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

Abstract

Antrodia camphorata (AC) is well known in Taiwan as a traditional Chinese medicine. The aim of this study was to investigate whether a fermented culture broth of AC could inhibit melanoma proliferation and progression via suppression of the Wnt/β-catenin signaling pathway. In this study, we observed that AC treatment resulted in decreased cell viability and disturbed Wnt/β-catenin cascade in B16F10 and/or B16F1 melanoma cells. This result was accompanied by a decrease in the expression of Wnt/β-catenin transcriptional targets, including c-Myc and survivin. Furthermore, treatment of melanoma cells with AC resulted in a significant increase in apoptosis, which was associated with DNA fragmentation, cytochrome c release, caspase-9 and -3 activation, PARP degradation, Bcl-2/Bax dysregulation, and p53 expression. We also observed that AC caused G₁ phase arrest mediated by a downregulation of cyclin D1 and CDK4 and increased p21 and p27 expression. In addition, we demonstrated that non- and subcytotoxic concentrations of AC markedly inhibited migration and invasion of highly metastatic B16F10 cells. The antimetastatic effect of AC was further confirmed by reductions in the levels of MMP-2, MMP-9, and VEGF expression. These results suggest that Antrodia camphorata may exert antitumor activity by downregulating the Wnt/β-catenin pathways.

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1. Introduction

Recent studies have found that activation of the Wnt/β-catenin signaling pathway plays an important role in both the initiation and progression of cancers in various tissues and organs, including the skin [1], liver [2], prostate [3], breast [4], esophagus [5], and lung [6]. Earlier studies suggested the involvement of the Wnt/β-catenin signaling pathway in the pathogenesis of malignant melanoma [7]. Aberrant activation of the Wnt/β-catenin pathway has been observed in approximately one-third of melanomas, and this subset has a very poor prognosis, suggesting that targeting Wnt/β-catenin signaling may be a promising target for chemoprevention and chemotherapy [8].

The Wnt extracellular signaling pathway controls multiple aspects of development, including proliferation, fate specification, and cell migration. In addition, aberrant activation of Wnt signaling by mutations is a major factor in oncogenesis in a variety of human tissues [9]. The Wnt ligand directly binds to its Frizzled (Fz) receptor, resulting in activation of both canonical and noncanonical pathways [10]. In the canonical pathway, Wnt binds to its Fz receptor to inactivate the β-catenin destructive complex comprising APC, Axin, and GSK3β, through the activation of the dishevelled (Dvl) protein. In this event, β-catenin is not phosphorylated at specific serine and threonine residues. Instead, β-catenin disassociates from the complex, translocates into the nucleus, and binds to T-cell factor family of Tcf/Lef transcription factors to form a heterodimeric complex that activates the transcription of Wnt target genes [11]. Many important genes, including c-Myc, survivin, cyclin D1, and metalloproteinase (MMP), which are involved in oncogenesis, are regulated by the activation of β-catenin/Tcf-mediated transcription [12]. Noncanonical pathways, which are Wnt signaling pathways that signal independently of β-catenin may signal through calcium flux, c-Jun NH₂-terminal kinase (JNK), and G proteins [10].

Increasing evidence indicates that the Wnt/β-catenin pathway promotes proliferation and cell survival in various normal and cancer cell types, including melanoma cells [1]. Previous studies have demonstrated that down-regulation of the Wnt/β-catenin or Wnt-1 pathway by small interfering RNAs (siRNA) or Wnt-1-targeted monoclonal antibodies induces apoptosis in a variety of human cancer cells. Meanwhile, activation of this pathway is inhibited by chemotherapy-induced apoptosis [12–15], suggesting that the Wnt/β-catenin pathway may be associated with cellular apoptosis. Melanoma metastasis is often associated with activation of the Wnt/β-catenin signaling pathway [16]. Vaid et al. reported that melanoma cells derived from Wnt, acting through Fz receptors induce MMP-2 and MMP-9 expression, which plays a major role in cell migration and invasion [17]. In addition, a crucial role for Wnt/β-catenin signaling in angiogenesis is further supported by the identification of Wnt-β-catenin target genes that encode angiogenic regulators, including vascular endothelial growth factor (VEGF) [18]. Another study suggested that the Wnt/β-catenin pathway may indirectly regulate angiogenesis via transcriptional activation of the VEGF-A gene in nonendothelial cells or tumors [19].

Antrodia camphorata (AC), an indigenous medicinal mushroom that is popularly known as “Niu Cheng Zhi” in Taiwan, is a newly discovered basidiomycete of the family Polyporaceae that only grows in the inner sap of the native Taiwanese tree Cinnamomum kanehira Hay (Lauraceae) [20]. AC has been used in traditional Chinese medicine for the treatment of food poisoning, drug intoxication, diarrhea, abdominal pain, hypertension, skin irritation, and cancer [21]. There is increasing evidence that AC possesses an extensive range of biological activities, including anticancer, antioxidant, antimetastasis, hepatoprotective, antihypertensive, antihyperlipidemic, immunomodulatory, and anti-inflammatory properties [20–23]. Both the fruiting bodies and mycelium of AC exerted potent anticancer activity against a variety of cancer cells, including breast, liver, bladder, prostate, oral, colon, lung, pancreatic, and leukemic cells [20]. In our earlier studies, we found that the fermented broth of AC induced significant apoptosis and cell cycle arrest in human breast cancer cells (MCF-7 and MDA-MB-231) in vitro or in vivo [24–27]. Despite the emerging evidence of its chemopreventive or chemotherapeutic importance, to date there have been no studies reporting the anticancer potential of AC against melanoma cells. Therefore, we investigated whether AC induced cell cycle arrest and apoptosis in melanoma B16F10 and B16F1 cells in vitro. Because metastasis of melanoma is the leading cause of skin-cancer-related death in humans, in the present study we also assessed the chemotherapeutic effect of AC on the migration and invasion potential of melanoma cells.

