Resveratrol suppresses human glioblastoma cell migration and invasion via activation of RhoA/ROCK signaling pathway

Abstract

Numerous studies have demonstrated that resveratrol has a potential use in cancer prevention and treatment. However, the effects of resveratrol on cancer cell motility and invasiveness remain unclear. The current study aimed to examine the effects of resveratrol on cell migration and invasion in human glioblastoma cells, and to explore the underlying molecular mechanisms. In wound-healing and Matrigel transwell assays, resveratrol was found to significantly inhibit the migration and invasion of U87MG, T98G and U251 glioblastoma cells in vitro. Results from western blot analysis and gelatin zymography revealed that resveratrol also suppressed the expression and activity of matrix metalloproteinase 2 (MMP-2; P<0.05), an important mediator of cell migration and invasion. Furthermore, using a pull-down assay, increased activation of RhoA was observed in glioblastoma cells treated with resveratrol vs. controls (P<0.05). Notably, inhibition of the RhoA/Rho-associated kinase (ROCK) pathway by C3 transferase or Y-27362 was found to attenuate the resveratrol-induced reductions in cell migration and invasion (P<0.05), and also partially rescued the decreased expression and activity of MMP-2 induced by resveratrol (P<0.05). Taken together, the results suggest that resveratrol may inhibit glioblastoma cell motility and invasiveness via activating the RhoA/ROCK signaling pathway.

Keywords: RhoA; glioblastoma; invasion; migration; resveratrol.

Figure 1.

Effects of resveratrol on cell viability of glioblastoma. An MTT assay was performed to measure the cell viability of U87MG, T98G and U251 cells treated with increasing concentrations of resveratrol (20–100 µM) for 48 h. Results are representative of three independent experiments (mean ± standard deviation).*P<0.05 vs. control group (untreated cells).

Figure 2.

Resveratrol suppresses human glioblastoma cell migration and invasion in vitro. (A) The effect of resveratrol on cell migration was measured by wound healing assay. Photographs were captured and the width of the wound was measured in five random fields following incubation for 48 h (original magnification, ×100). Bar graphs represent the mean migration distance relative to the control cells. (B) Matrigel transwell invasion assays were used to measure cell invasiveness. Following incubation with resveratrol for 48 h, cells that invaded through the membrane were stained and representative fields were photographed (original magnification, ×200). Bar graphs represent the mean number of invaded cells from five random fields, and all results are representative of three independent experiments (mean ± SD). *P<0.05 vs. control group (untreated cells).

Figure 3.

Resveratrol inhibits activity and expression of MMP-2 in glioblastoma cells. (A) Western blotting was used to analyze MMP-2 expression in glioblastoma cells treated by resveratrol. The bar graph represents the relative MMP-2 protein expression level in each cell type (mean ± SD). (B) Gelatin zymography was performed to detect the activity of MMP-2 in glioblastoma cells treated with resveratrol. The bar graph represents the relative activity level of MMP-2 in each cell type (mean ± SD). All results are representative of three independent experiments. *P<0.05 vs. control group (untreated cells). MMP-2, matrix metalloproteinase 2; SD, standard deviation.

Figure 4.

Resveratrol increases activation of RhoA in glioblastoma cells. (A) Cells were treated as indicated and harvested, and RhoA activation was analyzed by GST-Rhotekin pull-down, followed by western blotting with anti-RhoA antibody. Bar graph represents the quantified mean RhoA activation values (GTP-RhoA/total RhoA). (B) Resveratrol induced MYPT1 phosphorylation downstream of ROCK. Bar graph represents protein levels of phosphorylated (p) MYPT1 relative to β-actin. All results are representative of three independent experiments. *P<0.05 vs. control group (untreated cells). MYPT1, myosin phosphatase target subunit 1.

Figure 5.

Blocking of RhoA/ROCK pathways attenuated the resveratrol-inhibited migration and invasion of glioblastoma cells. (A) Wound-healing and (B) Matrigel transwell invasion assays revealed that the resveratrol-induced inhibition of U87MG cell migration and invasion were attenuated by treatment with a RhoA inhibitor (C3 transferase; 0.25 µg/ml) or a ROCK inhibitor (Y-27362; 5 µg/ml). (C) Western blot analysis was used to examine MMP-2 expression and MYPT1 phosphorylation (p) in U87MG cells treated with resveratrol alone or together with C3 transferase (0.25 µg/ml), or Y-27362 (5 µg/ml); bar graphs represent protein levels relative to β-actin. (D) Gelatin zymography was used to examine MMP-2 activity in U87MG cells treated with resveratrol alone, or in combination with C3 transferase or Y-27362; bar graphs represent relative activity levels. All results are presented as the mean ± standard deviation of three independent experiments. *P<0.05. ROCK, Rho-associated kinase; MMP-2, matrix metalloproteinase 2; MYPT1, myosin phosphatase target subunit 1.