Research Article

The Antitumor Activity of Antrodia camphorata in Melanoma Cells: Modulation of Wnt/β-Catenin Signaling Pathways

Figure 2

AC downregulates β-catenin and upregulates GSK3β in melanoma cells. (a) B16F10 and B16F1 cells were incubated with control vehicle or AC (40, 80, and 120 μg/mL) for 24 h. Western blot results showing the effects of AC on the total protein contents of β-catenin, p-β-catenin, Dvl, GSK3β, and p-GSK3β. (b) Immunocytochemistry was performed to measure the β-catenin expression in B16F10 melanoma cells. B16F10 cells were grown on 8-well Lab-Tek chambers and treated with or without AC (40–120 μg/mL) for 24 h. Cells were fixed with 2% paraformaldehyde and incubated with specific β-catenin antibodies, followed by a fluorescein isothiocyanate-conjugated secondary antibody (green), and visualized under a confocal microscope. The photomicrographs shown in this figure are from one representative experiment performed in triplicate, with similar results. (c) B16F10 and B16F1 cells were pretreated with MG132 (25 μM) or GSK3β inhibitor (SB216763 50 μM) for 30 min, followed by AC (80 μg/mL) for 24 h; the total protein levels of β-catenin were determined by Western blotting. Denatured proteins of each sample (50 μg) were separated by 8–15% SDS-PAGE and immunoblotted with specific antibodies. β-actin was used as an internal loading control. Relative changes in protein bands were measured by densitometric analysis with the control being 1.00-fold as shown just below the gel data. Typical results from three independent experiments are shown. (d) Western blotting was performed to measure the expression levels of β-catenin transcriptional target genes such as c-Myc and survivin in B16F10 and B16F1 cells.
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