Anti-proliferative effect of tryptanthrin on human microvascular endothelial cells.

<p>HMEC-1 cells (3×10³ cells/well) were cultured overnight in 96-well plates and treated with solvent control (0.1% DMSO) or various concentrations of tryptanthrin for 24 h, 48 h or 72 h. After treatment, (A) the relative cell proliferation was measured using the CyQUANT® NF Cell Proliferation Assay Kit and (B) LDH release was determined using the CytoTox 96® Non-Radioactive Cytotoxicity Assay Kit. The relative cell proliferation and LDH release for the solvent control treatment was set as 100%. Data are expressed as mean ± SD.</p

Tryptanthrin suppressed the Matrigel plug <i>in vivo</i> angiogenesis.

<p>Matrigel mixed with VEGF and heparin or tryptanthrin was injected into the flanks of 6-week-old BALB/c mice (five mice per group). Seven days later, the Matrigel plugs were removed for analysis. (A) Representative appearance of Matrigel plugs. (B) Hemoglobin content of Matrigel plugs from groups of mice was quantified by using QuantiChrom™ Hemoglobin Assay Kit. Data are expressed as mean ± SD. *** p<0.001.</p

Effect of tryptanthrin on the VEGFR2 signalling pathways in human microvascular endothelial cells.

<p>(A) The phosphorylation activity of VEGFR2 in the Tyr1175 phosphorylation site was measured after treatment with tryptanthrin for 4 h using the PathScan® Phospho-VEGFR-2 (Tyr1175) Sandwich ELISA Kit. (B) Protein levels of phosphorylated and unphosphorylated ERK, AKT, JNK and p38 in HMEC-1 cells were measured after treatment with various concentrations of tryptanthrin for 4 h using β-actin as an internal control. (C) The protein intensity was quantified using Quantity One software and the relative protein levels of p-ERK1/2, p-AKT, p-JNK1/2 and p-p38 compared to the corresponding total protein levels were determined. (D) The relative protein levels of p-ERK1 after tryptanthrin treatment. (E) The relative protein levels of p-ERK2 after tryptanthrin treatment. Results represent mean ± SD. ** p<0.01; *** p<0.001.</p

Tryptanthrin inhibited the capillary-like tube formation of human microvascular endothelial cells.

<p>HMEC-1 cells (1.5×10⁵cells/well) were seeded onto Matrigel-coated 24-well plates and incubated with solvent control (0.1% DMSO) or various concentrations of tryptanthrin for 16 h. (A) Representative appearance of HMEC-1 tube formation. (B) The tube formation was quantified by counting the number of branch points of the capillary network. Data are expressed as mean ± SD. *** p<0.001.</p

Sequences of primers used for RT-PCR are based on human genes and shown from 5′ to 3′.

<p>Sequences of primers used for RT-PCR are based on human genes and shown from 5′ to 3′.</p

Chemical structure of tryptanthrin.

<p>Chemical structure of tryptanthrin.</p

The favourable binding position of tryptanthrin with lowest binding free energy in the ATP-binding site of VEGFR2 (PDB code 1YWN) as analyzed by molecular docking study.

<p>(A) The three-dimensional diagram displays the interaction of tryptanthrin (the white stick) to the ATP-binding site of VEGFR2 with the labelled amino acid residue Cys917 which significantly contributed to the binding. (B) The two-dimensional diagram shows the interactions of tryptanthrin to the amino acid residues in the ATP-binding site. Colors of the residues indicate the forms of interactions as follows: van der Waals forces, green; polarity, magenta. Green arrow represents H-bonding with the amino acid main chain.</p