 3 μM LG268 for 48 h. (A) Cells were harvested for analysis of cell cycle distribution by flow cytometry. (B) Effect of LG268 on the expression of c-Myc and cyclin D1 determined by western blot analysis. Quantitative analysis was performed by measuring the relative expression level of c-Myc and cyclin D1 to that of β-Actin. Data are expressed as means ± SD. *P < 0.05.

Figure 3

LG268 induced apoptosis of NB4 cells. (A) Cells were treated with LG268 for 24 h or 48 h, and apoptosis was analyzed by flow cytometry using double staining with FITC-labeled annexin-V and propidium iodide. Cells undergoing early apoptosis are Annexin V-FITC⁺/PI^- , whereas cells undergoing late apoptosis are Annexin V-FITC⁺/PI⁺. The percentages of late and early apoptotic cells were summed to give the total number of apoptotic cells. (B) Cells were treated with LG268 for 48 h, and the effect of LG268 on the expression of cleaved-PARP and Survivin were determined by western blot analysis. Quantitative analysis was performed by measuring the relative expression level of cleaved-PARP and Survivin to β-Actin. Data are expressed as means ± SD. *P < 0.05.

Figure 4

LG268 affected multiple signaling molecules. Western blot analysis was used to measure the expressions of phosphorylated ERK and p38 MAPK. Quantitative analysis was performed by measuring the relative expression level of p-p38 to p38 or p-ERK to ERK. Data are expressed as means ± SD. *P < 0.05.