Figure 1.
The chemical structure of isoalantolactone and its growth-inhibiting effect on UM-SCC-10A cells and mouse splenocytes.
(A) The chemical structure of isoalantolactone. (B) UM-SCC-10A cells were pretreated with 0.1% DMSO or various concentrations of isoalantolactone for 24 and 48 h, and cell growth inhibition assays were performed using the MTT method. The data are expressed as the mean ± SEM of three independent experiments. (C) Isoalantolactone-induced morphologic changes in the UM-SCC-10A cells, and these changes were observed using inverted phase contrast microscopy. The control cells adhered well and displayed normal UM-SCC-10A cell morphology. After isoalantolactone treatment, many cytoplasmic vacuoles were observed in the cells. The vacuoles became progressively larger and denser with increasing concentrations of isoalantolactone. (D) Mouse splenocytes were treated with 25 and 50 µM isoalantolactone for 24 h and stained with 0.4% trypan blue, after which they were examined for dead and living cells microscopically. The dead cells stained blue. (E) Cell viability after 25 or 50 µM isoalantolactone treatment in UM-SCC-10A cells and mouse splenocytes for 24 h. The results are the mean ± SEM from three independent experiments. *P<0.05 and **P<0.01 compared to the control.
Figure 2.
Isoalantolactone-induced apoptosis in UM-SCC-10A 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 UM-SCC-10A cells incubated with different concentrations of isoalantolactone (25 and 50 µM) or caspase inhibitor (Z-VAD-FMK 50 µM). (B) Isoalantolactone induced apoptosis in the UM-SCC-10A cells in a dose-dependent manner. Z-VAD-FMK markedly reduced apoptosis in UM-SCC-10A cells treated with high-dose isoalantolactone. The data are expressed as the means ± SEM of three independent experiments with the similar results. *P<0.05 and **P<0.01 compared to the control. (C) The morphological nuclear changes in UM-SCC-10A cells treated with isoalantolactone at different concentrations. The cells were stained with Hoechst33258 for 30 min in the dark to examine the cleaved nuclei, which is a sign of apoptosis.
Figure 3.
The effect of isoalantolactone on the cell cycle in UM-SCC-10A cells.
(A) The DNA content in each cell cycle phase in UM-SCC-10A cells was analyzed by flow cytometry. The representative histograms are from UM-SCC-10A cells incubated with different concentration of isoalantolactone (25 and 50 µM) or caspase inhibitor (Z-VAD-FMK50 µM). (B) Isoalantolactone 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 control. Z-VAD-FMK treatment did not prevent cell cycle arrest following high-dose isoalantolactone treatment. The results are represented as the mean ± SEM for three independent experiments with similar results. *P<0.05 and **P<0.01 compared to the control.
Figure 4.
The effect of isoalantolactone on the expression of cell cycle regulators in UM-SCC-10A cells.
(A) Representative pictures for p53, p21 and cyclin D protein expression by western blot analysis. β-actin was used as a control. (B) Isoalantolactone up-regulated p53 and p21 expression, while down-regulating cyclin D expression in a dose-dependent manner. The results are represented as the means ± SEM from three independent experiments with similar results. *P<0.05 and **P<0.01 compared to the control.
Figure 5.
The effect of isoalantolactone on the MMP in UM-SCC-10A cells.
(A) The MMP of UM-SCC-10A cells treated with isoalantolactone at different concentrations was analyzed by flow cytometry. (B) The loss of the MMP in UM-SCC-10A cells following isoalantolactone treatment in a dose-dependent manner. The data are expressed as the means ± SEM for three independent experiments with similar results. *P<0.05 compared to the control.
Figure 6.
The effect of isoalantolactone on the expression of caspase-dependent mitochondrial apoptosis pathway proteins in UM-SCC-10A cells.
(A) Representative images of cytochrome c, Bax, Bcl-2 and caspase 3 protein expression detected by western blot. β-actin was used as a control. (B) The data are represented as the means ± SEM from three independent experiments with similar results. *P<0.05 and **P<0.01 compared to the control.