Novel angiogenesis inhibitory activity in cinnamon extract blocks VEGFR2 kinase and downstream signaling

Abstract

As a critical factor in the induction of angiogenesis, vascular endothelial growth factor (VEGF) has become an attractive target for anti-angiogenesis treatment. However, the side effects associated with most anti-VEGF agents limit their chronic use. Identification of naturally occurring VEGF inhibitors derived from diet is a potential alternative approach, with the advantage of known safety. To isolate natural inhibitors of VEGF, we established an in vitro tyrosine kinase assay to screen for diet-based agents that suppress VEGFR2 kinase activity. We found that a water-based extract from cinnamon (cinnamon extract, CE), one of the oldest and most popular spices, was a potent inhibitor of VEGFR2 kinase activity, directly inhibiting kinase activity of purified VEGFR2 as well as mitogen-activated protein kinase- and Stat3-mediated signaling pathway in endothelial cells. As a result, CE inhibited VEGF-induced endothelial cell proliferation, migration and tube formation in vitro, sprout formation from aortic ring ex vivo and tumor-induced blood vessel formation in vivo. Depletion of polyphenol from CE with polyvinylpyrrolidone abolished its anti-angiogenesis activity. While cinnamaldehyde, a component responsible for CE aroma, had little effect on VEGFR2 kinase activity, high-performance liquid chromatography-purified components of CE, procyanidin type A trimer (molecular weight, 864) and a tetramer (molecular weight, 1152) were found to inhibit kinase activity of purified VEGFR2 and VEGFR2 signaling, implicating procyanidin oligomers as active components in CE that inhibit angiogenesis. Our data revealed a novel activity in cinnamon and identified a natural VEGF inhibitor that could potentially be useful in cancer prevention and/or treatment.

Fig. 1.

CE inhibits VEGFR2 kinase activity. (A) VEGFR2 was incubated with various concentrations of CE and substrate phosphorylation was monitored by enzyme-linked immunosorbent assay. Data are represented as percentage of control (not treated with CE) and are mean ± SD from four experiments. (B) Lineweaver–Burk plot and (C) Dixon plot of the inhibition of VEGFR2 by CE. Increased concentrations of adenosine triphosphate (ATP) were incubated with VEGFR2 and various concentration of CE. (D and E) CE inhibits VEGFR2 signaling. Quiescent HUVECs were incubated in the presence or absence of CE followed by stimulation with VEGF for another 5 min (for VEGFR2) or 2 h (for MAPK, Stat3, Jak2 and Src). Phosphorylation of VEGFR2, MAPK, Stat3, Jak2 and Src was assessed by western blot. β-Actin/β-tubulin level was used as a loading control. Results are representative of two to four experiments.

Fig. 2.

CE inhibits endothelial cell proliferation. (A) HUVECs were treated with various concentrations of CE, stimulated with VEGF then cell number was assessed 48 h later. Data are mean ± SD (n = 3). *P < 0.05 versus control with VEGF and ^#P < 0.05 versus control without VEGF. (B) MDA-MB-231, HT29 and MCF-10A cells were treated with various concentrations of CE and cells counted 48 h later. Data are represented as percentage of vehicle-treated control. (C) HUVEC were treated with various concentrations of CE and then labeled with BrdU. DNA synthesis was measured by BrdU cell proliferation enzyme-linked immunosorbent assay. Data are represented as percentage of vehicle control and are mean ± SD (n = 3). *P < 0.05 versus control. (D) CE-induced endothelial cell apoptosis. HUVECs were treated with various concentration of CE then labeled with annexin V. Data represent percentage of apoptotic cells and are mean ± SD (n = 3). *P < 0.05 versus control.

Fig. 3.

CE inhibits endothelial cell migration and tube formation. (A) CE inhibits endothelial cell migration. Migration assay of HUVEC treated with various concentrations of CE then treated with VEGF; percentage of migrating cells was examined by microscopy. Data represent percentage of control (without CE + VEGF) and are mean ± SD (n = 3). *P < 0.05 versus control (without CE treatment) with VEGF present. (B) CE inhibits endothelial tube formation. HUVECs were plated on Matrigel in the presence of VEGF with or without CE and photographed under phase-contrast after 16 h. Results are representative of three preparations. Scale bar, 100 μm.

Fig. 4.

CE inhibits sprout formation from chick aorta. (A) Chick aortic rings were placed in Matrigel and treated with various concentrations of CE in the presence or absence of ECGS. The effect of CE on formation of vessel sprout from various aorta samples was examined on day 3. (B) Inhibitory effect of CE on vessel sprout formation is reversible. Aortic rings were first incubated with ECGS and CE (30 μg/ml) for 48 hr (left panel) and continued to culture with ECGS for another 48 hr after cinnamon was removed (right panel). Scale bar, 100 μm. The images shown here are representative of four experiments.

Fig. 5.

CE inhibits tumor-induced blood vessel formation in mice. (A) MDA-MB-231 cells were mixed with Matrigel in the presence and absence of CE and injected into both flank sites of male severely combined immunodeficient mice (SCID). Hemoglobin levels in the Matrigel plug were quantified after 10 days. Data are mean ± SD (n = 10). *P < 0.05 versus control treatment. (B) Blood vessels in the Matrigel plugs, either without CE (left panel) or with CE (75 μg/ml, right panel), were stained with CD31 antibody. The images shown here are representative of five samples for each condition. Scale bar, 20 μm.

Fig. 6.

Oligomeric procynanidins inhibit VEGFR2 kinase activity. (A–C) PVPP treatment depletes anti-angiogenesis activity in CE. PVPP depletion of procynanidins from CE (see Materials and Methods) resulted in (A) inhibited kinase activity of purified VEGFR2, and data are mean ± SD (n = 3), (B) phosphorylation of VEGFR2 in HUVEC and (C) sprout formation from aortic ring. Scale bar, 100 μm. Experiments were repeated at least twice. (D) Oligomeric procyanidins suppressed kinase activity of purified VEGFR2. Cinnamaldehyde, trimer or tetramer of procyanidins, was incubated with VEGFR2. Data represent the percentage of kinase control (without CE treatment) and are mean ± SD (n = 3). (E) Oligomeric procyanidins inhibited VEGFR2 signaling. HUVECs were incubated in the presence or absence of trimer and tetramer of procyanidins, followed by VEGF stimulation. Phosphorylation of VEGFR-2 or MAPK was assessed by western blot using anti-phospho-VEGFR2 antibody, anti-phospho-MAPK antibody, as well as anti-total VEGFR2 and anti-total MAPK antibody. Experiments were repeated twice.