2. Materials and Methods

2.1. Reagents

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), L-glutamine and penicillin/streptomycin/neomycin were obtained from GIBCO BRL/Invitrogen (Carlsbad, CA, USA). Anti-rabbit MMP-2, anti-goat MMP-9, anti-mouse VEGF, anti-mouse β-actin, anti-rabbit c-Myc, anti-mouse Bax, anti-rabbit Bcl-2, anti-rabbit p21, anti-mouse p27, anti-mouse β-catenin, anti-rabbit survivin, anti-rabbit p53, anti-rabbit cytochrome c, anti-rabbit caspase-3, and anti-goat Dvl antibodies were purchased from Santa Cruz Biotechnology, Inc. (Heidelberg, Germany). Anti-mouse cyclin D1, anti-mouse caspase-9, anti-mouse CDK4, anti-rabbit PARP, anti-rabbit GSK3β, anti-rabbit phos-GSK3β, anti-rabbit phos-β-catenin, and anti-rabbit histone H3 antibodies were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Protease inhibitor MG132 and GSK3β inhibitor SB216763 were purchased from Merk KGaA (Darmstadt, Germany). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA). All other chemicals were of the highest grade commercially available and were supplied either by Merck or Sigma.

2.2. Preparation of the Fermented Culture Broth of Antrodia Camphorata from Submerged Cultures.

Fermented culture broth of Antrodia camphorata was extracted as described before [26]. Briefly, Antrodia camphorata culture was inoculated on potato dextrose agar and incubated at 30°C for 15–20 days. The whole colony was then put into a flask containing 50 mL sterile water. After homogenization, the fragmented mycelial suspension was used as an inoculum. The seed culture was prepared in a 20 L fermentor (BioTop) agitated at 150 rpm with an aeration rate of 0.2 vvm at 30°C. A five-day culture of 15 L of mycelium inoculum was inoculated into a 250 L agitated fermentor (BioTop). The fermentation conditions were the same as those used for the seed fermentation, but the aeration rate was 0.075 vvm. The fermentation product was harvested at hour 331 and poured through a nonwoven fabric on a 20-mesh sieve to separate the deep-red fermented culture broth and the mycelia, and then centrifuged at 3000 ×g for 10 min followed by passage through a 0.2 μm filter. The culture broth was concentrated under vacuum and freeze-dried to a powder. The yield of dry matter from the culture broth was 18.4 g/L. For preparation of the stock solution, the powder samples were solubilized with DMEM containing 1% FBS (pH 7.4). The stock solution (1.6 mg/mL) was stored at −20°C before evaluation of its antimetastatic properties. The experiments were done using 2~4 different batches of the fermented culture of Antrodia camphorata. The HPLC profile of the fermented culture broth of Antrodia camphorata was performed as previously described [28]. Hereafter, we refer the fermented culture broth of Antrodia camphorata as AC throughout the paper.

2.3. Cell Culture

The B16F1 and B16F10 melanoma cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). These cells were grown in DMEM supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, and 1% penicillin-streptomycin-neomycin at 37°C in a humidified incubator with 5% CO₂. Cultures were harvested and monitored for changes in cell number by counting cell suspensions using a hemocytometer with phase contrast microscopy.

2.4. MTT Assay

Cell viability was determined by the MTT colorimetric assay. Briefly, cells (5 × 10⁴ cells/well in 24-well plates) were treated with various concentrations of AC (40, 80, 120, and 160 μg/mL) for 24 and 48 h, before 400 μL 0.5 mg/mL MTT in PBS was added to each well. After incubation at 37°C for 4 h, an equal volume of 90% isopropanol and 0.5% SDS mixture (400 μL) was added to dissolve the MTT formazan crystals, and the absorbance was measured at 570 nm ($A_{570}$) using an ELISA microplate reader (μ-Quant, Winoosky, VT, USA). The percentage (%) of cell viability was calculated as ($A_{570}$ of treated cells/$A_{570}$ of untreated cells) $\times$ 100.

2.5. Colony Formation Assay

Anchorage-independent growth was determined by colony formation using the soft-agar method. The assay was performed in 6-well plates with a base layer containing 0.5% agar in DMEM containing 10% FBS, 1 mM glutamine, and 100 units of penicillin plus 100 μg/mL of streptomycin. This layer was overlaid with a second layer of 1 mL of 0.35% agar (in DMEM containing 10% FBS, 1 mM glutamine, and 100 units of penicillin plus 100 μg of streptomycin) with a suspension of 1 × 10⁴ cells/well. Fresh medium with AC (0–40 μg/mL) was then added to the plates for 24 h. The plates were incubated at 37°C for 5 days, and the tumor colonies were determined with a microscope. The number of colonies >200 μm in size was counted using an electron microscope (40x magnification). Colonies were subsequently stained with p-iodonitrotetrazolium violet (1 mg/mL), and colonies larger than 200 μm were counted. The percentage of colony formation was calculated by defining the number of colonies in the absence of AC as 100%.

