7,8-Dihydroxyflavone (7,8-DHF) was purchased from Tokyo Chemical Industry (Tokyo, Japan). Alpha-melanocyte stimulating hormone (α-MSH), Matrigel^®, and Transwell^® chamber systems were obtained from Sigma-Aldrich (St. Louis, MO, USA), BD Biosciences (San Jose, CA, USA) and Corning Costar (Acton, MA, USA), respectively. L-3,4-dihydroxyphenylalanin (L-DOPA) was obtained from Acros Organics (Geel, Belgium). Anti-MITF, anti-HIF-1α, and anti-VEGF antibodies were purchased from Abcam (Cambridge, MA, USA), BD Bioscience (Bedford, MA, USA) and Santa Cruz Biotechnology (Santa Cruz, CA, USA), respectively. Anti-phospho-cMET, anti-c-MET, anti-phospho-AKT, anti-AKT, anti-phospho-ERK1/2, anti-ERK1/2, and anti-β-actin antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA).

B16F10 melanoma cells were grown in Dulbecco's modified Eagle's medium (Invitrogen, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen). Cells were maintained at 37°C in a humidified 5% CO₂ incubator. For cell growth assay, B16F10 cells were plated at 2×10³ cells/well in 96-well culture plates (SPL Life Sciences Co., Ltd., Gyeonggi, Korea). 7,8-DHF (0–100 μM) was added to each well in the presence of α-MSH (10 nM) and the cells were incubated for 72 h. Cell growth was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay.

Cell viability assay was performed using the trypan blue exclusion method. B16F10 cells were seeded at 1.5×10⁴ cells/well in 24-well culture plates (SPL Life Sciences). 7,8-DHF (0–40 μM) was added to each well in the presence of α-MSH (10 nM) and the cells were incubated for up to 72 h. After 72 h, the cells were stained with trypan blue and counted using a hemocytometer.

B16F10 cells were seeded at 5×10² cells/well in 6-well culture plates (SPL Life Sciences). 7,8-DHF was treated to each well in the presence of α-MSH (10 nM) and the cells were incubated for 10 days until colonies were formed. Cells were fixed with 4% formaldehyde, stained with 0.25% crystal violet for 10 min and washed with double-distilled water.

The confluent monolayer B16F10 cells were scratched using a tip and each well was washed with PBS to remove non-adherent cells. The cells were treated with various concentrations of 7,8-DHF in the presence of α-MSH (10 nM) and then incubated for up to 48 h. The perimeter of the area with a central cell-free zone was confirmed under an optical microscope (Olympus, Center Valley, PA, USA).

Cell invasion was assayed using a Transwell^® chamber system with polycarbonate filter inserts with a pore size of 8.0 μm. The lower side of the filter was coated with 10 μl gelatin (1 mg/ml) and the upper side was coated with 10 μl Matrigel^® (3 mg/ml). B16F10 cells (1×10⁵) were placed in the upper chamber of the filter and 7,8-DHF was added to the lower chamber in the presence of α-MSH (10 nM) and 0.5% BSA. The chamber was incubated at 37°C for 24 h, and the cells were subsequently fixed with methanol and stained with hematoxylin and eosin (H&E). The total number of cells that invaded the lower chamber of the filter was counted using an optical microscope (Olympus).

B16F10 cells (15×10⁴ cells/well) were plated in 12-well culture plates (SPL Life Sciences) and then treated with various concentrations of 7,8-DHF in the presence or absence of α-MSH (10 nM) for 72 h. The cells were then washed with PBS and lysed in 150 μl of 1 M NaOH at 95°C. A total of 100 μl of the lysate was added in 96-well microplate and the absorbance was measured at 405 nm using a microplate spectrophotometer (Thermo Scientific Multiskan^® Spectrum).

Tyrosinase activity was estimated by measuring the rate of L-DOPA oxidation. B16F10 cells were plated in 12-well culture plates at a density of 15×10⁴ cells/well and then treated with various concentrations of 7,8-DHF in the presence or absence of α-MSH (10 nM) for 72 h. The cells were washed with PBS and lysed in 50 mM phosphate buffer (pH 6.8) containing 1% Triton X-100 and 0.1 mM phenylmethylsulfonyl fluoride. Cellular lysates were centrifuged at 12,000 rpm for 20 min at 4°C. The supernatant was collected and the protein content was determined by the Bradford method. The cellular extract was incubated with L-DOPA (1.25 mM) in 25 mM phosphate buffer (pH 6.8) and the absorbance at 475 nm was read until the reaction has finished.

B16F10 cells were treated with various concentrations of 7,8-DHF in the presence or absence of α-MSH (10 nM) and incubated for 15 min. The cells were lysed in 0.1 M HCl, and intracellular cAMP generation was determined using enzyme-linked immunosorbent assay (ELISA) with a cAMP direct immunoassay kit (Abcam, Cambridge, MA, USA) according to the manufacturer's instructions. The cAMP levels were normalized to total protein content.

Cell lysates were separated by 10% SDS-PAGE electrophoresis and the separated proteins were transferred to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA) using standard electroblotting procedures. The blots were blocked and immunolabeled with primary antibodies against MITF, HIF-1α, VEGF, phospho-cMET, c-MET, phospho-AKT, AKT, phospho-ERK1/2, ERK1/2 and β-actin overnight at 4°C. Immunolabeling was detected with an enhanced chemiluminescence (ECL) kit (Bio-Rad Laboratories, Hercules, CA, USA), according to the manufacturer's instructions.

The results are expressed as the mean ± standard error (SE). Student's t-test was used to determine statistical significance between the control and the test groups. A P-value of <0.05 was considered to indicate a statistically significant result.

