Silymarin induces inhibition of growth and apoptosis through modulation of the MAPK signaling pathway in AGS human gastric cancer cells

Abstract

Apoptosis is regarded as a therapeutic target because it is typically disturbed in human cancer. Silymarin from milk thistle (Silybum marianum) has been reported to exhibit anticancer properties via regulation of apoptosis as well as anti‑inflammatory, antioxidant and hepatoprotective effects. In the present study, the effects of silymarin on the inhibition of proliferation and apoptosis were examined in human gastric cancer cells. The viability of AGS human gastric cancer cells was assessed by MTT assay. The migration of AGS cells was investigated by wound healing assay. Silymarin was revealed to significantly decrease viability and migration of AGS cells in a concentration‑dependent manner. In addition, the number of apoptotic bodies and the rate of apoptosis were increased in a dose‑dependent manner as determined by DAPI staining and Annexin V/propidium iodide double staining. The changes in the expression of silymarin‑induced apoptosis proteins were investigated in human gastric cancer cells by western blotting analysis. Silymarin increased the expression of Bax, phosphorylated (p)‑JNK and p‑p38, and cleaved poly‑ADP ribose polymerase, and decreased the levels of Bcl‑2 and p‑ERK1/2 in a concentration‑dependent manner. The in vivo tumor growth inhibitory effect of silymarin was investigated. Silymarin (100 mg/kg) significantly decreased the AGS tumor volume and increased apoptosis, as assessed by the TUNEL assay, confirming its tumor‑inhibitory effect. Immunohistochemical staining revealed elevated expression of p‑JNK and p‑p38 as well as reduced expression of p‑ERK1/2 associated with silymarin‑treatment. Silymarin was revealed to reduce tumor growth through inhibition of p‑ERK and activation of p‑p38 and p‑JNK in human gastric cancer cells. These results indicated that silymarin has potential for development as a cancer therapeutic due to its growth inhibitory effects and induction of apoptosis in human gastric cancer cells.

Figures

Figure 1.

Effects of silymarin on the cell viability of AGS human gastric cancer cells. The results are revealed cell viability inhibitory effect of silymarin on AGS cells. Cell viability was measured by the MTT assay. The results are presented as the mean ± SD from three independent experiments performed in triplicate. Significance was determined by Dunnett's t-test with *P<0.05 considered as statistically significant compared with non-treated controls.

Figure 2.

Effect of silymarin on the cell migration of AGS human gastric cancer cells. (A) AGS gastric cancer cells were treated with silymarin 0, 40 and 80 µg/ml for 24 h, and cell migration was then measured by wound healing assays. (B) Width of the wound for any field of view was measured at 3–4 different locations. Data represent the means ± SD of at least three replicates relative to the control (Dunnett's t-test with *P<0.05).

Figure 3.

Effects of silymarin on apoptotic cell in AGS human gastric cancer cells. (A) AGS cells were treated with silymarin for 24 h and apoptotic bodies stained with DAPI. The arrows indicate chromatin condensation in the AGS cells. Fragmentation of nuclear DNA was examined by a fluorescence microscope (×200). Arrows indicate apoptotic bodies, which represent DNA fragments produced during apoptosis. (B) AGS cells were treated with silymarin for 24 h and nuclear condensation was examined by DAPI staining. Graphs reveal quantification of DNA fragmentation and nuclear condensation. Each bar represents the mean ± SD calculated from five independent experiments. *Pt-test).

Figure 4.

Effect of silymarin on apoptosis in AGS human gastric cancer cells. (A) The apoptotic effects of silymarin at different concentrations were evaluated by Annexin V-fluorescein and propidium iodide (PI) staining. AGS cells were treated with silymarin (0, 40, and 80 µg/ml) for 24 h followed by flow cytometry. The lower left quadrant includes viable cells, which were negative for Annexin V-FITC binding (Annexin V−) and exclude PI (PI−); the lower right quadrant includes early apoptotic cells, which are positive for Annexin V-FITC binding (Annexin V+) but PI−; the upper right quadrant includes late apoptotic cells, which are Annexin V+ and reveal PI uptake (PI+); and the upper left quadrant includes necrotic cells, which are Annexin V−/PI+. (B) Annexin V/PI staining was revealed alive cell/dead cell/early-late apoptotic cell fluorescence intensity ratios.

Figure 5.

Effects of silymarin on apoptosis-related protein in AGS human gastric cancer cells. (A) AGS cells were treated with silymarin (0, 40 and 80 µg/ml) for 24 h and cells were harvested to assess protein levels of Bax, Bcl-2, and PARP by western blotting. The blots were also probed with anti-β-actin antibody to confirm equal sample loading. (B) Each bar represents the mean ± SD calculated from three independent experiments. *Pt-test).

Figure 6.

Effect of silymarin on the activation of MAPK pathway in AGS human gastric cancer cells. (A) AGS cells were treated with silymarin (0, 40 and 80 µg/ml) for 24 h. Cell lysates were prepared as described in Materials and methods, and analyzed by 12% SDS-PAGE followed by western blotting. (B) Each bar represents the mean ± SD calculated from independent experiments. *Pt-test). MAPK, mitogen-activated protein kinase.

Figure 7.

Effects of silymarin on AGS gastric cancer tumor xenograft growth and apoptosis in tumor tissues. Nude mice bearing AGS cells as xenograft models were treated with silymarin for 14 days, and (A) tumor volume, (B) tumor weight, and (C) body weight were determined. (D and E) Apoptosis was measured in tumor tissues by TUNEL assay. Slides were observed under an optical microscope (×200). Scale bar, 10 µm. *Pt-test).

Figure 8.

Effects of silymarin on MAPK expression in AGS gastric cancer tumor xenografts. (A) Nude mice were administered silymarin (0, 100 mg/kg) for 2 weeks and examined by immunohistochemistry using antibodies to p-ERK, p-JNK, and p-p38. Tumor tissues were observed under an optical microscope and images were captured at an ×200 magnification. Paraffin-embedded tumor tissues were cut into sections 5-µm thick. Scale bar, 10 µm. (B) Graphs reveal quantification of each protein (p-ERK1/2, p-JNK and p-p38) with positive staining as determined by immunohistochemistry. Significance was determined by Dunnett's t-test with *P<0.05 considered as statistically significant compared with non-treated controls. MAPK, mitogen-activated protein kinase.

Figure 9.

Histological analysis of silymarin-treated nude mice. The microscopic pathology of the liver and kidney revealed no evidence of adverse systemic toxicity of silymarin treatment in the mice. Slides were observed under an optical microscope (×200). Scale bar, 10 µm.