2.6. Luciferase Activity Assay

To determine the transcriptional activity of β-catenin/TCF, a luciferase reporter assay was performed using the TCF reporter constructs TOPFlash and FOPFlash as previously described [15]. Briefly, B16F10 melanoma cells (5 $\times$ 10⁴ cells/well) were seeded in 24-well plates and transfected with either TOPFlash or FOPFlash (100 ng) and the initial control plasmid pRL-TK (5 ng) using lipofectamine 2000 reagent (Invitrogen). TOPFlash and FOPFlash contain wild-type and mutated β-catenin/TCF binding sites, respectively, as well as the thymidine kinase (TK) minimal promoter upstream of the firefly luciferase open reading frame. After transfection, cells were treated with AC (40–160 μg/mL) for 24 h. Cells were then lysed in 350 μL of Triton lysis buffer (50 mM Tris-HCl, 1% (v/v) Triton X-100, 1 mM dithiothreitol, pH 7.8) and centrifuged at 12,000 $\times$g for 2 min at 4°C. Luciferase activity was measured by mixing 20 μL of cell lysate with 20 μL of luciferase reagent (470 μM luciferin, 33.3 mM dithiothreitol, 270 μM coenzyme A, 530 μM ATP, 20 mM Tricine, 1.07 mM (MgCO₃)$\cdots$Mg(OH)₂, 2.67 mM MgSO₄, 0.1 mM EDTA, pH 7.8) and determined with a luminometer (FB15, Zylux Corp., Maryville, TN). Relative β-catenin activity was calculated by dividing the relative luciferase unit (RLU) of treated cells by the RLU of untreated cells.

2.7. Cell Cycle Analysis

Cellular DNA content was determined by flow cytometry using the propidium iodide (PI) labeling method as described previously [23]. Briefly, B16F10 cells were seeded at a density of 4 × 10⁵ cells/dish in 10 cm dishes, and the cell cycle was synchronized by the addition of double thymidine (3 mM) for 16 h. Cell-cycle-synchronized cells were then washed with PBS and re-stimulated to enter the G1 phase together by the addition of fresh medium, which also contained various concentrations of AC (40, 80 and 120 μg/mL). Cells were harvested at 24 h, and the cell cycle analysis was performed using a FAC-Scan cytometry assay kit (BD Biosciences, San Jose, CA, USA) equipped with a single argon ion laser (488 nm). The DNA content of 1 × 10⁴ cells/analysis was monitored using the FACScalibur system. Cell cycle profiles were analyzed with ModFit software (Verity Software House, Topsham, ME, USA).

2.8. Determination of Apoptosis

Apoptotic cell death was measured using terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick end labeling (TUNEL) with the fragmented DNA detection kit (Roche, Mannheim, Germany) as previously described [23]. Briefly, B16F1 or B16F10 cells at a density of 2 × 10⁴ cells/well in 24-well plates were treated with various concentrations of AC (40–80 μg/mL) for 24 h. After AC treatment, cells were fixed in 2% paraformaldehyde for 30 min and then permeabilized with 0.1% Triton X-100 for 30 min at room temperature. Cells were then incubated with TUNEL reaction buffer for 1 h in the dark and then incubated with 4′,6-diamidino-2-phenylindole (DAPI, 1 mg/mL) at 37°C for 5 min; stained cells were visualized under a fluorescence microscope.

2.9. In Vitro Wound-Healing Repair Assay

To assess cell migration, B16F1 or B16F10 cells were seeded into a 12-well culture dish and grown in DMEM containing 10% FBS to a nearly confluent cell monolayer. The cells were resuspended in DMEM medium containing 1% FBS, and the monolayers were carefully scratched using a 200 μL pipette tip. Cellular debris was removed by washing with PBS, and then the cells were incubated with a noncytotoxic concentration of AC (20 and 40 μg/mL) for 24 h. The migrated cells were photographed (100x magnification) at 0 and 24 h to monitor the migration of cells into the wounded area, and the closure of the wounded area was calculated.

2.10. Cell Invasion Assay

Invasion assays were performed using BD Matrigel invasion chambers (Bedford, MA, USA). For the invasion assay, 10 μL Matrigel (25 mg/50 mL) was applied to 8-μm polycarbonate membrane filters, 1 $\times$ 10⁵ cells were seeded to the matrigel-coated filters in 200 μL of serum-free medium containing various concentrations of AC (20 and 40 μg/mL) in triplicate. The bottom chamber of the apparatus contained 750 μL of complete growth medium. Cells were allowed to migrate for 24 h at 37°C. After 24 h incubation, the nonmigrated cells on the top surface of the membrane were removed with a cotton swab. The migrated cells on the bottom side of the membrane were fixed in cold 75% methanol for 15 min and washed 3 times with PBS. The cells were stained with Giemsa stain solution and then destained with PBS. Images were obtained using an optical microscope (200x magnification), and invading cells were quantified by manual counting.

