Galangin, MTT reagent and dimethyl sulfoxide (DMSO) were acquired from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). All the antibodies used against extracellular signal-regulated kinase (Erk)1/2, p-Erk1/2, protein kinase B (AKT), p-AKT and ADAM9, secondary antibodies, β-actin and small interfering (si)ERK were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). ADAM9 protein, MEK inhibitor (U0126) and the ARY021 human protease array kit were ordered from Abcam (Cambridge, UK).

The A172 human glioma cancer cell line was purchased from American Type Culture Collection (Manassas, VA, USA). These cell lines were cultured in RPMI-1640 medium supplemented with 10% FBS, penicillin and streptomycin (100 U/ml + 100 mg/ml) and then maintained in an incubator with humidified atmosphere (5% CO₂) at 37°C. Galangin (10 mM) stock solution was prepared using DMSO solvent, kept at −20°C for storage and then diluted further to carry out the other experiments.

MTT assay was performed to determine the cell cytotoxicity. In this experiment, the human glioma cancer cell line (A172) at a density of 4×10³ cells/well were cultured in 24-well plates and then treated with different galangin concentrations (5–25 µM) for different time periods, 24 and 72 h. Later, the cell lines were incubated with MTT reagent (0.4 mg/ml) for another ~5 h. Following discarding the medium, each well was added with ~50 µl isopropanol and then absorbance was measured at 590 nm using a spectrophotometer.

In the current assay, Matrigel was not used to coat the cell culture chambers. The experiment was carried out in such a way that the A172 cells at a density of 4×10⁴ per well were cultured in the upper compartment using serum free medium, whereas the lower compartment was added with 10% FBS containing Dulbecco's modified Eagle's medium (Thermo Fisher Scientific, Inc., Waltham, MA, USA). It was then treated with different galangin concentrations (5–25 µM) and incubated for 6 h. Following incubation, a cotton swab was used to clean the cells located inside the chambers and the migrated cells to the lower portion were stained using Giemsa (0.1%). The migration was carried out via poly-vinylpyrrolidine free polycarbonate membrane with a pore size of 8 mm. A light microscope at the magnification ×200 was used for counting and non-treated cells are treated as controls.

A cell invasion assay was performed similarly to that of the cell migration assay as mentioned in previous method. In this assay, initially the chamber membranes were coated using Matrigel (50 µg/ml) followed by cell culturing at a density of 4×10⁴ cells/well in the upper compartment. The cell invasion via Matrigel and polycarbonate membranes to the lower portion was recorded at 12 h incubation, the same way as mentioned in cell migration experiments.

In this experiment, TRIzol plus RNA extraction kit (Invitrogen; Thermo Fisher Scientific, Inc.) was used to extract total RNAs and the synthesis of cDNAs (from 4×10⁴ A 172 cells) was performed using cDNA synthetic kit/Superscript III Platinum One Step RTqPCR kit (Thermo Fisher Scientific, Inc.) based on previous methods presented in the reported literature (20). Briefly, TaqMan probe reaction mixture (SYBR-Green I dye from Invitrogen; Thermo Fisher Scientific, Inc.) in a standard cycling program were used along with specific forward and reverse primers (flurogenic primers) of ADAM9 (forward, 5′-GCTACGCACCTCCAAATTGT-3′ and reverse 5′-GGCCTCAAGTCATTGGAA-3′) and β-actin (forward 5′-AGTTTAGGACTTGACC-3′ and reverse, 5′-TTAAGCCCTGAAATCCT-3′-designed using Primer3 program) were run in a Step One Plus RT PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) to amplify the mRNAs of ADAM9 and β-actin. The relative fold change in mRNA expression levels were quantified using the 2^−ΔΔCq method (20).

To carry out western blotting, lysis buffer [50 mM Tris-HCl (pH 7.5), 0.5% sodium deoxycholate, 150 mM NaCl, 0.1% SDS, 1 mM dithiothreitol, 5 mM EDTA, 50 mM sodium fluoride] was used to extract the cells total protein and estimated using Bradford method. Equal concentration of protein extracts (50 µg) were added to each well for SDS-PAGE (10%) separation electrotransferred onto polyvinylidene difluoride (PVDF) membranes. The membrane was blocked with TBS containing Tween-20 and 5% skimmed milk for 2 h at 37°C and incubated with primary antibodies anti-rabbit monoclonal ADAM9 (cat. no. 2099S; 1:1,000), Erk1/2 (cat. no. 4376S; 1:1,000), p-Erk1/2 (cat. no. 4370S; 1:1,000), AKT (cat. no. 4685S; 1:1,000), p-AKT (cat. no. 4060S; 1:1,200) antibodies and β-actin (cat. no. 4970S; 1:1,000) for 2 h at 37°C and again incubated with a secondary antibody with conjugated to anti-rabbit immunoglobulin G horseradish peroxidase (cat. no. 7074S; 1:10,000) for 2 h at 37°C. All the antibodies (primary/secondary) are bought from Cell Signaling Technology, Inc. The visualization of bounded proteins was determined using the enhanced chemiluminescence detection system (GE Healthcare Life Sciences, Chalfont, UK). The image/band intensity was detected using a LAS 4000 gel documentation system from GE Healthcare Life Sciences (Chalfont, UK) using Image J software from the National Institutes of Health (version 2.8; Bethesda, MD, USA).

