Figure 1.
The chemical structure of abieslactone and its growth-inhibiting effect on HepG2, SMMC7721 and QSG7701 cells.
(A) The chemical structure of abieslactone. (B and C) Viability of HepG2 and SMMC7721 cells after exposure to 0.1% DMSO or various concentrations of abieslactone for 24, 48, and 72 h. The data are expressed as the means ± SEM of three independent experiments. (D) Cell viability after 5, 10, and 20 µM abieslactone treatment in HepG2, SMMC7721 and QSG7701 cells for 24 h. The results are the mean ± SEM from three independent experiments. *P<0.05; **P<0.01 vs. the untreated control. (E) The morphological nuclear changes in QSG7701 cells treated with abieslactone at different concentrations (5, 10, and 20 µM). The cells were stained with Hoechst 33258 for 30 min in the dark to examine the cleaved nuclei, which is a sign of apoptosis.
Figure 2.
Abieslactone-induced apoptosis in HepG2 and SMMC7721 cells.
(A) Apoptosis was evaluated using an annexin V-FITC apoptosis detection kit and flow cytometry. The X- and Y-axes represent annexin V-FITC staining and PI, respectively. The representative pictures are from HepG2 and SMMC7721 cells incubated with different concentrations of abieslactone or caspase inhibitor (Z-VAD-FMK 20 µM). (B) Abieslactone induced apoptosis in HepG2 and SMMC7721 cells in a dose dependent manner. Z-VAD-FMK markedly reduced apoptosis in HepG2 and SMMC7721 cells treated with high-dose abieslactone. The data are expressed as the means ± SEM of three independent experiments with the similar results. *P<0.05; **P<0.01 vs. the untreated control.
Figure 3.
Nuclear morphology of HepG2 and SMMC7721 cells treated with 5, 10, and 20 µM abieslactone or 20 µM Z-VAD-FMK for 24 h was determined by staining with Hoechst 33258.
Figure 4.
The effect of abieslactone on the cell cycle and the expression of cell cycle regulators in HepG2 and SMMC7721 cells.
(A) Abieslactone treatment induced a dose dependent increase in the proportion of cells in the G1 phase and a decrease in cells in the S and G2 phases compared to the untreated control. Z-VAD-FMK treatment did not prevent cell cycle arrest following high-dose abieslactone treatment. The results are represented as the mean ± SEM for three independent experiments with similar results. *P<0.05; **P<0.01 vs. the untreated control. (B) Representative pictures for p53, p21 CDK2 and cyclin D1 protein expression by western blot analysis. β-actin was used as a control.
Figure 5.
The effect of abieslactone on the MMP in HepG2 and SMMC7721 cells.
(A) The MMP of HepG2 and SMMC7721 cells treated with abieslactone at different concentrations was analyzed by flow cytometry. (B) The loss of the MMP in HepG2 and SMMC7721 cells following abieslactone treatment in a dose dependent manner. The data are expressed as the means ± SEM for three independent experiments with similar results. *P<0.05 vs. the untreated control.
Figure 6.
The effect of abieslactone on the expression of caspase-dependent mitochondrial apoptosis pathway proteins in HepG2 and SMMC7721 cells.
Representative images of cytochrome c, Bax, Bcl-2, PARP, cleaved caspase 9 and cleaved caspase 3 protein expression detected by western blot. β-actin was used as a control.
Figure 7.
The involvement of ROS/Akt pathway in abieslactone-induced HepG2 cell apoptosis.
(A) HepG2 and SMMC7721 cells were treated with different concentrations of abieslactone or ROS scavenger (NAC 10 mM), and then ROS was measured by DCF fluorescence analysis. The increased fluorescence of DCF was determined as the increased intracellular ROS accumulation. (B) HepG2 and SMMC7721 cells pretreated with 10 mM NAC for 1 h were incubated with 20 µM abieslactone for 24 h, and cell viability was determined by MTT assay. *P<0.05 vs. the untreated control. (C) The effect of abieslactone on the expression of p-Akt, Akt and NF-kB p65 proteins in HepG2 cells. β-actin was used as a control.
Figure 8.
Schematic form of the proposed mechanisms for abieslactone-induced G1 arrest and apoptosis in HepG2 cells.