2.11. Protein Isolation and Western Blot Analysis

B16F1 or B16F10 cells were seeded in a 6 cm dish at a density of 1 × 10⁵ cells/dish. Next, the cells were incubated with or without various concentrations of AC for 24 h, detached and washed once in ice-cold PBS, and then resuspended in 100 μL lysis buffer containing 10 mM Tris-HCl [pH 8], 0.32 M sucrose, 1% Triton X-100, 5 mM EDTA, 2 mM dithiothreitol, and 1 mM phenylmethyl sulfonyl fluoride. The suspension was kept on ice for 20 min and then centrifuged at 15,000 $\times$g for 30 min at 4°C. Total protein content was determined using the Bio-Rad protein assay reagent, with bovine serum albumin (BSA) as the standard. Protein extracts were reconstituted in sample buffer (0.062 M Tris-HCl, 2% sodium dodecylsulfate (SDS), 10% glycerol, and 5% β-mercaptoethanol), and the mixture was boiled for 5 min. Equal amounts (50 μg) of the denatured proteins were loaded onto each lane, separated on 8%–15% SDS polyacrylamide gels, followed by transfer of the proteins to polyvinylidene difluoride (PVDF) membranes overnight. Membranes were blocked with 0.1% Tween 20 in PBS containing 5% nonfat dried milk for 20 min at room temperature, and the membranes were reacted with primary antibodies for 2 h. The membranes were then incubated with a horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse antibody for 2 h before development using a chemiluminescence substrate (Millipore, Billerica, MA, USA). Densitometry analyses were performed using commercially available quantitative software (AlphaEase, Genetic Technology Inc. Miami, FL) with the control representing 1.0-fold as shown below the data.

2.12. Fluorescent Imaging of β-Catenin

B16F10 or B16F1 cells at a density of 2 × 10⁴ cells/well were seeded in an 8-well Lab-Tek chamber and treated with various concentrations of AC (40–120 μg/mL) for 24 h. After treatment, cells were fixed in 2% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 10 min, and then incubated for 1 h with anti-β-catenin primary antibodies in 1.5% FBS. FITC (488 nm) secondary antibodies were incubated for another 1 h in 6% BSA. 1 μg/mL DAPI was stained for 5 min. Stained cells were washed with PBS and visualized using a fluorescence microscope at 400x magnification.

2.13. Statistical Analyses

The results are presented as the mean ± standard deviation (mean ± SD). All study data were analyzed using analysis of variance followed by Dunnett’s test for pair-wise comparison. An asterisk indicates that the experimental values were significantly different from those of the controls (*$P<0.05$).

3. Results

3.1. AC Reduces the Viability of Melanoma B16F10 and B16F1 Cells.

The effects of AC on the proliferation of murine melanoma cell lines (B16F10 and B16F1) were investigated. Cells were treated with various concentrations of AC (10–160 μg/mL) for 24 and 48 h. To varying extents, a dose-dependent increase in the rate of growth inhibition was observed with 40–160 μg/mL of AC (Figure 1(a)). AC treatment for 24 and 48 h resulted in a significant ($P<0.05$) cytotoxic effect on both B16F10 and B16F1 melanoma cell lines with an IC₅₀ of 103 and 74 μg/mL, respectively, (Figure 1(a)). At 160 μg/mL for 48 h, AC inhibited >95% of growth in B16F10 and >90% in B16F1 melanoma cells (Figure 1(a)). Furthermore, the colony formation ability (a characteristic of tumor cells that is closely correlated with tumorigenesis in vivo) was assessed to determine the long-term impact of AC on melanoma cell growth. The colony-forming ability of B16F10 and B16F1 cells was significantly ($P<0.05$) as well as dose-dependently suppressed by AC relative to the controls (Figure 1(b)). The reductions in colony number were accompanied by a reduction in colony size in both types of melanoma cells. These data indicate that treatment of melanoma cells with AC may decrease their rate of proliferation and tumor forming ability.

[figures omitted; refer to PDF]

3.2. AC Downregulates the Wnt/β-Catenin Signaling Pathway in Melanoma Cells

Abnormal activation of the Wnt/β-catenin signaling pathway and subsequent upregulation of β-catenin-driven downstream targets including c-Myc, survivin, cyclin D1, CDK4, and metalloproteinases has been detected in a wide range of tumor types, including melanoma [29]. Therefore, we investigated the mechanism of action of growth inhibition by AC in B16F10 and B16F1 melanoma cells. The involvement of Wnt/β-catenin was examined by Western blot and RT-PCR analysis. As shown in Figure 2(a), AC treatment caused a dose-dependent reduction in the total protein content of β-catenin. However, at a concentration of 120 μg/mL of AC 3-fold increased β-catenin phosphorylation at serine 33/34 residues, which eventually lead to proteasomal degradation. A similar pattern of results was also observed from the immunofluorescence assay, indicating that AC treatment dose-dependently inhibited β-catenin expression in B16F10 melanoma cells (Figure 2(b)). In contrast, the gene expression pattern of β-catenin was not affected by AC in either B16F10 or B16F1 cells within the test concentrations (data not shown).

