Figure 1.
Effect of aciculatin on cell viability and cell cycle in human cancer cells.
A, Structure of aciculatin: 8-(2,6-dideoxy-β-ribo-hexopyranosyl)-5-hydroxy- 2-(4-hydroxyphenyl)-7-methoxy-4H-1-benzopyran-4-one sesquihydrate. B, HCT116 cells were incubated with the indicated concentrations of aciculatin (5–10 µM) for 48 h. Cell viability was then determined by MTT assay. C, HCT116 cells were starved overnight, and then incubated with vehicle (0.1% DMSO) or aciculatin (10 µM) for the indicated period. The DNA content was subsequently analyzed by PI staining using a FACScan flow cytometric assay. The curve chart shows that aciculatin increased the G1 population of cells in a time-dependent manner. Mean ± SE values from 3 independent experiments. *P<0.05 and ***P<0.001, compared with non-treated cells. D, HCT116 cells were treated with vehicle (0.1% DMSO) or aciculatin (7.5 µM) and then double stained with TUNEL and DAPI. Increased green fluorescence indicated that the cells underwent apoptosis after aciculatin treatment (TUNEL, right panel). The nuclei were stained with DAPI (left panel). The bar chart shows the proportions of TUNEL positive cells in each treatment normalized to DAPI. ***P<0.001.
Figure 2.
Effect of aciculatin on G0/G1-related proteins and apoptotic factors in HCT116 cells.
A, HCT116 cells were treated with aciculatin (10 µM) for the indicated periods and then harvested for detection of G0/G1 arrest-related proteins (p21, pRb, and p27) by immunoblotting. B, HCT116 cells were treated with aciculatin (10 µM) for the indicated periods and then harvested for detection of caspase activation and PARP cleavage. C, HCT116 cells were treated with various concentrations of aciculatin (5, 7.5 and 10 µM) for 24 h. After aciculatin treatment, apoptotic proteins were detected for each treatment concentrations. The star marks a non-specific band.
Figure 3.
Accumulation of p53 is observed following aciculatin treatment in HCT116 and A549 cells via a proteasome degradation pathway.
A, HCT116 and A549 cells were treated with aciculatin (10 µM) for the indicated periods and then harvested for detection of p53-related proteins. The levels of p53, phospho-ser15-p53, γ-H2AX, and the p53 downstream target MDM2 were then determined in HCT116 cells. The levels of p53, MDM2, γ-H2AX, cleaved caspase-9 and PARP were determined in A549 cells. The star marks a non-specific band. B, Nuclear extraction of HCT116 cells was performed after aciculatin treatment at the indicated time points. Aciculatin-induced nuclear accumulation of p53 was shown to be time-dependent. C, HCT116 cells were treated with aciculatin (10 µM) at different time points followed by extraction of total RNA. The p53 mRNA was co-amplified with GAPDH. D, HCT116 cells were pretreated with aciculatin (10 µM) for 3 h, followed by treatment with cycloheximide (20 µg/ml) with or without aciculatin (10 µM) for the indicated periods. E, HCT116 and A549 cells were co-treated with 10 µM MG132 and 10 µM aciculatin for 6 h and then harvested for p53 detection by immunoblotting.
Figure 4.
Aciculatin treatment attenuates MDM2 mRNA, contributing to p53 accumulation.
A, HCT116 and A549 cells were co-treated with 10 µM MG132 and 10 µM aciculatin for 6 h (HCT116) and 3 h (A549) then harvested for detection of MDM2 by immunoblotting. B, HCT116 and p53-KO HCT116 cells were treated with aciculatin (10 µM) at different time points followed by extraction of total RNA. The MDM2 mRNA was co-amplified with GAPDH. C, The MDM2 plasmid was introduced into the HCT116 cells to induce over-expression of the MDM2 protein; the cells were then treated with aciculatin for 3 h. Protein levels of p53 and MDM2 were detected by western blotting.
Figure 5.
p53 is required for aciculatin to trigger cell cycle arrest and apoptosis.
A, p53-KO HCT116 cells were incubated with aciculatin (5–10 µM) for 48 h. Cell viability was then determined by MTT assay. The results were compared with wild-type HCT116 cells. Mean ± SE from 3 independent experiments. *P<0.05 and **P<0.01. B, p53-KO HCT116 cells were starved overnight and then incubated with vehicle (0.1% DMSO) or aciculatin (10 µM) for different periods. The DNA proportion was subsequently analyzed by PI staining. The cell cycles of p53-WT and p53-KO HCT116 cells after treatment of 18 h are shown. The G1 proportions of both cell lines were compared. Mean ± SE values from 3 independent experiments. *P<0.05 and ***P<0.001. C, p53-WT and p53-KO HCT116 cells were incubated with aciculatin (10 µM) for 24 h and 48 h and then harvested for detection of p53, caspase activation, and cleavage of PARP (upper). p53-WT and p53-KO cells were treated with vehicle (0.1% DMSO) or aciculatin (10 µM) for 24 h and were subsequently analyzed by PI staining. The induction of sub-G1 phase of each group was determined (lower bar chart). **P<0.005. D, HCT116 cells were treated with various concentrations of aciculatin (5, 7.5, and 10 µM). After 24 h and 48 h aciculatin treatment, The levels of p53, p21, pRb and PUMA were then determined (upper). p53 siRNA was used to knock down the p53 level of HCT116 and A549 cells, which were then treated with aciculatin for 24 h. The cells were harvested for detection of p53 and apoptotic related proteins (lower). E, p53 knock-down HCT116 and A549 cells were treated with aciculatin (10 µM) for 40 h and then double stained with annexin V-FITC and PI. The percentages of fluorescently labeled cells were determined by flow cytometry.
Figure 6.
Aciculatin anti-cancer activity in the HCT116 xenograft models.
HCT116 cells were injected subcutaneously into the flanks of severe combined immunodeficient mice. The mice were divided into two groups (n = 5) and when the tumors reached the average volume (90 mm3) the treatment was initiated. The mice were sacrificed on day 12 thereafter. A, The curves show the mean (±SE) tumor sizes measured within each group. The differences in tumor size between the control and treated mice were statistically significant (*P<0.05). Bodyweight was measured every day from day 1 of administration. There were no significant differences after treatment in any of the groups (data not shown). B, Treated and untreated tumor slices were assessed by H&E (a, b) and IHC staining for p53 protein (c, d) and Ki-67 protein (e, f). Tumor samples were observed under 100× (for H&E) and 200× (for IHC) magnification. C, The tumor tissue slices were assessed by TUNEL assay kit and the bar charts indicates the fold change of sub-G1 phase. **P<0.01.