Sulforaphane inhibits invasion via activating ERK1/2 signaling in human glioblastoma U87MG and U373MG cells

Abstract

Background: Glioblastoma has highly invasive potential, which might result in poor prognosis and therapeutic failure. Hence, the key we study is to find effective therapies to repress migration and invasion. Sulforaphane (SFN) was demonstrated to inhibit cell growth in a variety of tumors. Here, we will further investigate whether SFN inhibits migration and invasion and find the possible mechanisms in human glioblastoma U87MG and U373MG cells.

Methods: First, the optimal time and dose of SFN for migration and invasion study were determined via cell viability and cell morphological assay. Further, scratch assay and transwell invasion assay were employed to investigate the effect of SFN on migration and invasion. Meanwhile, Western blots were used to detect the molecular linkage among invasion related proteins phosphorylated ERK1/2, matrix metalloproteinase-2 (MMP-2) and CD44v6. Furthermore, Gelatin zymography was performed to detect the inhibition of MMP-2 activation. In addition, ERK1/2 blocker PD98059 (25 µM) was integrated to find the link between activated ERK1/2 and invasion, MMP-2 and CD44v6.

Results: The results showed that SFN (20 µM) remarkably reduced the formation of cell pseudopodia, indicating that SFN might inhibit cell motility. As expected, scratch assay and transwell invasion assay showed that SFN inhibited glioblastoma cell migration and invasion. Western blot and Gelatin zymography showed that SFN phosphorylated ERK1/2 in a sustained way, which contributed to the downregulated MMP-2 expression and activity, and the upregulated CD44v6 expression. These molecular interactions resulted in the inhibition of cell invasion.

Conclusions: SFN inhibited migration and invasion processes. Furthermore, SFN inhibited invasion via activating ERK1/2 in a sustained way. The accumulated ERK1/2 activation downregulated MMP-2 expression and decreased its activity and upregulated CD44v6. SFN might be a potential therapeutic agent by activating ERK1/2 signaling against human glioblastoma.

Figure 1. SFN inhibited cell viability.

An in(0, 10, 20, 30, 40, 50, 60, 70, 80 and 90 µM) for 24 h. The viability of the SFN-treated cells was measured using the MTS assay. Results were expressed as a percentage of control, which was considered as 100%. Data were reported as mean ± SD and at least three separate experiments were performed.

Figure 2. SFN changed cell morphology.

(A) Cellular morphological changes in U87MG and U373MG cell lines were done in a dose-dependent manner after treated with SFN for 24 h when observed by a Leica DMIRB Microscope at ×100 magnification. (B) Cellular morphological changes in U87MG and U373MG cell lines were done in a time-dependent manner after treated with 20 µM SFN when observed by a Leica DMIRB Microscope at ×100 magnification.

Figure 3. SFN inhibited migration in U87MG and U373MG cell lines.

Confluent U87MG and U373MG cells were scratched and incubated at different concentrations of SFN (µM). The area covered by migrating cells was recorded by phase-contrast microscopy connected to a digital camera at time 0 and 24 h. The wound closure area was calculated by measuring the diminution of the wound bed surface upon time using Image J software. Representative pictures of three independent experiments were shown. *, indicates P<0.05 versus no SFN group.

Figure 4. SFN inhibited invasion in a dose-dependent manner in U87MG and U373MG cell lines.

Approximately 1×10⁵ cells were seeded in the 24-well plate with cell culture inserts, the cells were treated with different concentrations of SFN (µM) for 24 h to test invasion. Assays were performed as described in Materials and Methods. The results showed that SFN inhibited significantly cell invasion in a dose-dependent manner. *, indicates P<0.05 versus no SFN group. Data were shown as means ± SD from three independent experiments.

Figure 5. SFN phosphorylated ERK1/2 in a sustained way.

(A) We have demonstrated that SFN inhibited cell migration and invasion in a dose-dependent manner. Thus, here we treated the U87MG and U373MG cells with different doses of 0, 10, 20, and 30 µM SFN for 24 h. Western blot showed that SFN phosphorylated ERK1/2 in a dose-dependent manner. At the concentration of 20 µM, ERK1/2 activation was significantly increased. (B) The cells were treated with 20 µM SFN for 0, 3, 6, 12, 24 and 48 h. Western blot showed that SFN activated ERK1/2 in a sustained way. At the time point of 24 h, ERK1/2 phosphorylation reached the peak. *, indicates P<0.05 versus no SFN group, #, indicates P<0.05 versus other groups. Data were shown as means ± SD from three independent experiments.

Figure 6. SFN Inhibited invasion via sustained activation of ERK1/2.

(A) The U87MG and U373MG cells were respectively treated with SFN (20 µM) without or with PD98059 (25 µM) for 24 h, western blot showed that SFN activated ERK1/2 significantly. PD98059 decreased ERK1/2 phosphorylation. *, indicates P<0.05 versus the control group. Data were shown as means ± SD from three separate tests. (B) We seeded 1×10⁵ cells in a 24-well plate with cell inserts, the cells were added with SFN (20 µM) without or with PD98059 (25 µM) for 24 h to detect cell invasion. Results showed that SFN inhibited cell invasion significantly versus control. SFN plus PD98059 reduced cell invasion inhibition compared with SFN-only. All procedures were performed as described in Methods. *, indicates P<0.05 versus control, # indicates P<0.05 versus SFN-only group. Data were shown as means ± SD from three separate tests.

Figure 7. SFN decreased MMP-2 expression and activity in U87MG and U373MG cells.

(A) SFN decreased MMP-2 expression via activated ERK1/2. We treated the cells with SFN (20 µM) without or with PD98059 (25 µM) for 24 h. Western blot showed that SFN significantly downregulated MMP-2 expression. SFN plus PD98059 reduced the downregulation of MMP-2 expression. The results indicated that SFN regulated MMP-2 expression via ERK1/2 activation. *, indicates P<0.05 versus control. #, indicates P<0.05 versus the SFN group. Data were shown as means ± SD from three separate tests. (B) SFN decreased MMP-2 activity via activated ERK1/2. We collected the conditioned medium from the above treatment for gelatin zymography assay as the methods. The results showed that SFN significantly decreased MMP-2 activity. SFN plus PD98059 significantly reduced MMP-2 activity inhibition versus the SFN-only. *, indicates P<0.05 versus control. #, indicates P<0.05 versus the SFN-only group. Data were shown as means ± SD from three separate tests.

Figure 8. SFN upregulated CD44v6 expression in U87MG and U373MG cells.

We treated the cells with SFN (20 µM) without or with PD98059 (25 µM) for 24 h. Western blot showed that SFN significantly upregulated CD44v6 expression. After PD98059 and SFN were added into the medium, CD44v6 expression was reduced significantly versus SFN-only group. That indicated SFN regulated CD44v6 expression via sustained ERK1/2 activation. *, indicates P<0.05 versus control. Data were shown as means ± SD from three separate tests.