[figure omitted; refer to PDF]

3.3. AC Promotes GSK3β-Mediated Phosphorylation and Subsequent Degradation of β-Catenin

The upstream components of β-catenin, including GSK3β, APC, and axin, form a large multimeric complex that induces phosphorylation and subsequent proteasomal degradation of β-catenin [30]. To further delineate the role of GSK3β and its inhibitor, Dvl, in AC-induced down-regulation of β-catenin, the expression levels of GSK3β and Dvl were evaluated. As shown in Figure 2(a), compared to control cells, AC treatment significantly increased the expression of GSK3β in both B16F10 and B16F1 melanoma cells. However, phosphorylation of GSK3β was significantly inhibited by AC treatment, perhaps due to proteasomal degradation. In addition, AC treatment dose-dependently inhibited the expression of 85 kDa Dvl (Dvl-2), an up-stream regulator of GSK3β in both B16F10 and B16F1 melanoma cells (Figure 2(a)). Furthermore, to examine whether the degradation of β-catenin by AC is 26S proteasome dependent, B16F10 and B16F1 melanoma cells were incubated with a proteasome-specific inhibitor (MG132) in the absence or presence of AC (80 μg/mL). Western blot analyses showed that cells preincubated with MG132 significantly prevented AC-induced β-catenin degradation in both B16F10 and B16F1 melanoma cells (Figure 2(c)). Previous studies demonstrated that proteasomal degradation of β-catenin was mediated by GSK3β in melanoma cells [31]. Therefore, we examined whether AC-induced proteasomal degradation of β-catenin was mediated by GSK3β, B16F10, and B16F1 melanoma cells preincubated with GSK3β-specific inhibitor SB216763 with or without AC (80 μg/mL). Figure 2(c) shows that preincubation of cells with GSK3β inhibitor significantly increased β-catenin expression in B16F10, and B16F1 cells by 1.4-fold and 1.5-fold, respectively. However, the GSK3β inhibitor-induced upregulation of β-catenin was significantly inhibited by AC. Thus, these results suggested that AC-induced degradation of β-catenin occurred through the proteasome pathway.

3.4. AC Inhibits Expression of c-Myc and Survivin in Melanoma Cells

Because β-catenin expression was significantly inhibited by AC, it is logical to speculate that down-regulation of β-catenin’s transcriptional targets, including c-Myc and survivin, may be significant evidence of AC-induced growth inhibition in melanoma cells. To test this hypothesis, the expression levels of c-Myc and survivin were monitored using Western blot analysis. As shown in Figure 2. To further evaluate AC-induced melanoma growth inhibition, the proliferation regulatory proteins and Wnt/β-catenin transcriptional targets c-Myc and survivin were monitored by Western blot analysis. As shown in Figure 2(d), AC downregulated the expression levels of c-Myc and survivin in a dose-dependent manner. In addition, a greater inhibition of c-Myc by AC was observed in B16F10 cells than of B16F1 cells (Figure 2(d)).

3.5. AC Suppressed Transcriptional Activation and Nuclear Translocation of β-Catenin in Melanoma Cells

Transcriptional activation followed by the nuclear translocation of β-catenin is a hallmark of Wnt signaling and is responsible for the transcription of cell growth regulatory genes, including c-Myc and survivin in melanoma cells [32]. Therefore, we performed Western blot and luciferase reporter assays to determine whether the transcriptional activation followed by the nuclear translocation of β-catenin was associated with AC-induced inhibition of c-Myc and survivin. As shown in Figure 3(a), treatment of B16F10 melanoma cells with AC significantly inhibited the transcriptional activation of β-catenin compared with control cells. Furthermore, results of Western blot analyses showed that control cells expressed a greater quantity of β-catenin in both nuclear and cytoplasmic fractions, whereas AC treatment dose-dependently inhibited the accumulation of β-catenin in the nucleus (Figure 3(a)). The reduction of β-catenin in cytoplasmic fraction was also observed in response to AC treatment (Figure 3(a)). To further demonstrate that AC modulated the transcriptional activity of β-catenin in melanoma cells, we used the TOP/FOP luciferase reporter system. As shown in Figure 3(b), the luciferase activity in B16F10 cells transfected with TOP reporter vector was significantly decreased by AC in a dose-dependent manner, whereas cells transfected with the negative control FOP reporter vector were not affected by AC. Taken together, the above results demonstrate that β-catenin is a bona fide target of AC in melanoma cells and that AC downregulated melanoma proliferation by inhibition of β-catenin-mediated transcriptional activity.

[figures omitted; refer to PDF]

3.6. AC Induced G₁ Cell Cycle Arrest in Melanoma Cells

Hence, we hypothesized that the reduced cell proliferation in melanoma cells by AC may be caused by cell cycle arrest. To test this hypothesis, the effect of AC on melanoma cell cycle progression was determined from cellular distribution in the various posttreatment phases. Figure 4(a) shows that exposure of cells to AC resulted in a dose-dependent, progressive, and sustained accumulation of cells in the G₁ phase. Furthermore, in response to AC treatment, the percentage of cells in the G₁ phase gradually increased from 61.7% to 73.9% in B16F10 cells, and from 49.6% to 57.5% in B16F1 cells, whereas the percentage of those in the S and G₂/M phases was significantly decreased (Figure 4(a)).

[figures omitted; refer to PDF]

3.7. AC Downregulates Cyclin D1 and CDK4 and Upregulates p27 and p21 in Melanoma Cells

To further examine the molecular mechanism(s) and underlying changes in cell cycle patterns caused by AC treatment, the expression profile of G₁/S transition phase regulatory proteins, including cyclin D1 and their kinase CDK4, was examined by using Western blot. As shown in Figure 4(b), AC treatment (40–120 μg/mL) for 24 h caused a dose-dependent reduction of cyclin D1 and its up-stream kinase, CDK4, in both B16F10 and B16F1 melanoma cells. Because uncontrolled CDKs activity is often the cause of human cancers, their function is tightly regulated by cell cycle inhibitors such as p27 and p21 proteins. Interestingly, we observed that AC treatment caused a significant increase in both p27 and p21 protein levels in a dose-dependent manner (Figure 4(b)). These data suggest that AC treatment also promotes cell growth inhibition by inducing G₁/S transition phase arrest, followed by the down-regulation of cyclin D1 and CDK4 and upregulation of p27 and p21 expression in melanoma cells.

