Inhibition of proliferation and migration of melanoma cells by ketoconazole and Ganoderma immunomodulatory proteins

Abstract

Ketoconazole, an antifungal agent, has been used to inhibit hormone synthesis in types of prostate and breast cancer. Immunomodulatory proteins of Ganoderma microsporum (GMI) inhibit the tumor necrosis factor-α- and epidermal growth factor-induced metastatic ability of lung cancer cells. Cutaneous malignant melanoma is a highly invasive and metastatic skin cancer. However, to the best of our knowledge, there is limited understanding regarding the effects of ketoconazole and GMI on melanoma. The current study aimed to investigate the inhibitory effects of GMI combined with ketoconazole on melanoma survival and metastasis. The effects of GMI combined with ketoconazole on the viability, migration and protein expression of melanoma cells were determined by MTT assay, Boyden chamber assay and western blot analysis, respectively. The expression of monocyte chemoattractant protein-1 (MCP-1) was investigated by enzyme-linked immunoabsorbent assay. The present results indicate that ketoconazole enhances the GMI-induced decrease in proliferation and migration of A375.S2 melanoma cells in a concentration-dependent manner. Ketoconazole was identified to reduce the level of GMI-induced phosphorylated-adenosine monophosphate-activated protein kinase (p-AMPK)-α and autophagy; however, ketoconazole did not affect p-AMPK-β levels in A375.S2 cells. In addition, ketoconazole and dorsomorphin dihydrochloride, an AMPK inhibitor, were revealed to reduce MCP-1 secretion in A375.S2 cells. In summary, the present study revealed that ketoconazole enhances GMI-inhibited proliferation and migration of A375.S2 melanoma cancer cells, and inhibits the secretion of MCP-1.

Keywords: Ganoderma microsporum; adenosine monophosphate-activated protein kinase; ketoconazole; melanoma; monocyte chemoattractant protein-1.

Figure 1.

Effects of ketoconazole combined with GMI in A375.S2 cells. A375.S2 cells were treated with various concentrations of ketoconazole (0, 5, 10 and 20 µM) and GMI (0, 0.3, 0.6 and 1.2 µM) for (A), 24 or (B) 48 h, and cell survival was analyzed by MTT assay. (C) A375.S2 cells were treated with GMI and ketoconazole for 24 h and apoptotic cells were detected by Annexin V/propidium iodide staining and flow cytometry. Data are presented as the mean ± standard deviation. *P<0.05 vs. 0 µM GMI; ^#P<0.05 vs. 1.2 µM GMI. GMI, Ganoderma microsporum.

Figure 2.

GMI inhibits the migration of A375.S2 cells in vitro. (A) A375.S2 cells were treated with various concentrations of ketoconazole (0, 10 or 20 µM) and GMI (0, 0.6 or 1.2 µM) for 16 h. The migratory capacity of the cells was then determined in vitro by a Boyden chamber assay. Scale bar, 100 µm. (B) The numbers of migrated cells were quantified relative to the control. (C) A375.S2 cells were treated with ketoconazole (0 or 20 µM) and GMI (0, 0.3 or 0.6 µM) for 16 h. The migratory capacity of the cells was then determined in vitro by a Boyden chamber assay. (D) The numbers of migrated cells were quantified relative to the control. Data are presented as the mean ± standard deviation. GMI, Ganoderma microsporum. *P<0.05 vs. 0 µM GMI; ^#P<0.05 vs. 1.2 µM GMI.

Figure 3.

Effects of GMI and dorsomorphin dihydrochloride on the protein expression levels of proteins associated with the AMPK signaling pathway. (A) A375 cells were treated with GMI (0 or 0.6 µM) for 0, 1, 3 or 6 h. (B) A375.S2 cells were treated with various concentrations of dorsomorphin dihydrochloride for 24 h. GMI, Ganoderma microsporum; AMPK, adenosine monophosphate-activated protein kinase; p-, phosphorylated; ACC, acetyl-CoA carboxylase.

Figure 4.

Effects of GMI and dorsomorphin dihydrochloride on the protein expression levels of proteins associated with cell death and the AMPK signaling pathway. (A) A375.S2 cells were pretreated with 40 mM dorsomorphin dihydrochloride for 1 h and then treated with ketoconazole (0 or 20 µM) combined with GMI (0 or 0.6 µM) for 48 h. The expression levels of p-AMPK, AMPK, p-ACC, ACC, LC3B (LC3B-I and LC3B-II) and survivin were measured by western blot analysis. GMI and ketoconazole increased the expression level of LC3B and reduced the expression of survivin. (B) A375.S2 cells were treated with ketoconazole (0 or 20 µM) combined with GMI (0 or 0.6 µM) for 3 h. The expression levels of p-AMPK, AMPK and β-actin were then measured by western blot analysis. GMI, Ganoderma microsporum; AMPK, adenosine monophosphate-activated protein kinase; p-, phosphorylated; ACC, acetyl-CoA carboxylase; LC3B, dihydrosphingosine 1-phosphate phosphatase LCB3.

Figure 5.

Effects of ketoconazole combined with GMI on the level of MCP-1. A375.S2 cells were pretreated with various concentrations of dorsomorphin dihydrochloride (0 or 40 µM) for 1 h followed by treatment with ketoconazole (0 or 20 µM) and GMI (0 or 0.6 µM) for 48 h. The conditioned media were then subjected to an enzyme-linked immunoabsorbent assay to measure the levels of secreted MCP-1. Data are presented as the mean ± standard deviation from three independent experiments. NS, no significance; GMI, Ganoderma microsporum; MCP-1, monocyte chemoattractant protein-1. *P<0.05 vs. GMI 0 µM.