A375.S2 human melanoma cells and Hs68 fibroblast were purchased from the Food Industry Research and Development Institute (Hsinchu, Taiwan). A375.S2 cells were incubated in minimum essential medium (MEM) (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 2 mM L-glutamine, 0.1 mM non-essential amino acids, 1.5 g/l sodium bicarbonate and 1.0 mM sodium pyruvate. The medium also contained 10% heat-inactivated fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, Inc) and antibiotics, including 100 U/ml penicillin and 100 µg/ml streptomycin. The cells were cultured in an incubator with a humidified atmosphere of 5% CO₂ at 37°C.

MTT was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). Dorsomorphin dihydrochloride (catalog no. 3093) and ketoconazole (catalog no. 1103) were obtained from Tocris Bioscience (Bristol, UK).

GMI, kindly provided by Mycomagic Biotechnology Co., Ltd. (Taipei, Taiwan), was expressed and generated from G. microsporum. GMI was cloned and expressed as described previously (4).

A375.S2 cells grown to 80% confluency were washed twice with PBS and trypsinized with 1 ml 0.25% trypsin-0.03% EDTA. The cells were then seeded at a density of 5×10³ cells/well into 96-well microtiter tissue culture plates. The seeded plates were incubated for 24 h at 37°C in 5% CO₂. The original media was then aspirated and fresh media containing varying concentrations of GMI (0, 0.3, 0.6 and 1.2 µM) and/or ketoconazole (0, 10 and 20 µM) was added to the 96-well plates, followed by incubation at 37°C for 24 or 48 h. A cytotoxicity assay was then performed using MTT and dimethyl sulfoxide was used to dissolve the purple formazan. The absorbance was recorded at a wavelength of 570 nm using a microtiter plate reader.

A375.S2 cell lysates were extracted by RIPA buffer (catalog no., 9806; Cell Signaling Technology, Inc.). The protein concentrations were determined by Bio-Rad Protein Assay (catalog no. 5000006; Bio-Rad Laboratories, Inc.). Total lysates (50 µg) were loaded per lane and resolved by 10% SDS-PAGE, and transferred onto polyvinylidene difluoride (PVDF) membranes using a transblot system (Bio-Rad Laboratories, Inc.). The PVDF membranes were then incubated with blocking buffer [0.01 M Tris-HCl buffer (pH 7.5), 0.1 M NaC, 0.1% Tween-20 and 3% BSA] for 1 h at 25°C and washed. Subsequently, the membranes were incubated overnight at 4°C with the following primary antibodies: Anti-phosphorylated (p)-adenosine monophosphate-activated protein kinase (AMPK)α (catalog no. 2535; dilution 1:500), anti-AMPKα (catalog no. 2603; dilution 1:1,000), anti-p-AMPKβ1 (catalog no. 4181; dilution 1:1,000), anti-AMPKβ1/2 (catalog no. 4150; dilution 1:1,000), anti-p-acetyl-CoA carboxylase (ACC; catalog no. 3661; dilution 1:500), anti-dihydrosphingosine 1-phosphate phosphatase LCB3 (LC3B; catalog no. 3868; dilution 1:2,000), anti-survivin (catalog no. 2808; dilution 1:1,000), anti-β-actin (catalog no. 3700; dilution 1:5,000) and anti-ACC (catalog no. 3676; dilution 1:1,000), all obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Primary antibodies were diluted to the appropriate volume in blocking buffer. The membranes were then incubated with a horseradish peroxidase-conjugated anti-rabbit secondary antibody (catalog no. 7074; dilution 1:5,000) for 1 h at room temperature. Immunoreactive bands were visualized using an enhanced chemiluminescent system (NEN Life Science Products, Inc., Boston, MA, USA). The band density was analyzed by ImageJ version 1.34e software (National Institutes of Health).

A375.S2 cells (5×10⁵ cells) were plated onto a 6 cm dish (Nalge Nunc International, Penfield, NY, USA) with or without 40 µM dorsomorphin dihydrochloride following treatment with GMI (0 and 0.6 µM) and/or ketoconazole (0 and 20 µM) for 48 h incubated at 37°C. The conditioned media were assayed for MCP-1 secretion by solid-phase enzyme-linked immunoabsorbent assay (ELISA) using a MCP-1/CCL2 Human Uncoated ELISA kit (catalog no. 88-7399; eBioscience; Thermo Fisher Scientific, Inc., Waltham, MA, USA), according to the manufacturer's protocol.

Cell migration assays were performed using modified Boyden chambers. MEM with 10% FBS was added to the lower chamber. A375.S2 cells were pretreated with GMI (0, 0.6, and 1.2 µM) for 24 h at 37°C and collected, followed by resuspension at a density of 2×10⁵ cells/well in MEM with 0.5% FBS in the upper chamber. Following incubation for 24 h, cells on the membrane were fixed with methanol and stained with 20% Giemsa solution (Merck KGaA) for 2 h at 25°C. The stained cells were then counted under a light microscope (magnification, ×100).

