Effects of portulacerebroside a on apoptosis of human leukemia HL60 cells and p38/JNK signaling pathway

Abstract

Acute myeloid leukemia is known as one of the most malignant diseases. We aimed at exploring the effect of portulacerebroside A (PCA) on the apoptosis in human leukemia HL60 cells and clarify the possible mechanisms involved in. By MTT analysis, we found that PCA (1-100 μM) inhibited the cell viability in a time- and dose-dependent manner, and cell cycle was arrested at G0/G1 period. PCA treatment from 5 to 50 μM dose-dependently induced apoptosis from 12.7 ± 1.56% to 52.7 ± 6.214% of HL60 cells. Mitochondrial membrane potential (MMP) was decreased and reactive oxygen species (ROS) accumulated obviously. mRNA expressions and protein levels of Bax/Bcl-2, caspase-3 and caspase-9 were elevated significantly. ERK1/2, JNK1/2 and p38 MAPK pathway were blocked detected by western blot analysis. In conclusion, PCA can act as a new agent for leucocythemia treatment.

Keywords: Portulacerebroside A; apoptosis; leucocythemia; p38/JNK.

Figure 1

PCA inhibited the proliferation of HL60 cells. After treated with PCA (1, 2, 5, 10, 20, 50 and 100 μM) for 12, 24 and 48 h, cell viability was detected by MTT. Data was presented as mean ± SD (n = 6). *,#,ΔP<0.05, **,##,ΔΔP<0.01, compared with the control group.

Figure 2

PCA caused cell cycle arrest of HL60 cells HL60 cells were treated with PCA (5, 10 and 50 μM) for 24 h, cell cycle distribution was identified by flow cytometry. Data was presented as mean ± SD (n = 6). **P<0.01, compared with the control group.

Figure 3

PCA induced apoptosis in HL60 cells. HL60 cells were treated with PCA PCA (5, 10 and 50 μM) for 24 h, and cell apoptosis was assessed by flow cytometry. Data was presented as mean ± SD (n = 6). **P<0.01, compared with the control group.

Figure 4

Effects of PCA on MMP and ROS in HL60 cells. A, B. Cells were treated with PCA for 24 h at 5, 10, and 50 μM respectively, then incubated with Rhodamine 123 and analyzed by flow cytometry. C, D. Cells were treated with PCA for 24 h at 5, 10, and 50 μM respectively, and fluorescence probe DCFH-DA was used to determine the levels of ROS production. Data was presented as mean ± SD (n = 3). *P<0.05, **P<0.01, compared with the control group.

Figure 5

PCA surpressed the Bax/Bcl-2, caspase-3 and caspase-9 expression. A, B. HL60 cells were treated with PCA (5, 10, and 50 μM) for 12 h, mRNA expressions of Bax/Bcl-2, caspase-3 and caspase-9 were detected by real time PCR. C-F. HL60 cells were treated with PCA (5, 10, and 50 μM) for 24 h, protein levels of Bax/Bcl-2, caspase-3 and caspase-9 were detected by western blot analysis. Data was presented as mean ± SD (n = 6). *P<0.05, **P<0.01, compared with the control group.

Figure 6

PCA blocked the JNK and p38 signaling. A, B. HL60 cells were treated with PCA (5, 10, and 50 μM) for 3 h, protein levels of P-JNK, JNK, P-p38 and p38 were identified by western blot analysis. Data was presented as mean ± SD (n = 6). *P<0.05, **P<0.01, compared with the control group.