Fig 1.
Andrographolide inhibits migration of RAS-transformed cells.
Wound healing assay was evaluated after incubation with or without 10 uM andrographolide (Andro). Migration distance was measured at 0 and 12 hours after cells were scratched. (A) Wound healing assay under microscope. (B) The graphical data represented the percentage of migration area. Data are presented as the means± SD of three experiments. P values indicate the statistical significance between control and andrographolide -treated cells for 12 hours.
Table 1.
Lung metastatic rate in mice.
Fig 2.
Effects of andrographolide and radiation on the expression EMT-related markers (E-cadherin, vimentin) and matrix metalloproteinases MMP-2 and -9.
(A) RAS-transformed cells were treated with 10 uM andrographolide for 6 hours with and without 2 or 4 Gy radiation. After 24 hours, the cells were harvested for preparation of whole-cell protein lysates followed by Western blotting to detect the given proteins. (B) Quantitative analyses of MMP-2 expression evaluated by MMP-2/GAPDH ratio. The results are shown as mean ± SD (n = 3). *p < 0.05 for andrographolide combined with 4 Gy vs. andrographolide alone.
Fig 3.
Andrographolide and radiation reduces MMP-2 expression.
Gelatin zymography was performed using the conditioned media that were harvested after 48 hours in the presence or absence of 10 uM andrographolide, and then treated with/without radiation (2 or 4Gy) for 24 hours. The samples were applied without reduction to a 10% polyacrylamide gel containing gelatin, and proteolytic activity was demonstrated by digestion of the gelatin and clearing of the gel.
Fig 4.
Andrographolide with radiation enhanced downregulation of radiation-induced MMP-2 leading to the suppression of ERK signaling.
The cells were treated with 10 uM andrographolide with/without 2Gy or 4 Gy radiation for 24 hours. The figure displays typical data of MMP-2, phosphor-ERK1/2, total-ERK1/2 and NF-κB activity by Western blotting. Relative band intensities were analyzed by the Image J software. β-actin was used as an internal control for normalization. The numbers beneath the blots indicate the relative expression of each band when compared to the respective untreated control.
Fig 5.
Andrographolide plus radiation reduced angiotensin II-induced MMP-2 activation and invasion.
The Ras-transformed cells were incubated with/without 300 nM angiotensin II (Ang II) for 1 hour, and then treated with andrographolide, or radiation, or both for 24 hours. (A) The typical data show the gelatin zymography analysis band of the MMP-2 in conditioned media. (B) Invasion assays were performed using Transwell inserts of 8-micron pore size membrane and matrigel. After treatment, the cells were seeded in Transwell plates for 72 hours. The invaded cells were stained and quantified at an optical density of 560 nm. The experiments have been repeated three times; representative results of three independent experiments were shown. Quantification of cell invasion expressed as the percentage of control; one-way ANOVA was used for statistical analysis.
Fig 6.
Andrographolide with radiation suppressed angiotensin II-induced MMP-2 expression through inhibition of ERK1/2 signaling.
Cells were incubated with 300 nM Ang II for 1 hour and then treated with andrographolide, irradiation, or both for 24 hours. Protein level was determined by Western blotting.