The Ashitaba (Angelica keiskei) Chalcones 4-hydroxyderricin and Xanthoangelol Suppress Melanomagenesis By Targeting BRAF and PI3K

Abstract

Malignant melanoma is an aggressive tumor of the skin and still lacks effective preventive and therapeutic treatments. In melanoma, both the BRAF/MEK/ERK and PI3-K/AKT signaling pathways are constitutively activated through multiple mechanisms, which result in cell-cycle progression and prevention of apoptosis. Therefore, the development of novel strategies for targeting BRAF and PI3K are of utmost importance. In this study, we found that Ashitaba (Angelica keiskei) chalcones, 4-hydroxyderricin (4HD) and xanthoangelol (XAG), suppressed melanoma development by directly targeting both BRAFV600E and PI3K, which blocked the activation of downstream signaling. This led to the induction of G₁ phase cell-cycle arrest and apoptosis in melanoma cells. Importantly, 4HD or XAG dramatically attenuated tumor incidence and volume in the BRAF-activated Pten-deficient melanoma mouse model. Our findings suggest that 4HD and XAG are promising chemopreventive or potential therapeutic agents against melanomagenesis that act by targeting both BRAF and PI3K, providing hope for rapid clinical translation. Cancer Prev Res; 11(10); 607-20. ©2018 AACR.

Figure 1. 4HD or XAG binds to and effectively suppresses BRAFV600E and PI3-K activity

(A) The structures of 4HD and XAG. Computer docking model for 4HD or XAG and target kinases, including (B) BRAFV600E and (C) PI3-K. The binding was further confirmed by an in vitro binding assay. (D) SK-MEL-28 cell lysates (500 µg) were incubated with 4HD- or XAG-conjugated Sepharose 4B beads or Sepharose 4B beads alone and the pulled-down proteins were analyzed by Western blotting. (E) 4HD or XAG inhibits BRAFV600E activity in vitro. Reactions were performed in the presence of 10 µCi [γ³²P] ATP with active MEK1, 4HD or XAG (0, 5, 10, or 20 µM) in 40 µL of reaction buffer at 30°C for 30 min. (F) 4HD or XAG inhibits PI3-K kinase activity in vitro. Active kinase PI3-K (p110) and different doses of 4HD, XAG or LY294002, a PI3-K kinase inhibitor, were mixed with the substrate phosphatidylinositol and then incubated with a [γ³²P] ATP mixture. The relative amounts of incorporated radioactivity were visualized by autoradiography.

Figure 2. Effects of 4HD or XAG on growth and target protein expression levels in melanoma cells

(A) 4HD or XAG inhibits anchorage-independent cell growth. Cells (8 × 10³/well) were seeded into 6-well plates with 0.3% Basal Medium Eagle agar containing 10% FBS and different concentrations of 4HD or XAG and then cultured for 2 weeks. Colonies were scored under a microscope using the Image J software program. (B) 4HD or XAG inhibits proliferation of melanoma cells. SK-MEL-5 and SK-MEL-28 cells (1 × 10³ cells/well) were seeded into 96-well plates. The cells were treated with 4HD or XAG. After incubation for 1, 2, 3 or 4 days, viability was estimated using the MTS assay as described in Materials and Methods. (C) SK-MEL-28 (70–80% confluent) melanoma cells were incubated with the indicated concentrations of 4HD or XAG for 24 h and then the expression of target proteins was determined by Western blotting. Data are represented as mean values ± S.D. as determined from 3 independent experiments. The asterisks indicate a significant decrease compared with untreated control cells (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

Figure 3. 4HD or XAG induces G1 arrest in melanoma cells

SK-MEL-28 melanoma cells (2.5 ×10⁴/well) were cultured in 60-mm plates and then synchronized in the G0-phase by serum deprivation. (A) The cells were treated with 15 µM 4HD or XAG or vehicle control for 6, 12, 18, 24 or 48 h. (B) The cells were treated with various concentrations of 4HD, XAG or vehicle control for 18 h. After fixing with ethanol, cells were stained with propidium iodide and the cell-cycle phase was determined by flow cytometry. (C) To determine the effect of 4HD or XAG on G1 phase-associated cyclin D1 protein expression, Western blotting was performed using cyclin D1 and β-actin antibodies.

Figure 4. 4HD or XAG induces apoptosis in melanoma cells

(A and B) SK-MEL-28 cells (2.5 × 10⁵/well) were incubated with 4HD or XAG (5, 10, or 20 µM), or vehicle control for 72 h. Cells were collected and apoptosis was detected using flow cytometry and Annexin V staining. Data are represented as mean values ± S.D. as determined from 3 independent experiments and the asterisks indicate a significant decrease compared with untreated control cells (*, p < 0.05; **, p < 0.01; ***, p < 0.001). (C) The cells were incubated with 20 µM 4HD or XAG or vehicle control for 72 h, then the effect of 4HD or XAG on apoptosis-associated protein expression was determined by western blotting.

Figure 5. Prevention of BRAFV600E/PTEN-null induced melanoma by 4HD or XAG

(A) BRAFV600E/PTEN-null mice (6–8 weeks old) were treated with 2.5 µL of 5 mM 4-HT by local application to the dorsal surface. Mice were randomly assigned to be administered 4HD (10 mg/kg, n = 10), 4HD (50 mg/kg, n = 10), XAG (10 mg/kg, n = 10), XAG (50 mg/kg, n = 10) or solvent control (n = 10). (B) Representative images of solvent control, 4HD- or XAG-treated mice immediately after drug administration are shown at day 47. Tumor size in each of the treatment groups was measured every week. Tumor weight was measured at day 47. (C) Mice were euthanized and melanoma specimens were disrupted and then the expression of p-ERKs, ERKs, GAPDH, p-AKT, and AKT, as well as β-actin was assessed by Western blotting. (D) Melanoma specimens were prepared for staining to detect PCNA, cyclin D1 or Bcl-2 by immunohistochemistry. Scale bars, 25 µm. Data are shown as mean values ± S.E. as determined from 3 independent experiments and the asterisks indicate a significant decrease compared with vehicle control mice (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

Figure 6. 4HD or XAG suppresses melanoma growth in BRAFV600E/PTEN-null mice

(A) Melanoma was initiated in BRAFV600E/PTEN-null mice (6–8 weeks old) by local administration of 2.5 µL of 5 mM 4-HT by local injection to the dorsal skin. At 23 days later, when the animals had readily measurable melanoma lesions, mice were randomly divided into 5 groups that were administered 4HD (10 mg/kg, n = 10), 4HD (50 mg/kg, n = 10), XAG (10 mg/kg, n=10), XAG (50 mg/kg, n = 10) or solvent control (n = 10). (B) Representative images of solvent control, 4HD- or XAG-treated mice immediately after drug administration are shown at day 50. Tumor size in each of the treatment groups was measured every week. Tumor weight was measured at day 50. (C) Mice were euthanized and melanoma specimens were disrupted and then the expression of p-ERKs, ERKs, GAPDH, p-AKT, and AKT as well as β-actin was assessed by Western blotting. (D) Melanoma specimens were prepared for staining for PCNA, cyclin D1 or Bcl-2 by immunohistochemistry. Scale bars, 25 µm. Data are represented as mean values ± S.E. as determined from 3 independent experiments and the asterisks indicate a significant decrease compared with vehicle control mice (*, p < 0.05; **, p < 0.01; ***, p < 0.001).