3.8. AC Induces Apoptosis in Melanoma Cells

The induction of apoptosis (programmed cell death) is a hallmark of the cellular response of many cancer cells to treatment with anticancer drugs. To assess whether AC promotes apoptosis in melanoma cells, AC-induced DNA fragmentation (an apoptotic biomarker) was examined by TUNEL assay. The fragmented DNA can be detected by 3′-OH end labeling of fragmented DNA with dUTP-fluorescein, and TUNEL-positive cells are counted as apoptotic cells [23]. As shown in Figure 5(a), AC caused a dose-dependent induction of apoptosis in melanoma cells. At an AC concentration of 80 μg/mL, apoptotic cells increased by more than 200%, which increased to 250% at 120 μg/mL. This finding directly correlated with the inhibition of cell growth.

[figures omitted; refer to PDF]

3.9. AC-Induced Apoptosis is Mediated by the Caspase-Dependent Mitochondrial Pathway

To investigate the signaling cascade, which mediates AC-induced apoptosis, the mitochondrial release of cytochrome $\text{c}$ was determined by Western blot analysis. As shown in Figure 5(b), AC treatment resulted in a dose-dependent increase in cytoplasmic cytochrome $\text{c}$ while the amount of cytochrome $\text{c}$ in mitochondria was significantly decreased, which indicates that AC caused apoptosis in the mitochondrial pathway. To further delineate the activation of caspases, a downstream effector of cytochrome $\text{c}$ was examined. Figure 5(b) shows that AC treatment caused a significant decrease in the proform of caspase-2 and caspase-9 in both B16F10 and B16F1 melanoma cells. Treatment of melanoma cells with AC also resulted in a dose-dependent reduction in PARP (Figure 5(b)). Moreover, Bcl-2 family proteins, including Bcl-2 and Bax, play an important role in the regulation of apoptosis. Thus, we investigated the effect of AC on the expression of antiapoptotic Bcl-2 and proapoptotic Bax in melanoma cells. After 24 h of treatment, AC (120 μg/mL) caused a significant 3.8-fold increase in Bax protein and a 1.5-fold increase in B16F10 and B16F1 melanoma cells, respectively, whereas a dose-dependent reduction in Bcl-2 protein was observed (Figure 5(b)). Moreover, increased p53 (a proapoptotic protein) expression was also noted in AC-induced B16F10 and B16F1 melanoma cells (Figure 5(b)).

3.10. AC Inhibits Melanoma Migration and Invasion In Vitro

We previously reported that AC potentially blocks tumor cell migration in human breast cancer cells in vitro and in vivo [26, 27]. In this study, we examined whether AC inhibited metastasis of melanoma cells. The highly metastatic murine melanoma B16F10 and B16F1 cells lines were subjected to an in vitro wound healing assay. As shown in Figure 6(a), the migration ability of melanoma cells was significantly restricted by AC (20 and 40 μg/mL). Moreover, the migration potential of B16F10 melanoma cells was comparatively higher than that of B16F1 cells. To further examine the possible role of AC in the prevention of melanoma invasion, B16F10 and B16F1 cells were treated with AC (20 and 40 μg/mL) for 24 h, and the matrigel-based transwell invasion assay was performed. As with the migratory potential, the invasive ability of B16F10 melanoma is also greater than that of B16F1, as directly indicated by the number of invasive cells in the control group. However, treatment of melanoma cells with AC significantly inhibited melanoma invasion (Figure 6(b)). It must be noted that the melanoma migration and invasion assays were performed with noncytotoxic or subcytotoxic concentrations of AC.

[figures omitted; refer to PDF]

3.11. AC Downregulates MMP-2, MMP-9, and VEGF Expression in Melanoma Cells

Aberrant activation of MMPs including MMP-2 and MMP-9 plays an important role in melanoma migration and invasion by stimulating degradation of the extracellular matrix (ECM). Here, we examined whether the anti-invasive potential of AC (10–50 μg/mL) was associated with down-regulation of MMP-2 and MMP-9 expression. As shown in Figure 6(c), AC treatment significantly inhibited the expression of MMP-2 and MMP-9 in a dose-dependent manner. We previously reported that AC treatment significantly blocked VEGF expression in human breast cancer cells [27]. Therefore, it was of interest to examine whether AC (10–50 μg/mL) treatment inhibited VEGF expression in melanoma cells. Figure 6(c) shows that control cells expressed higher amounts of VEGF, whereas AC treatment significantly as well as dose-dependently reduced VEGF expression in both B16F10 and B16F1 melanoma cells.

4. Discussion

No data are currently available that address the possibility of alternative pathways that substitute for β-catenin activity to promote tumor growth and thereby circumvent the attack of therapeutics targeting the Wnt/β-catenin pathway. Tumors are marked by a proliferation disorder and an apoptosis obstacle. The inhibition of proliferation and the induction of apoptosis and suppression of metastasis are currently being employed as criteria by which the efficacy of new therapeutic agents is assessed in preclinical studies [33, 34]. The results of the present study indicate that AC may be an antimelanoma agent in vitro. Our data demonstrated that the efficacy of AC in the inhibition of cell proliferation, G₁ cell cycle arrest, induction of apoptosis, and suppression of metastasis may be through the suppression of the β-catenin pathway in melanoma cells and may provide a new strategy for melanoma treatment.

