Myricetin inhibits UVB-induced angiogenesis by regulating PI-3 kinase in vivo

Abstract

Myricetin is one of the principal phytochemicals in onions, berries and red wine. Previous studies showed that myricetin exhibits potent anticancer and chemopreventive effects. The present study examined the effect of myricetin on ultraviolet (UV) B-induced angiogenesis in an SKH-1 hairless mouse skin tumorigenesis model. Topical treatment with myricetin inhibited repetitive UVB-induced neovascularization in SKH-1 hairless mouse skin. The induction of vascular endothelial growth factor, matrix metalloproteinase (MMP)-9 and MMP-13 expression by chronic UVB irradiation was significantly suppressed by myricetin treatment. Immunohistochemical and western blot analyses revealed that myricetin inhibited UVB-induced hypoxia inducible factor-1alpha expression in mouse skin. Western blot analysis and kinase assay data revealed that myricetin suppressed UVB-induced phosphatidylinositol-3 (PI-3) kinase activity and subsequently attenuated the UVB-induced phosphorylation of Akt/p70(S6K) in mouse skin lysates. A pull-down assay revealed the direct binding of PI-3 kinase and myricetin in mouse skin lysates. Our results indicate that myricetin suppresses UVB-induced angiogenesis by regulating PI-3 kinase activity in vivo in mouse skin.

Fig. 1.

Effects of myricetin on UVB-induced blood vessel formation in SKH-1 hairless mice. Myricetin inhibits UVB-induced neovascularization in SKH-1 hairless mouse skin. (a) Vehicle-treated controls, (b) UVB-irradiated (0.18 J/cm²) mice and UVB plus (c) mice treated with 8 nmol myricetin or (d) mice treated with 20 nmol myricetin. Fifteen mice were treated topically with myricetin (8 or 20 nmol in 200 μl of acetone/mouse) or vehicle as described in Materials and Methods and then irradiated with UVB light 3× a week for 27 weeks. Photographs of blood vessels in the mice were taken after euthanization at the end of the experiment using a Samsung digital camera.

Fig. 2.

The effect of myricetin on UVB-induced VEGF expression in SKH-1 hairless mice. (A and B) Myricetin significantly inhibits UVB-induced VEGF expression in SKH-1 hairless mice. VEGF or β-actin expression was assessed by western blotting using antibodies against VEGF or β-actin as described in Materials and Methods. (B) Each band was densitometrically quantified by image analysis. The results are shown as means ± SEMs (n = 3). The pound (#) symbol indicates a significant difference (P < 0.05) between the control group and the UVB-treated group, whereas the asterisk (*) indicates a significant difference (P < 0.05) between the UVB-irradiated/myricetin-treated and UVB-only irradiated groups.

Fig. 3.

Effects of myricetin on UVB-induced MMP-9 activity and MMP-13 expression in SKH-1 hairless mice. (A) Myricetin inhibits UVB-induced MMP-9 activity in SKH-1 hairless mice. Proteins were extracted from isolated mouse skin samples as described in Materials and Methods. MMP-9 activity was assessed by gelatin zymography as described in Materials and Methods. (B) Myricetin inhibits UVB-induced MMP-13 expression in SKH-1 hairless mice as shown by western blotting using antibodies against MMP-13 or β-actin. Each band was densitometrically quantified by image analysis. The results are shown as means ± SEMs (n = 3). The pound (# and ##) symbols indicate a significant difference (P < 0.05 and P < 0.01, respectively) between the control and UVB-irradiated groups, whereas the asterisks (* and **) indicate a significant difference (P < 0.05 and P < 0.01, respectively) between the UVB-irradiated/myricetin-treated and UVB-only irradiated groups.

Fig. 4.

Effects of myricetin on UVB-induced HIF-1α expression in SKH-1 hairless mice. (A and B) Myricetin inhibits UVB-induced HIF-1α expression in SKH-1 hairless mice. (a) Vehicle-treated controls, (b) UVB-irradiated (0.18 J/cm²) mice and mice treated with UVB plus (c) 8 nmol of myricetin or (d) 20 nmol of myricetin. The mice were treated as described in Figure 1B. Serial sections were mounted on silane-coated slides and immunostained for HIF-1α as described in Materials and Methods. The photos are representative of results from five or six tissue samples. HIF-1α appears brown. HIF-1α or β-actin expression was assessed by western blotting as described in Materials and Methods. Each band was densitometrically quantified by image analysis. The results are shown as means ± SEMs (n = 3). The pound (#) symbol indicates a significant difference (P < 0.05) between the control and UVB-irradiated groups, whereas the asterisk (*) indicates a significant difference (P < 0.05) between the UVB-irradiated/myricetin-treated and UVB-only irradiated groups.

Fig. 5.

Effect of myricetin on UVB-mediated signaling in SKH-1 hairless mouse skin. (A) Myricetin inhibits the phosphorylation of Akt and p70^S6K in SKH-1 hairless mouse skin. (B) Myricetin inhibits the UVB-induced phosphorylation of MEK and ERKs in SKH-1 hairless mouse skin. Proteins were extracted from isolated mouse skin samples as described in Materials and Methods. The phosphorylation of Akt, p70^S6K, MEK and ERKs and level of total Akt, p70^S6K, MEK and ERKs was assessed by western blotting. The results are shown as means ± SEMs (n = 3). The pound (#) symbol indicates a significant difference (P < 0.05) between the control and UVB-irradiated groups, whereas the asterisk (*) indicates a significant difference (P < 0.05) between the UVB-irradiated/myricetin-treated and UVB-only irradiated groups.

Fig. 6.

Effect of myricetin on UVB-induced PI-3 kinase activity in SKH-1 hairless mice. (A) Myricetin inhibits UVB-induced PI-3 kinase activity in a dose-dependent manner. To assay for PI-3 kinase activity, dorsal skin lysates were prepared from the epidermis and subjected to immunoprecipitation and a PI-3 kinase assay as described in Materials and Methods. The results are shown as means ± SEMs (n = 3). The pound (##) symbols indicate a significant difference (P < 0.01) between the control and UVB-irradiated groups; the asterisk (*) indicates a significant difference (P < 0.05) between the UVB-irradiated/myricetin-treated and UVB-irradiated groups. (B) Myricetin binds PI-3 kinase directly in mouse skin lysates. In vivo myricetin binding was confirmed by immunoblotting using an antibody against subunit p110: lane 1 (input control), whole lysate from mouse dorsal skin; lane 2 (control), mouse dorsal skin lysate precipitated with Sepharose 4B beads (see Materials and Methods) and lane 3, whole-cell lysate from mouse dorsal skin precipitated by myricetin–Sepharose 4B affinity beads (see Materials and Methods). (C) Simplified view of the proposed anti-angiogenic mechanism of myricetin.