The human malignant melanoma cell line WM451 was purchased from the Cell Bank of Central South University. The cells were incubated in DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) at 37°C in a humidified incubator with 5% CO₂. Subsequently, different concentrations of tepotinib (1, 2, 5, 10, 20 and 50 ng/ml; Selleck Chemicals) were used to treat WM451 cells for 48 h (13). For tepotinib + hepatocyte growth factor (HGF; R&D Systems, Inc.)-treated group, 50 ng/ml of HGF (a natural agonist of MET) and 10 ng/ml tepotinib were concurrently administered to WM451 cells for 48 h.

The proliferative capacity of WM451 cells was evaluated using the MTT assay. WM451 cells were incubated in 96-well plates at a density of 5×10⁴ cells/ml/well for 48 h at 37°C. Subsequently, tepotinib was used at different concentrations (1, 2, 5, 10, 20 and 50 ng/ml) to treat WM451 cells. Following exposure to a 10 µl MTT solution for 4 h, the cells were incubated with formazan lysis solution (DMSO) until the purple crystals were completely dissolved. Finally, the absorbance was detected at 570 nm with a spectrophotometer.

WM451 cells were seeded in six-well plates (6×10⁴ cells/well) and cultured to 90–100% confluence. A straight scratch was made in the cell monolayer with a 200-µl pipette tip, followed by PBS washing three times. During the wound healing assay, WM451 cells were cultured in serum-free DMEM in an incubator at 37°C with 5% CO₂ for 48 h. The width of the wound was recorded and images were captured at 0 and 48 h time points by using an inverted light microscope (Olympus Corporation). Finally, the area occupied by the migratory cells in the linear scratch was detected by ImageJ Software (version 6.0; National Institutes of Health).

The invasive ability of WM451 cells was detected using a Transwell plate with an 8-µm pore insert precoated with Matrigel (BD Biosciences) at room temperature for 1 h. The cells were plated at a density of 4×10⁴ cells into the upper compartment of the Transwell chamber with serum-free DMEM. Subsequently, the lower compartment of the Transwell chamber was filled with 500 µl DMEM supplemented with 10% FBS; the cells were incubated for 24 h at 37°C. Subsequently, the invaded cells were fixed with 4% paraformaldehyde for 15 min at room temperature, and stained with 0.1% crystal violet at room temperature for 5 min. Finally, the visualization of the invasive cells was observed with an inverted light microscope (Olympus Corporation).

The effects of tepotinib (10 ng/ml) on the induction of WM451 cell apoptosis of were assessed with a TUNEL assay kit (Invitrogen; Thermo Fisher Scientific, Inc.) in accordance with the manufacturer's protocol. A total of 2×10⁵ cells/well in a 24-well plate were treated with tepotinib for 48 h and subsequently fixed with 4% paraformaldehyde for 15 min at room temperature and then permeabilized with 0.25% Triton X-100 for 2 min at 4°C. Subsequently, the cells were labeled with TUNEL at 37°C for 60 min and counterstained with 0.01 mg/ml DAPI for 10 min in the dark. Slides were mounted using glycerol, and images of the TUNEL-positive apoptotic cells in six randomly selected fields of view were captured with a fluorescence microscope (Nikon Corporation) and quantified by ImageJ Software (version 6.0; National Institutes of Health).

Total protein from WM451 cells was isolated using RIPA lysis buffer (Beyotime Institute of Biotechnology) and quantified using the bicinchoninic acid method (Beyotime Institute of Biotechnology). The proteins (40 µg per lane) were separated with 10% SDS-PAGE and transferred on polyvinylidene fluoride membranes. Following blocking with 5% skimmed milk for 2 h at room temperature, specific primary antibodies were incubated with the membranes at 4°C overnight. Following the primary incubation, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody (1:3,000; cat. no. 7074S; Cell Signaling Technology, Inc.) for 2 h at room temperature. Finally, an enhanced chemiluminescence kit (Beyotime Institute of Biotechnology) was applied to determine the expression levels of the protein bands. The intensities of the bands were detected by ImageJ Software (version 6.0; National Institutes of Health). GAPDH was used as an internal control. The following primary antibodies were diluted to 1:1,000 in TBS + 0.1% Tween-20 buffer and used: E-cadherin (cat. no. 3195T), N-cadherin (cat. no. 13116T), vimentin (cat. no. 5741T), Bcl-2 (cat. no. 4223T), Bax (cat. no. 5023T), cleaved caspase 3 (cat. no. 9664T), caspase 3 (cat. no. 14220T), phospho (p)-MET (cat. no. 3077T), MET (cat. no. 4560S), PI3K (cat. no. 4249T), p-AKT (cat. no. 4060T), AKT (cat. no 4685S) GAPDH (cat. no. 5174T; all from Cell Signaling Technology, Inc.) and p-PI3K (cat. no. ab278545; Abcam).

