Table 1.
The experimental design and specifications.
Fig 1.
Schematic representation of the bioassay-guided isolation of annomuricin E from EEAML.
The cytotoxic effect of each fraction was examined against HT-29 cells for 48 h using an MTT assay. The IC50 values (μg/ml) represent the means ± SEM of three independent experiments.
Table 2.
1H NMR (500 MHz) and 13C NMR (125 MHz) spectral data of annomuricin E in CDCl3 (δ in ppm, J in Hz).
Fig 2.
The number of ACF formed in proximal and distal parts of the colon.
Tissue specimens were collected from five groups of rats: (A) negative control, (B) cancer control, (C) low dose of EEAML, (D) high dose of EEAML and (E) treatment control. Data are expressed as the means ± SEM of (n = 6/group). *P<0.05 compared with cancer control.
Table 3.
Distribution of aberrant crypt categories (1, 2, 3, 4 and more) in the colons of five groups of rats:
(A) negative control, (B) cancer control, (C) low dose of EEAML, (D) high dose of EEAML and (E) treatment control.
Fig 3.
Topographical views of the colon mucosa.
Tissue specimens were collected from five groups of rats: (A) negative control, (B) cancer control, (C) low dose of EEAML, (D) high dose of EEAML and (E) treatment control and were stained with methylene blue dye. The red arrows depict ACF in the colon mucosa. Scale bar: 500 μm.
Fig 4.
Immunohistochemical analysis of colon tissue sections for PCNA.
Tissue specimens were collected from five groups of rats: (A) negative control, (B) cancer control, (C) low dose of EEAML, (D) high dose of EEAML and (E) treatment control. Quantitative analysis of immunopositivity shown as brown staining demonstrated a significant down-regulation of PCNA in groups C-E compared with the cancer control group. Data are expressed as the means ± SEM of (n = 6/group). *P<0.05 compared with the cancer control group. Scale bar: 10 μm.
Fig 5.
Expression of Bax in colon tissue sections.
Tissue specimens were collected from five groups of rats (n = 6/group) and were analyzed using immunohistochemistry: (A) negative control, (B) cancer control, (C) low dose of EEAML, (D) high dose of EEAML and (E) treatment control. The up-regulation of Bax in groups C-E is shown as brown staining. Scale bar: 10 μm.
Fig 6.
Immunohistochemical analysis of colon tissue sections for Bcl-2.
Tissue specimens were collected from five groups of rats (n = 6/group): (A) negative control, (B) cancer control, (C) low dose of EEAML, (D) high dose of EEAML and (E) treatment control. Immunopositivity shown as brown staining revealed the down-regulation of Bcl-2 in groups C-E. Scale bar: 10 μm.
Fig 7.
Level of CAT, GPx, MDA and SOD in colon tissue homogenates.
Samples were collected from five groups of rats: (A) negative control, (B) cancer control, (C) low dose of EEAML, (D) high dose of EEAML and (E) treatment control. Data are expressed as the means ± SEM of (n = 6/group). *P<0.05 compared with cancer control.
Fig 8.
Chemical structure of annomuricin E.
Table 4.
Cytotoxic effects of annomuricin E and 5-FU on the proliferation of CCD841 and HT-29 cells after 12, 24 and 48 h of treatment.
Fig 9.
Effects of annomuricin E on LDH leakage formation in HT-29 cells.
Cells were exposed to 0.1% vehicle DMSO (control) and annomuricin E at different concentrations for 24 h. The treated HT-29 cells showed a significant LDH release at 4 to 16 μg/ml concentrations compared with the control. The data represent the means ± SEM of three independent experiments. *P<0.05 compared with the control.
Fig 10.
Effect of annomuricin E on cell cycle distribution in HT-29 cells.
Cells were treated with (A) 0.1% vehicle DMSO (control) for 48 h and annomuricin E at the IC50 concentration for (B) 12, (C) 24 and (D) 48 h. After staining the cells with PI, the DNA contents were monitored using flow cytometry. (E) The representative bar chart shows the significant induction of G1 cell cycle arrest by annomuricin E after 12 h of treatment. The data represent the means ± SEM of three independent experiments. *P<0.05 compared with the control.
Fig 11.
Effect of annomuricin E on apoptosis in HT-29 cells via quadrant statistics.
After treatment with annomuricin E (IC50 concentration) for (B) 12, (C) 24 and (D) 48 h, the cells were double stained with Annexin V-FITC/PI and monitored using flow cytometry. Cells treated with 0.1% vehicle DMSO were employed as the (A) control treatment. (E) The representative bar chart depicted the percentages of early apoptotic, late apoptotic and necrotic cells. The data represent the means ± SEM of three independent experiments. *P<0.05 compared with the control.
Fig 12.
Effect of annomuricin E on caspase 3/7 and caspase 9 activities in HT-29 cells using bioluminescent analysis.
Cells were treated with the IC50 concentration of annomuricin E for 3, 6, 12, 24 and 48 h. The activities of both caspase were significantly elevated after 12 h of treatment. Cells treated with 0.1% vehicle DMSO were employed as the control treatment. The data represent the means ± SEM of three independent experiments. *P<0.05 compared with the control.
Fig 13.
Images of HT-29 cells treated with annomuricin E at the IC50 concentration for 24 h.
The treated cells were stained with different and specific dyes for the detection of total nuclear intensity, cell membrane permeability, MMP and cytochrome c release. Cells treated with 0.1% vehicle DMSO were employed as the control treatment.
Fig 14.
Representative bar charts of the multiple cytotoxicity assay.
After 12 h of treatment with annomuricin E at the IC50 concentration, the total nuclear intensity, MMP and cytochrome c release were significantly elevated compared with the control. However, cell membrane permeability showed a significant increase only after 24 h. Cells treated with 0.1% vehicle DMSO were employed as the control treatment. The data represent the means ± SEM of three independent experiments. *P<0.05 compared with the control.
Fig 15.
Effect of annomuricin E on Bax and Bcl-2 mRNA expression was assessed using Q-PCR analysis.
The housekeeping gene β-actin was used for the normalization of the mRNA expression. The result depicted a time-dependent upregulation of Bax and down-regulation of Bcl-2 after treatment with annomuricin E at the IC50 concentration. The data represent the means ± SEM of three independent experiments. *P<0.05 compared with the control.
Fig 16.
Immunofluorescence analysis of (A) Bax and (B) Bcl-2 protein expression in HT-29 cells.
Cells were treated with annomuricin E at the IC50 concentration for 12, 24 and 48 h and were stained with DAPI and Bax/Bcl-2 antibodies conjugated to FITC. Cells treated with 0.1% vehicle DMSO were employed as the control treatment. As the number of cells reduced in a time-dependent manner, the fluorescent intensity showed a marked upregulation and down-regulation for Bax and Blc-2 proteins, respectively.
Fig 17.
Representative bar charts of Bax and Bcl-2 immunofluorescence analysis.
Annomuricin E at IC50 concentration induced significant upregulation of Bax and down-regulation of Bcl-2 after 12 h. Cells treated with 0.1% vehicle DMSO were employed as the control treatment. The data represent the means ± SEM of three independent experiments. *P<0.05 compared with the control.