The apoptosis assay was performed by Annexin V and propidium iodide staining using the FITC Annexin V Apoptosis Detection kit (catalog no. 556547; BD Biosciences, San Jose, CA, USA). Following GMI (0, 0.6 and 1.2 µM) and ketoconazole (0, 10 and 20 µM) treatment for 24 h at 37°C, A375.S2 cells were trypsinized and stained with Annexin V and propidium iodide solution, according to the manufacturer's protocol. Following staining, the cells were analyzed using a flow cytometer and CellQuest 5.1 software (BD Biosciences).

Comparisons between two groups were performed by one-way analysis of variance followed by Tukey's post hoc test using Predictive Analytics 18 software (IBM Corporation). All data are presented as the mean ± standard deviation. Each experiment was performed in triplicate. P<0.05 was considered to indicate a statistically significant difference.

The cell viability of A375.S2 cells treated with varying concentrations of ketoconazole and GMI for 24 and 48 h was analyzed by MTT assay. GMI was identified to mediate cytotoxicity in A375.S2 cells. As presented in Fig. 1A and B, ketoconazole enhanced GMI-induced inhibition of cell viability in a concentration-dependent manner in A375.S2 cells. Using Annexin V/propidium iodide staining, it was revealed that GMI induced apoptosis in a dose-dependent manner and ketoconazole increased GMI-activated apoptosis in A375.S2 cells (Fig. 1C). In addition, it was identified that treatment with 0.3 and 0.6 µM GMI did not decrease cell viability in human skin fibroblast Hs68 cells (data not shown), which suggests ketoconazole does not induce cytotoxicity in Hs68 cells.

The effects of ketoconazole on GMI-inhibited cell migration were then investigated using a modified Boyden chamber assay to quantify the migratory potential of A375.S2 cells. The results revealed that GMI induced a dose-dependent decrease in migration (Fig. 2A). As presented in Fig. 2B, following treatment with 0.6 µM GMI the proportion of migrated cells reduced to 39% and with 1.2 µM GMI with or without 20 µM ketoconazole the proportion of migrated cells was <20%. Ketoconazole induced a dose-dependent decrease in GMI-inhibited migration to <15%. Furthermore, low-toxic concentrations (0.3 µM) of GMI and ketoconazole were used for a migration assay. GMI (0.3 and 0.6 µM) significantly inhibited the migratory ability of A375.S2 cells (Fig. 2C and D). This indicates that GMI inhibits the migratory ability of A375.S2 cells in a cytotoxic-independent manner.

To investigate the effects of ketoconazole and GMI on p-AMPK signaling and autophagy, the expression levels of dihydrosphingosine 1-phosphate phosphatase LCB3 (LC3B), p-AMPKα, p-AMPKβ1 and downstream p-ACC were measured by western blot analysis. Activation of AMPK inhibits the metastatic potential of tumor cells by reducing the activity of molecules associated with the ERK signaling pathway, particularly berberine and 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (14). To investigate which p-AMPK isoform is regulated by GMI, the expression levels of p-AMPKα, p-AMPKβ1 and p-ACC were determined. Treatment with 0.6 µM GMI for 6 h increased p-AMPKα and p-ACC expression levels but did not affect the expression level of p-AMPKβ1 (Fig. 3A). The AMPK inhibitor dorsomorphin dihydrochloride is a cell-permeable compound that inhibits AMPK kinases, vascular endothelial growth factor receptor 2 and activin-like kinase 2 (9). In the present study, treatment with 40 µM dorsomorphin dihydrochloride for 24 h reduced the expression level p-AMPKα (Fig. 3B).

As presented in Fig. 4A, treatment with GMI for 48 h increased the expression levels of LC3B and p-AMPK in A375.S2 cells. Treatment with ketoconazole and GMI for 48 h decreased the expression level of survivin but did not affect the expression of LC3B, p-AMPK or p-ACC. In addition, treatment with 40 µM dorsomorphin dihydrochloride inhibited GMI-induced p-AMPK expression. The LC3B expression level increased following treatment with dorsomorphin dihydrochloride. In addition, dorsomorphin dihydrochloride inhibited the ketoconazole and GMI-induced decrease in survivin expression level. Furthermore, treatment with GMI and ketoconazole for 3 h increased the expression level of activated p-AMPK (Fig. 4B), which suggests that treatment for a short time period can increase AMPK activity.

In addition, MCP-1 is an important chemokine that attracts macrophages to tumors (15). The present study used ELISA to evaluate the secretion of MCP-1 following treatment with GMI and ketoconazole. Ketoconazole was identified to significantly reduce the level of MCP-1, whereas 0.6 µM GMI did not affect the level of MCP-1. In addition, dorsomorphin dihydrochloride significantly reduced the level of MCP-1 (Fig. 5).