chrysin and Skin-Neoplasms

Numerous evidences have shown that chrysin induced cytotoxic effects via induced cell cycle arrest and induction of cell apoptosis in human cancer cell lines, however, no information showed that chrysin inhibited skin cancer cell migration and invasion. In this study, we investigated anti-metastasis mechanisms of chrysin in human melanoma cancer A375.S2 cells in vitro. Under sub-lethal concentrations of chrysin (0, 5, 10, and 15 μM) which inhibits cell mobility, migration and invasion of A375.S2 cells that were assayed by wound healing and Transwell filter. That chrysin inhibited MMP-2 activity in A375.S2 cells was investigated by gelatin zymography assay. Western blotting was used to examine protein expression and results indicated that chrysin inhibited the expression of GRB2, SOS-1, PKC, p-AKT (Thr308), NF-κBp65, and NF-κBp50 at 24 and 48 hours treatment, but only at 10-15 μM of chrysin decreased Ras, PI3K, p-c-Jun, and Snail only at 48 hours treatment and only decrease p-AKT(Ser473) at 24 hours treatment. Furthermore, chrysin (5-15 μM) decreased the expression of uPA, N-cadherin and MMP-1 at 24 and 48 hours treatment but only decreased MMP-2 and VEGF at 48 hours treatment at 10-15 μM and 5-15 μM of chrysin, respectively, however, increased E-cadherin at 5-15 μM treatment. Results of confocal laser microscopy systems indicated that chrysin inhibited expression of NF-κBp65 in A375.S2 cells. Based on these observations, we suggest that chrysin can be used in anti-metastasis of human melanoma cells in the future.

Topics: Apoptosis; Cell Culture Techniques; Cell Line, Tumor; Cell Movement; Cell Survival; Flavonoids; Humans; MAP Kinase Signaling System; Matrix Metalloproteinase 2; Melanoma; Neoplasm Invasiveness; NF-kappa B; Skin Neoplasms

Mitogen- and stress-activated kinase 1 (MSK1) is a nuclear serine/threonine protein kinase that acts downstream of both extracellular signal-regulated kinases and p38 mitogen-activated protein kinase in response to stress or mitogenic extracellular stimuli. Increasing evidence has shown that MSK1 is closely associated with malignant transformation and cancer development. MSK1 should be an effective target for cancer chemoprevention and chemotherapy. However, very few MSK1 inhibitors, especially natural compounds, have been reported. We used virtual screening of a natural products database and the active conformation of the C-terminal kinase domain of MSK1 (PDB id 3KN) as the receptor structure to identify chrysin and its derivative, compound 69407, as inhibitors of MSK1. Compared with chrysin, compound 69407 more strongly inhibited proliferation and 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced neoplastic transformation of JB6 P+ cells with lower cytotoxicity. Western blot data demonstrated that compound 69407 suppressed phosphorylation of the MSK1 downstream effector histone H3 in intact cells. Knocking down the expression of MSK1 effectively reduced the sensitivity of JB6 P+ cells to compound 69407. Moreover, topical treatment with compound 69407 before TPA application significantly reduced papilloma development in terms of number and size in a two-stage mouse skin carcinogenesis model. The reduction in papilloma development was accompanied by the inhibition of histone H3 phosphorylation at Ser10 in tumors extracted from mouse skin. The results indicated that compound 69407 exerts inhibitory effects on skin tumorigenesis by directly binding with MSK1 and attenuates the MSK1/histone H3 signaling pathway, which makes it an ideal chemopreventive agent against skin cancer.

Topics: Animals; Anticarcinogenic Agents; Cell Cycle; Cell Proliferation; Cell Transformation, Neoplastic; Flavones; Flavonoids; Histones; Male; Mice; p38 Mitogen-Activated Protein Kinases; Papilloma; Phenethylamines; Phosphorylation; Protein Structure, Tertiary; Ribosomal Protein S6 Kinases, 90-kDa; Signal Transduction; Skin; Skin Neoplasms; Tetradecanoylphorbol Acetate

Chrysin (5,7-dihydroxyflavone), a natural flavonoid widely distributed in plants, reportedly has chemopreventive properties against various cancers. However, the anticancer activity of chrysin observed in in vivo studies has been disappointing. Here, we report that a chrysin derivative, referred to as compound 69407, more strongly inhibited EGF-induced neoplastic transformation of JB6 P(+) cells compared with chrysin. It attenuated cell cycle progression of EGF-stimulated cells at the G1 phase and inhibited the G1/S transition. It caused loss of retinoblastoma phosphorylation at both Ser-795 and Ser-807/811, the preferred sites phosphorylated by Cdk4/6 and Cdk2, respectively. It also suppressed anchorage-dependent and -independent growth of A431 human epidermoid carcinoma cells. Compound 69407 reduced tumor growth in the A431 mouse xenograft model and retinoblastoma phosphorylation at Ser-795 and Ser-807/811. Immunoprecipitation kinase assay results showed that compound 69407 attenuated endogenous Cdk4 and Cdk2 kinase activities in EGF-stimulated JB6 P(+) cells. Pulldown and in vitro kinase assay results indicated that compound 69407 directly binds with Cdk2 and Cdk4 in an ATP-independent manner and inhibited their kinase activities. A binding model between compound 69407 and a crystal structure of Cdk2 predicted that compound 69407 was located inside the Cdk2 allosteric binding site. The binding was further verified by a point mutation binding assay. Overall results indicated that compound 69407 is an ATP-noncompetitive cyclin-dependent kinase inhibitor with anti-tumor effects, which acts by binding inside the Cdk2 allosteric pocket. This study provides new insights for creating a general pharmacophore model to design and develop novel ATP-noncompetitive agents with chemopreventive or chemotherapeutic potency.

Topics: Allosteric Regulation; Animals; Binding Sites; Carcinoma, Squamous Cell; Cell Line, Tumor; Crystallography, X-Ray; Cyclin-Dependent Kinases; Epidermal Growth Factor; Flavonoids; G1 Phase; Humans; Mice; Mice, Inbred BALB C; Mice, Nude; Models, Molecular; Neoplasm Transplantation; Protein Kinase Inhibitors; Retinoblastoma Protein; S Phase; Skin Neoplasms