Mechanism of inhibition of tumor angiogenesis by beta-hydroxyisovalerylshikonin.
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.
Komi
Yusuke
Y
Molecular Ligand Biology Research Team, Chemical Genomics Research Group, Chemical Biology Department, RIKEN Advanced Science Institute, 2-1 Hiroshima, Wako, Saitama, Japan.
Suzuki
Yasuhiro
Y
Shimamura
Mariko
M
Kajimoto
Sachiko
S
Nakajo
Shigeo
S
Masuda
Michitaka
M
Shibuya
Masabumi
M
Itabe
Hiroyuki
H
Shimokado
Kentaro
K
Oettgen
Peter
P
Nakaya
Kazuyasu
K
Kojima
Soichi
S
D020533:Angiogenesis Inhibitors / Q000494:pharmacology*; D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D018827:Carcinoma, Lewis Lung / Q000098:blood supply* / Q000188:drug therapy; D002448:Cell Adhesion; D002465:Cell Movement; D049109:Cell Proliferation; D002478:Cells, Cultured; D002645:Chickens; D049033:Chorioallantoic Membrane / Q000378:metabolism / Q000473:pathology; D004365:Drugs, Chinese Herbal / Q000494:pharmacology*; D024202:Electrophoretic Mobility Shift Assay; D005260:Female; D007124:Immunoenzyme Techniques; D047468:Immunoprecipitation; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D009285:Naphthoquinones / Q000494:pharmacology*; D009389:Neovascularization, Pathologic / Q000517:prevention & control*; D010766:Phosphorylation; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D014162:Transfection; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism; D040281:Vascular Endothelial Growth Factor Receptor-1 / Q000235:genetics / Q000378:metabolism; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000235:genetics / Q000378:metabolism
D020533:Angiogenesis Inhibitors; D004365:Drugs, Chinese Herbal; D009285:Naphthoquinones; D012333:RNA, Messenger; D042461:Vascular Endothelial Growth Factor A; C118460:beta-hydroxyisovalerylshikonin; C467480:vascular endothelial growth factor A, mouse; C016101:shikonin; D008156:Luciferases; D040281:Vascular Endothelial Growth Factor Receptor-1; D040301:Vascular Endothelial Growth Factor Receptor-2