All data collected from the experiments are displayed as the mean ± standard deviation and assessed with GraphPad Prism 8.0 (GraphPad Software, Inc.). The comparisons between two groups were conducted with the unpaired Student's t-test. One-way analysis of variance followed by Tukey's post hoc test was applied to determine the statistical significance among multiple groups. P<0.05 was considered to indicate a statistically significant difference.

Initially, the effects of tepotinib were investigated on the cell proliferation of WM451 cells. The proliferation of WM451 cells was significantly decreased by tepotinib treatment in comparison with the control group (Fig. 1A). In addition, the inhibitory effects of tepotinib on the proliferation of WM451 cells occurred in a concentration-dependent manner. A total of 10 ng/ml tepotinib presented ~50% inhibitory effect to WM451 cell proliferation, therefore, this concentration was selected to perform the subsequent experiments. The migratory and invasive activities of tepotinib-treated WM451 cells were detected with the application of wound healing and Transwell assays, respectively. The results from Fig. 1B demonstrated that treatment with 10 ng/ml tepotinib significantly diminished the migration of WM451 cells compared with that of the control group. Similarly, the invasive activity of WM451 cells was also reduced by tepotinib (Fig. 1C). These results suggested that tepotinib could suppress the proliferation, migration and invasion of melanoma cells.

The expression levels of EMT-related proteins were assessed with western blot analysis. Tepotinib treatment caused an apparent upregulation of E-cadherin expression levels, while downregulating N-cadherin and vimentin expression levels compared with those of the control group (Fig. 2). In summary, the aforementioned results suggested that tepotinib exerted inhibitory effects on the EMT process of malignant melanoma cells.

The induction of apoptosis of tepotinib-treated WM451 cells was evaluated using TUNEL staining. The induction of apoptosis of WM451 cells was significantly increased following tepotinib (10 ng/ml) treatment compared with that of the control group, revealing that this compound could induce apoptosis of WM451 cells (Fig. 3A and B). In addition, the expression levels of the apoptosis-related proteins were detected by western blot analysis and the results indicated that tepotinib significantly downregulated Bcl-2 expression, while upregulating the expression levels of Bax and cleaved caspase 3 in comparison with the corresponding effects noted in the control group (Fig. 3C and D). In conclusion, the data indicated that tepotinib induced apoptosis of melanoma cells.

To investigate the effects of tepotinib on MET and PI3K/AKT signaling pathways, western blot analysis was utilized to measure the expression levels of MET, phosphorylated (p)-MET, PI3K, p-PI3K, AKT and p-AKT. Tepotinib diminished the expression levels of MET, p-MET, p-PI3K and p-AKT compared with those noted in the control group, suggesting that it exhibited inhibitory effects on activation of MET and PI3K/AKT signaling pathways (Fig. 4).

In order to assess the detailed mechanism of action of tepotinib, HGF (50 ng/ml), which is a natural agonist of MET, was administered to WM451 cells for 48 h. As exhibited in Fig. 5, the addition of HGF significantly upregulated the expression of p-MET, p-PI3K and p-AKT when compared with the tepotinib group. Moreover, tepotinib inhibited the proliferative ability of WM451 cells, which was partially reversed by HGF, indicating the beneficial effects of this factor on the proliferation of tepotinib-treated WM451 cells (Fig. 6A). Tepotinib considerably diminished the relative migration rate of WM451 cells, while HGF administration partially abolished the suppressive effects of tepotinib, as demonstrated by the increased migration of the tepotinib + HGF cell group in comparison with that noted in the tepotinib cell group (Fig. 6B). Similarly, the declined invasive activity of tepotinib-treated WM451 cells was subsequently enhanced following HGF administration (Fig. 6C). Besides, tepotinib increased E-cadherin expression, while reducing the expression levels of N-cadherin and vimentin, whereas HGF administration exhibited the opposite effects, as determined by the decreased expression levels of E-cadherin and the increased expression levels of N-cadherin and vimentin in the tepotinib + HGF group (Fig. 6D).

According to the results obtained from the TUNEL assay, the increased apoptotic levels caused by tepotinib treatment were significantly diminished by HGF administration (Fig. 7A and B). In addition, the expression levels of the apoptosis-related proteins were measured by western blot analysis and the results revealed that tepotinib significantly downregulated Bcl-2 expression levels, while causing a significant upregulation in the expression levels of Bax and cleaved caspase 3. Nevertheless, HGF administration exerted the opposite effects on these proteins, as determined by the increased Bcl-2 expression levels as well as the decreased expression levels of Bax and cleaved caspase 3 in the tepotinib + HGF group (Fig. 7C and D).