Resveratrol inhibits the hedgehog signaling pathway and epithelial-mesenchymal transition and suppresses gastric cancer invasion and metastasis

Abstract

The hedgehog (Hh) signaling pathway is vital to vertebrate development, the homeostatic process and tumorigenesis. Epithelial-mesenchymal transition (EMT) is a cellular process during which epithelial cells become mesenchymal-appearing cells, which in turn promotes cancer metastasis and invasion. Resveratrol is a natural polyphenolic compound found in grapes, a variety of berries, peanuts and other plants. Numerous studies have demonstrated that the Hh signaling pathway is able to regulate the EMT, and that resveratrol can suppress carcinoma invasion and metastasis. In addition, certain studies have indicated that resveratrol can inhibit the Hh signaling pathway and EMT in cancers other than gastric cancer. The purpose of the present study was to investigate the inhibitory effect of resveratrol on the Hh signaling pathway and EMT in gastric cancer in vitro. Gastric cancer SGC-7901 cells were treated with resveratrol or cyclopamine at different concentrations. The viability of the cells was assessed using an MTT assay. The expression of Gli-1, a key component of the Hh signaling pathway, and Snail, E-cadherin and N-cadherin, key components of EMT, was detected by reverse transcription polymerase chain reaction (RT-PCR) and western blotting. The invasion and metastasis of the cells were observed by performing a cell scratch test. The RT-PCR and western blotting showed a decrease in Gli-1, Snail and N-cadherin expression, and an increase in E-cadherin expression in the resveratrol and cyclopamine group compared with the control group, suggesting that resveratrol inhibited the Hh pathway and EMT, as did cyclopamine. The MTT assay indicated that the viability of the SGC-7901 cells was significantly decreased in a concentration-dependent manner following resveratrol and cyclopamine treatment. The cell scratch test showed slower cell invasion and metastasis in the resveratrol and cyclopamine groups. These findings indicated that resveratrol was able to inhibit the Hh signaling pathway and EMT, and suppress invasion and metastasis in gastric cancer in vitro.

Keywords: cyclopamine; gastric cancer epithelial-mesenchymal transition; hedgehog signaling pathway; resveratrol.

Figure 1.

MTT assays were used to examine the inhibitory effect of resveratrol and cyclopamine on cell proliferation. The SGC-7901 cells were exposed to varying concentrations of (A) resveratrol (3.125–300 µmol/l) or (B) cyclopamine (3.125–150 µmol/l). Each data point is the result of more than three independent experiments.

Figure 2.

Effect of resveratrol and cyclopamine on the metastasis and invasion of SGC-7901 cells. The cells were cultured with RPMI 1640 medium (Ctrl), resveratrol (Res) and cyclopamine (Cyc) at their respective IC₅₀ values for 48 h. Scratches of the same width were made for each group at 0 h, and then the width of the scratched was measured at 12, 24 and 48 h.

Figure 3.

Various effects of resveratrol exposure (IC₅₀ value) on the target genes for different times. The SGC-7901 cells were cultured with resveratrol (55 µmol/l) for 0, 3, 6, 12, 24 and 48 h. (A) The expression levels of the mRNA of Gli-1, Snail, E-cadherin and N-cadherin were detected by reverse transcription-polymerase chain reaction assay. GAPDH was used as a loading control. (B) The relative mRNA expression of the target genes. **P<0.01 vs. Gli-1 0 h; ^##P<0.01 vs. Snail 0 h; ^▲▲P<0.01 vs. N-cadherin 0 h; ^♦♦P<0.01 vs. E-cadherin 0 h. E-cad, E-cadherin; N-cad, N-cadherin.

Figure 4.

Different effects of resveratrol exposure for different times at its IC₅₀ concentration. The human gastric cancer SGC-7901 cells were treated in complete medium containing resveratrol (55 µmol/l) for 0, 3, 6, 12, 24 and 48 h. Subsequent to treatment, the proteins of Gli-1 and EMT markers were analyzed by western blotting analysis with corresponding antibodies. β-tubulin was used as a loading control. **P<0.01 vs. Gli-1 0 h; ^##P<0.01 vs. Snail 0 h; ^▲▲P<0.01 vs. N-cadherin 0 h; ^♦♦P<0.01 vs. E-cadherin 0 h. E-cad, E-cadherin; N-cad, N-cadherin.

Figure 5.

Expression of Gli-1, Snail, E-cadherin and N-cadherin mRNA in untreated (Ctrl), resveratrol-treated (55 µmol/l; Res) and cyclopamine-treated (25 µmol/l; Cyc) SGC-7901 cells was assessed by reverse transcription-polymerase chain reaction analysis. The expression of each gene was standardized using GAPDH as a reference gene. The relative intensity for each band was measured using ImageJ software, dividing the absolute intensity of each sample band by the absolute intensity of the standard. **P<0.01 vs. control (Gli-1); ^##P<0.01 vs. control (Snail); ^▲▲P<0.01 vs. control (N-cadherin); ^♦♦P<0.01 vs. control (E-cadherin). E-cad, E-cadherin; N-cad, N-cadherin.

Figure 6.

Proteins expression level of Gli-1, Snail, E-cadherin and N-cadherin in untreated (Ctrl), resveratrol-treated (Res) and cyclopamine-treated (Cyc) SGC-7901 cells with respective IC₅₀ values, assessed by western blotting analysis. Proteins were extracted with radioimmunoprecipitation assay buffer. Specific antibodies against Gli-1, Snail, E-cadherin and N-cadherin were used. Bound proteins were detected using appropriate horseradish peroxidase-conjugated secondary antibodies. Analysis of the western blot images was performed by calculating the relative intensity of the immunoreactive bands following analysis by means of the ImageJ software. The relative densities of the bands were expressed as arbitrary units. **P<0.01 vs. control (Gli-1); ^##P<0.01 vs. control (Snail); ^▲▲P<0.01 vs. control (N-cadherin); ^♦♦P<0.01 vs. control (E-cadherin). E-cad, E-cadherin; N-cad, N-cadherin.