shikonin and Carcinoma--Lewis-Lung

Shikonin and beta-hydroxyisovalerylshikonin (beta-HIVS) from Lithospermum erythrorhizon inhibit angiogenesis via inhibition of vascular endothelial growth factor receptors (VEGFR) in an adenosine triphosphate-non-competitive manner, although the underlying molecular mechanism has not been fully understood. In the present study, we found that beta-HIVS inhibited angiogenesis within chicken chorioallantoic membrane approximately threefold more efficiently than shikonin. beta-HIVS also significantly inhibited angiogenesis in two other assays, induced either by Lewis lung carcinoma cells implanted in mouse dorsal skin or by VEGF in s.c. implanted Matrigel plugs and metastasis of Lewis lung carcinoma cells to lung. Therefore, using beta-HIVS as a bioprobe, we investigated the molecular mechanism of shikonin's anti-angiogenic actions. beta-HIVS inhibited the phosphorylation and expression of VEGFR2 and Tie2 without affecting VEGFR1 and fibroblast growth factor receptor 1 levels. beta-HIVS suppressed the phosphorylation but not the expression of extracellular signal-regulated kinase, and an Sp1-dependent transactivation of the VEGFR2 and Tie2 promoters, thereby suppressing the proliferation of vascular endothelial and progenitor cells. This was mimicked by an Sp1 inhibitor mithramycin A and partially rescued by Sp1 overexpression. These results implicate potential use of shikonin and beta-HIVS as leading compounds for clinical application in the future by virtue of their unique properties including: (i) inhibition of VEGFR2 and Tie2 phosphorylation in an adenosine triphosphate-non-competitive manner; (ii) simultaneous inhibition of the phosphorylation and expression of VEGFR2 and Tie2; and (iii) bifunctional inhibition of the growth in endothelial cells and vascular remodeling.

Topics: Angiogenesis Inhibitors; Animals; Apoptosis; Blotting, Western; Carcinoma, Lewis Lung; Cell Adhesion; Cell Movement; Cell Proliferation; Cells, Cultured; Chickens; Chorioallantoic Membrane; Drugs, Chinese Herbal; Electrophoretic Mobility Shift Assay; Female; Immunoenzyme Techniques; Immunoprecipitation; Luciferases; Mice; Mice, Inbred C57BL; Naphthoquinones; Neovascularization, Pathologic; Phosphorylation; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Transfection; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factor Receptor-1; Vascular Endothelial Growth Factor Receptor-2

Lithospermum erythrorhizon has been used for treatment of inflammatory diseases and cancer as a folk remedy. Based on the evidences that anti-inflammatory agents frequently exert antiangiogenic activity, thus we examined comparatively the antiangiogenic activities of three naphthoquinone derivatives (shikonin, acetylshikonin, and isobutyroylshikonin) isolated from the plant. Three derivatives exhibited weak cytotoxicity against human umbilical vein endothelial cells (HUVECs) with IC50 of over 20 microM. Shikonin had more specific inhibitory effects on proliferation and vascular endothelial growth factor (VEGF) production by VEGF compared with different derivatives. All of derivatives significantly suppressed the migration of VEGF treated HUVECs at different optimal concentrations. Also, shikonin and acetylshikonin significantly disrupted VEGF-induced tube formation. Furthermore, three derivatives effectively downregulated the expression of urokinase-type plasminogen activator (uPA), but not its receptor uPAR. Additionally, shikonin significantly inhibited tumor growth in LLC-bearing mice, whereas its derivatives had relatively mild effects. Taken together, our findings suggest that shikonin and its derivatives exhibit the antiangiogenic and antitumorigenic effects by suppressing proliferation and angiogenic factors.

Topics: Animals; Anthraquinones; Anti-Inflammatory Agents, Non-Steroidal; Carcinoma, Lewis Lung; Cell Movement; Cells, Cultured; Depression, Chemical; Dose-Response Relationship, Drug; Down-Regulation; Endothelial Cells; Female; Humans; Lithospermum; Mice; Naphthoquinones; Neovascularization, Pathologic; Urokinase-Type Plasminogen Activator; Vascular Endothelial Growth Factor A