Initially, the human A172 cells were transfected using siErk with the help of Lipofectamine 2000 (Thermo Fisher Scientific, Inc.) and kept for next 2 days to undergo transfection. Once the cells were transfected, galangin was added and harvested to extract total RNA/protein. Later, the above discussed methods (RT-qPCR and western blot section) were used to measure the mRNA/protein levels.

All the given experimental data were expressed as mean ± standard error of the mean. All the experiments in this work were performed independently in triplicate. Measurement of statistical differences between the groups were recorded using one-way analysis of variance followed by Dunnett's multiple comparison post hoc test with the help of GraphPad Prism software (version, 5; GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered to indicate a statistically significant difference.

The investigation results of cell migration and invasion assays on A172 cells is presented in Fig. 1. The results indicated that galangin treatment reduced the A172 cell migration and invasion. In order to study the galangin effect on cell viability, MTT assay (Fig. 1B) was carried out on the A172 glioma cancer cell line. Upon treatment of the A172 cell line with galangin at different concentrations (5–25 µM), no significant inhibition on A172 cell viability was observed. To elucidate the galangin potentials on A172 cell migration and invasion, a specific galangin concentration was chosen in such a way that it did not show significant effects on A172 cell growth. The data indicated that treatment of various concentrations of galangin (5–25 µM) for ~24 h on the A172 cell line markedly decreased the migration potential of the A172 cell line in a concentration dependent fashion when compared to that of non-treated cells. The percentage of migration was from 16 to 84% in A172 cells, whereas the percentage of cell invasion was from 14 to 81% (Fig. 1C and D). All these results indicated that galangin strongly inhibited A172 cell migration and invasion under non-toxic doses.

The authors conducted an experiment using a human protease inhibitor array kit in order to exhibit the molecular mechanisms underlying the reducing effects of galangin on A172 cell migration and invasion. This investigation helps to display the invasion associated protein actions inside the cells. An earlier report indicated that ADAM9 has a special role in cell migration and cell invasion in glioma cancer cell lines (21). The study observed ADAM9 expression following treatment with different doses of galangin and the results clearly indicated that galangin reduced ADAM9 expression (Fig. 2A). To further elucidate the protease array results, western blot analysis and RT-qPCR was conducted using galangin and demonstrated that it hindered mRNA and ADAM9 protein expression in the A172 cell line (Fig. 2B and C). Results of these studies revealed that galangin hinders the A172 cell migration and invasion potential, which could be due to the decrease of ADAM9 protein expression. Before investigating the pharmacological properties of galangin on reduced ADAM9 expression, the authors checked whether galangin could hinder RhADAM9-induced A172 cell migration and invasion. Results presented in Fig. 2D and E clearly indicated that galangin hindered both RhADAM9-induced A172 cell migration and invasion in the A172 glioma cell line. These investigations revealed that the anti-metastatic ability of galangin results from repressing the ADAM9 expression in the A172 cell line.

A previous report clearly indicated that Erk1/2 activation is associated functions of cell migration and invasion in human glioma cells (22). Hence, the authors tested galangin against the A172 glioma cell line for Erk1/2 activation in addition to the AKT signaling pathway. The results obtained from ERK1/2 activation and the AKT signaling pathway demonstrated that galangin effectively induced Erk1/2 phosphorylation whereas it failed to report a significant effect on AKT phosphorylation (Fig. 3). Furthermore, no notable quantity of proteins (t-Erk1/2 and t-AKT) was obtained. All these data clearly evidenced the role of activation of Erk1/2 in the galangin inhibited the A172 cell migration and invasion mechanism.

In order to investigate the mechanistic pathway for galangin induced cell invasion inhibition, the authors first pre-treated the A172 glioma cell line with 15 µM MEK inhibitor, U0126, for ~3 h and then further added galangin for next 12 h before finally subjecting to western blot analysis, RT-qPCR and cell migration and invasion studies. Data obtained from these studies indicated that U0126 decreased galangin and triggered expression of Erk1/2, protecting galangin from protein hindrance and expressions of ADAM9 mRNA in A172 cells, as presented in Fig. 4A. Results from cell migration and cell invasion assays suggested that the pretreatment of MEK inhibitor completely blocked galangin-induced inhibition of A172 cell migration and invasions (Fig. 4B and C). Based on these results, ERK1/2 activation was identified to serve a specific function in galangin-mediated inhibition to cell migration and invasion. Moreover, A172 cells transiently transfected with siERK displayed effective reduction in total expression of ERK1/2 and terminated galangin-induced inhibition to ADAM9 expression (Fig. 5A). In addition, it was identified that siERK expression in A172 cells also blocked galangin-induced inhibition to cell migration and invasion (Fig. 5B and C). All the obtained results clearly indicated that galangin hinders A172 glioma cell invasion via activation of ERK1/2.