Increased GSK3β protein stability may hinder the degradation of β-catenin in melanoma cells [18]. Several phytocompounds or plant extracts have been shown to inhibit the proteasome activity of GSK3β and promote β-catenin degradation [20, 35]. This is consistent with our data, which indicate that AC treatment decreased GSK3β phosphorylation, thereby facilitating ubiquitin-dependent proteasomal degradation of β-catenin. A previous report demonstrated that treatment of specific GSK3β inhibitor (SB216763) increased β-catenin activity in melanoma cells [36]. The present data also provide evidence that GSK3β is an upstream target of β-catenin expression and is responsible for β-catenin inhibition by AC. The effects of AC on Wnt/β-catenin-dependent transcription may be important for AC-induced-antitumorigenesis.

In addition, c-Myc was identified as one of the transcriptional targets of β-catenin/Tcf in various cancer cells, suggesting that Wnt signaling functions in oncogenesis, in part, occur through the growth-promoting activity of c-Myc [10]. On the other hand, survivin, a member of the inhibitor of apoptosis (IAP) gene family, is an emerging and therapeutic target in most cancer cells [12]. In addition, survivin has been correlated with tumor aggression and a poor prognosis for many cancers including melanoma [37]. The importance of this effect is clearly indicated by the reduced expression of c-Myc, and survivin proteins play a critical role in tumor cell proliferation and survival [38].

Disturbance of the cancer cell cycle is one of the therapeutic targets for the development of new anticancer agents [39]. Eukaryotic cell cycle progression involves the sequential activation of CDKs, whose activation is dependent upon association with cyclins [40]. It is possible that the antiproliferative effects of AC are due to the inhibition of cyclin D1 expression in melanoma cells. AC inhibits the proliferation of various cancer cell lines, and this inhibition correlates with the down-regulation of the expression of cyclin D1 in most cells [20]. In this study, AC led to a sustained suppression of cyclin D1 levels, a result consistent with the inhibition of G₁/S transition. Moreover, the suppression of cyclin D1 by AC led to the inhibition of CDK4-mediated phosphorylation of Rb in melanoma cells. This result is in accordance with our previous report that AC induces cell cycle arrest through the suppression of cyclin D1 and CDK4 in human breast cancer (MDA MB 453 and BT-474) cell lines [23]. Cell cycle progression in eukaryotic cells is also regulated by the relative balance between the cellular concentrations of CDK inhibitors, including the p27 and p21 proteins of the Cip/Kip family [41]. In fact, p27 was originally identified in cells arrested by transforming growth factor-β. These researchers subsequently found that p27 inhibited the formation of the cyclin-CDK complex from phosphorylation of histone H1. Furthermore, overexpression of p27 was found to prevent CDK activation and entry into the S-transition phase [42]. In addition, p21 has been shown to function as an apoptosis-promoting protein, and the mechanisms by which p21 promotes apoptosis may be related to its interaction with the DNA repair machinery [43]. Previous studies reported that down-regulation of p21 and p27 expression is frequently observed in a variety of human cancers, including lung, breast, prostate, gastric, colon, ovarian, and skin cancers and is usually correlated with poor clinical outcomes [42]. Recently, we demonstrated that p21 and p27 expression was significantly increased in response to AC treatment in MDA-MB-453 human breast cancer cells [23]. Therefore, we suggest that the G₁/S cell cycle arrest and induction of apoptosis by AC may also be mediated by the activation of p21 and p27.

Conventional chemotherapy and radiotherapy may induce apoptosis, which is being investigated as a tool for the management of cancer treatment. Apoptosis is characterized by a number of well-defined features, including cellular morphological changes, chromatin condensation, internucleosomal DNA cleavage, and the activation of caspase cascades [44]. Apoptosis activation occurs mainly through separate but interlinked mitochondrial-dependent (intrinsic) and death-receptor-dependent (extrinsic) apoptosis signaling pathways [45]. Previous studies documented that many conventional therapeutic agents activate both intrinsic and extrinsic pathways in a variety of cancer cells [46]. Recently, we also demonstrated that the induction of apoptosis by AC treatment is mediated by the activation of both mitochondrial and death-receptor pathways [20]. In the present study, TUNEL assays demonstrated that treatment of melanoma cells with AC markedly induced internucleosomal DNA fragmentation, which directly indicates apoptotic cell death. Cells undergoing apoptosis were found to have elevated levels of cytochrome $\text{c}$ in the cytosol, with a corresponding decrease in cytochrome $\text{c}$ in the mitochondria [47]. Cytosolic cytochrome $\text{c}$ activates procaspase-9 by binding to Apaf1 in the presence of dATP, leading to the activation of caspase-9. Subsequently, downstream effector caspases (including caspase-3) are activated, which eventually trigger apoptosis. We observed that the AC-induced apoptosis in melanoma cells was mediated by the activation of caspase-9 cascades, followed by the activation of cytochrome $\text{c}$ release from the mitochondria. In mammalian cells, the Bcl-2 gene family contains a number of antiapoptotic proteins, including Bcl-2 and Bcl-xL, which are thought to be involved in resistance to conventional cancer treatment, while the proapoptotic proteins from the same gene family, including Bax, Bak and Bad, may induce apoptotic cell death [23]. Therefore, apoptosis largely depends on the balance between antiapoptotic and proapoptotic protein levels. We have previously demonstrated that the induction of apoptosis by AC in human breast cancers and leukemia is associated with the upregulation of Bax and down-regulation of Bcl-2 protein expression [23, 48]. Similarly, the present study indicates a dose-dependent inhibition of the antiapoptotic protein Bcl-2 and a concomitant increase in the expression of the proapoptotic protein Bax by AC in melanoma cells. Moreover, PARP, a nuclear protein, was shown to be required for apoptosis to proceed in various cell lines. Prolonged activation of PARP may lead to DNA damage by up-regulating cellular NAD and ATP levels [49]. In this study, we also found that AC treatment significantly reduced the proform of PARP, which indirectly indicates that AC treatment induces PARP activation in melanoma cells.

