Shikonin Suppresses Lymphangiogenesis via NF-kappa B/HIF-1 alpha Axis Inhibition

Lymphangiogenesis, the formation of lymphatic vessels from preexisting ones, promotes cancer growth and metastasis. Finding natural compounds with anti-lymphangiogenic activity will be useful for preventive treatment of lymphatic metastasis. Shikonin, an ingredient of a traditional Japanese and Chinese medicinal herb Lithospermum erythrorhizon, has been widely used in several pharmaceutical and cosmetic preparations, as well as in food colorants. Shikonin has been reported to inhibit lymphangiogenesis in vitro, but the mechanism of inhibition has not been determined. The aim of this study is to investigate the mechanism of anti-lymphangiogenesis of shikonin in primary human lymphatic endothelial cells (HMVEC-dLy). Shikonin, at non-toxic concentrations, significantly inhibited cord formation ability of lymphatic endothelial cells in a dose- and time-dependent manner. Western blotting analysis showed that shikonin decreased nuclear factor-kappaB (NF-kappa B) activation, as indicated by phosphorylation and nuclear translocation of NF-kappa B p65, and also reduced both mRNA and protein levels of hypoxia-inducible factor-1 (HIF-1)alpha. Use of an NF-kappa B inhibitor (BAY 11-7085) and HIF-1 alpha small interfering RNA (siRNA) transfection revealed that NF-kappa B activation was upstream of HIF-1 alpha expression, which controls cord formation by HMVEC-dLy. In addition, the reduction of vascular endothelial growth factor C (VEGF-C) and vascular endothelial growth factor receptor-3 (VEGFR-3) mRNA levels were also found in HMVEC-dLy that treated with shikonin. In conclusion, shikonin inhibits lymphangiogenesis in vitro by interfering the NF-kappa B/HIF-1 alpha pathway and involves in suppression of VEGF-C and VEGFR-3 mRNA expression.