α-MSH has been reported to elicit a variety of biological responses that bring about melanoma development as well as melanocyte differentiation (22–24). We thus assessed the anticancer activities of 7,8-DHF against malignant melanoma in α-MSH-stimulated B16F10 cells. To determine whether 7,8-DHF affects melanoma cell growth, B16F10 cells were treated with various concentrations of 7,8-DHF for 72 h and the MTT assay was performed. 7,8-DHF dose-dependently inhibited the proliferation of B16F10 with an IC₅₀ value of 9.04 μM (Fig. 2A). Notably, a trypan blue viability test revealed that 7,8-DHF treatment did not show cytotoxic effects up to 40 μM (Fig. 2B). These data indicate that the inhibition of melanoma cell growth by 7,8-DHF resulted from a cytostatic effect. To further evaluate the tumor suppressive effect of 7,8-DHF, we carried out clonogenic assay, which is an in vitro cell survival assay based on the ability of a single cell to grow into a colony. Treatment of B16F10 cells with 7,8-DHF resulted in a remarkable inhibition of the colony-forming ability (Fig. 2C). Taken together, these results suggest that 7,8-DHF may be considered a novel anticancer agent with potent antiproliferative activity against melanoma cells.

The most dangerous aspect of melanoma is its ability to spread to other parts of the body. To assess the inhibitory potential of 7,8-DHF on the metastatic ability of melanoma cells, we first confirmed its effect on migration of α-MSH-stimulated B16F10 cells using wound healing assay. As shown in Fig. 3A, relative to untreated control cells, treatment with 7,8-DHF for 48 h reduced the migration capacity of B16F10 cells in a dose-dependent manner. Next, the effect of 7,8-DHF on invasive potential of α-MSH-treated B16F10 cells was investigated by employing the Matrigel matrix-coated Transwell chamber assay. As shown in Fig. 3B, 7,8-DHF treatment significantly decreased the invasiveness of B16F10 cells. These data demonstrate that 7,8-DHF has the chemotherapeutic potential to suppress melanoma metastasis.

Malignant melanocytes tend to exhibit upregulated melanogenesis and defective melanosomes (25). Cellular tyrosinase activity is the major factor that stimulates melanin synthesis and ultimately induces melanogenesis (26). We thus determined the cellular tyrosinase activity and melanin content in order to investigate the effect of 7,8-DHF on melanogenesis of B16F10 cells. The cells were stimulated by α-MSH in the presence or absence of 7,8-DHF for 72 h. As shown in Fig. 4, treatment with 7,8-DHF clearly blocked the melanin formation and tyrosinase activity of B16F10 cells induced by α-MSH, indicating that 7,8-DHF downregulates the differentiation of melanoma cells associated with melanogenesis.

MITF is not only a key player in melanocyte development but is thought to also function as an oncogene in melanoma (7). α-MSH can increase intracellular cAMP levels and consequently activate MITF expression (14). To elucidate the anticancer mechanisms of 7,8-DHF against melanoma, we investigated the effect of 7,8-DHF on α-MSH/cAMP/MITF signaling network in melanoma cells. As shown in Fig. 5, treatment with 7,8-DHF significantly reduced the intracellular cAMP levels in B16F10 cells stimulated by α-MSH. We next examined the effect of 7,8-DHF on the expression of MITF and its downstream transcription targets. 7,8-DHF markedly decreased the protein levels of MITF in α-MSH-stimulated B16F10 cells (Fig. 6). In addition, the expression of HIF1α and c-MET, the major transcriptional targets of MITF which have been implicated in antiapoptotic and metastatic potential in melanoma, was prominently downregulated by 7,8-DHF treatment. Furthermore, the inhibition of HIF1α resulted in a remarkable reduction in expression of VEGF, its transcriptional target that is required for tumor angiogenesis. In addition, the diminished c-MET levels were also linked to the blockade of c-MET signaling such as inhibiting phosphorylation of c-MET and its downstream effector AKT. However, 7,8-DHF treatment rather increased the phosphorylation of extracellular signal-regulated kinases (ERK1/2), another c-MET downstream effector. Recent studies have revealed that activation of ERK induces phosphorylation and degradation of MITF and consequently downregulates α-MSH-induced melanogenesis by inhibiting tyrosinase activity and expression (27,28). Accordingly, 7,8-DHF-induced activation of ERK1/2 may partly account for the antimelanogenic effect of the compound. These results collectively suggest that 7,8-DHF may exhibit the potential anticancer activity against melanoma through the downregulation of cAMP levels and subsequent suppression of the expression of MITF and its key oncogenic target genes such as HIF1α and c-MET.

Resveratrol is a naturally-occurring compound that possesses anticancer capabilities (29). A recent study demonstrated that resveratrol inhibits MITF expression, viability, and invasiveness activated by α-MSH in melanoma cells (30). To further exploit promising combination therapies of 7,8-DHF, the anticancer effects of 7,8-DHF and resveratrol either alone or in combination were examined in α-MSH-stimulated B16F10 cells. At the concentrations tested, combined treatment with 7,8-DHF and resveratrol more efficiently inhibited the melanoma cell growth compared with single agent treatment (growth inhibition of 43, 31 and 65% with 7,8-DHF, resveratrol, and the 7,8-DHF/resveratrol combination, respectively) (Fig. 7A). Furthermore, combination of the two compounds greatly increased suppression of the melanoma cell invasion compared to either agent alone (invasion inhibition of 28, 27 and 80% with 7,8-DHF, resveratrol, and the 7,8-DHF/resveratrol combination, respectively) (Fig. 7B). Notably, the combined treatment resulted in an enhanced reduction in MITF expression levels compared with inhibition by single agent treatment (Fig. 7C). These findings suggest that 7,8-DHF might be used to treat melanoma growth and metastasis in combination with resveratrol.