The most important indicator of the prognosis of malignant melanoma is metastasis to the lymph node or distant organs through lymphatics and/or by hematogenous routes. Any organs may be involved in melanoma metastasis, but the lung and liver are the most common sites [50]. According to data of the American Joint Committee on Cancer (AJCC), nearly 50% of melanoma patients present pathological or clinical evidence of nodal metastases, and the 5- and 10-year survival rates are less than 49% and 37%, respectively, for patients with nodal metastases [51]. Increased expression of MMP-2, MMP-9, and angiogenic cytokine vascular endothelial growth factor (VEGF) in melanoma has been suggested to be associated with the highly metastatic potential of melanoma [52]. Our recent study clearly demonstrated that the anti-invasive and antimetastatic effects of AC against highly metastatic human breast cancer cells (MDA-MB-231) are due to the inhibition of invasion and metastasis regulatory proteins such as MMP-2, MMP-9, uPA, uPA receptor, and VEGF through down-regulation of the MAPK/NF-κB signaling pathway [27]. In the present study we show that AC treatment significantly inhibits melanoma migration and invasion by down-regulating MMP-2, MMP-9 expression. Therefore, AC may inhibit melanoma metastasis through the suppression of MPP-2, MMP-9, and VEGF expression.

Antrodia camphorata has been conventionally used as a traditional Chinese medicine for the prevention of liver diseases and cancers [21, 22]. In recent years, a number of studies have demonstrated the anticancer potential of AC in a variety of cancer cells including breast, lung, liver, ovarian, bladder, and leukemia [20]. The inhibitory effect against cancer cells by AC may be mediated by various cellular mechanisms of actions, such as regulation of oncogene and tumor suppressor gene expression inhibition of metastasis and angiogenesis regulatory proteins down-regulation of signal transduction pathways involving NF-κB, AP-1, Nrf2, and MAPK induction of cell cycle arrest and apoptosis involving the Wnt/β-catenin, p53, death ligands, Bcl-2, and caspase families [20, 22, 23].

Previous studies have shown that naturally derived phytocompounds inhibit the Wnt/β-catenin pathway at both the transcription and translation levels, eventually suppressing tumor growth and dissemination [13]. There are a number of compounds in AC that are predominantly polysaccharides, triterpenoids, steroids, benzenoids, and maleic/succinic acid derivatives ([53] Hseu et al.). The reported yields of polysaccharides, crude triterpenoids, and total polyphenols in the fermented AC broth were 23.2 mg/g, 47 mg/g, and 67 mg/g, respectively, whereas no polysaccharides, crude triterpenoids, or polyphenols were detected in the dry matter of the culture medium [26]. Five lanostanes (sulfuric acid, dehydroeburicoic acid, dehydrosulfurenic acid, 15α-acetyl dehydrosulfurenic acid, and 24-triene-21-oic acid) and three ergostane-type triterpenes (zhankuic acid, zhankuic acid-A, and zhankuic acid-C) were isolated from the fruiting bodies of AC, and they exerted antiproliferative effects against various cancer cell lines in vitro [54]. Lee et al. [55] reported that antroquinonol, a ubiquinone derivative isolated from the solid-state fermented mycelium of AC, showed cytotoxic effects against human breast cancer cells. A similar result was observed in another compound, antrocin, which is also isolated from the fruiting bodies of AC, which exhibited the highest antiproliferative effect against the human breast cancer MDA-MB-231 cell line [56]. In this study, we confirmed the antitumor activity of AC against melanoma cells through its ability to inhibit the Wnt/β-catenin pathway. It is reasonable to suggest that AC metabolizes the culture medium and releases active components during fermentation by submerged culture. The bioassay-directed fractionations leading to the identification and purification of the components responsible for the antimelanoma effect of AC are of further interest.

In conclusion, our data demonstrated that the efficacy of AC in cell growth inhibition, induction of apoptosis, and prevention of metastasis may be due to modulation of the Wnt/β-catenin signaling pathway in melanoma cells. Our results also highlight the importance of the Wnt/β-catenin inhibitors (GSK3β and Dvl) and their transcriptional targets (including c-Myc, survivin, cyclin D1, CDK4, and MMPs), which may serve as future targets for the development of therapeutic strategies against human melanoma. To the best of our knowledge, this is the first report that indicates the anticancer potential of effect of AC against malignant melanoma. However, in vivo studies are highly warranted to confirm the pharmacological efficacy and safety of AC.

Acknowledgments　

This work was supported by Grants NSC-97-2320-B-039-042, NSC-99-2320-B-039-035-MY3, DOH101-TD-C-111-002, NSYSUKMU101-006, and CMU100-ASIA-14 from the National Science Council, Kaohsiung Medical University, National Sun Yat-sen University, Asia University, and China Medical University of Taiwan.

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