Piceatannol, a Natural Analog of Resveratrol, Exerts Anti-angiogenic Efficiencies by Blockage of Vascular Endothelial Growth Factor Binding to Its Receptor

Abstract

Piceatannol is also named as trans-3,4,3',5'-tetrahydroxy-stilbene, which is a natural analog of resveratrol and a polyphenol existing in red wine, grape and sugar cane. Piceatannol has been proved to possess activities of immunomodulatory, anti-inflammatory, antiproliferative and anticancer. However, the effect of piceatannol on VEGF-mediated angiogenesis is not known. Here, the inhibitory effects of piceatannol on VEGF-induced angiogenesis were tested both in vitro and in vivo models of angiogenesis. In human umbilical vein endothelial cells (HUVECs), piceatannol markedly reduced the VEGF-induced cell proliferation, migration, invasion, as well as tube formation without affecting cell viability. Furthermore, piceatannol significantly inhibited the formation of subintestinal vessel in zebrafish embryos in vivo. In addition, we identified the underlying mechanism of piceatannol in triggering the anti-angiogenic functions. Piceatannol was proposed to bind with VEGF, thus attenuating VEGF in activating VEGF receptor and blocking VEGF-mediated downstream signaling, including expressions of phosphorylated eNOS, Erk and Akt. Furthermore, piceatannol visibly suppressed ROS formation, as triggered by VEGF. Moreover, we further determined the outcome of piceatannol binding to VEGF in cancer cells: piceatannol significantly suppressed VEGF-induced colon cancer proliferation and migration. Thus, these lines of evidence supported the conclusion that piceatannol could down regulate the VEGF-mediated angiogenic functions with no cytotoxicity via decreasing the amount of VEGF binding to its receptors, thus affecting the related downstream signaling. Piceatannol may be developed into therapeutic agents or health products to reduce the high incidence of angiogenesis-related diseases.

Keywords: VEGF; VEGFR2; herbal medicine; piceatannol; pure compound; vasculogenesis.

Figure 1

Piceatannol suppresses vascular endothelial growth factor A (VEGF)-induced cell proliferation and binding with VEGF. (A) Cultured endothelial cells (HUVECs) were incubated with a series of concentrations of piceatannol for 48 h and cytotoxicity detection kit plus (LDH) (left panel) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (right panel) assays were performed. HUVECs, seeded into each well of a 96-well plate with cell density set at 5000 cells/well, were treated with piceatannol for 48 h in the presence or absence of VEGF (10 ng/mL). Data are in percentage of control group, as mean ± SEM, where n = 4; p < 0.05 (*); p < 0.01 (**) vs. VEGF-treated group; (B) Chemical structure of piceatannol and protein structure, for performing molecular auto-docking, were downloaded from NCBI-PubChem database and PDB, respectively. The interaction of piceatannol binding to VEGF was demonstrated. VEGF: green; piceatannol: sticks, color of carbon: yellow, oxygen: red, hydrogen: silver; the proposed binding site: purple (right panel). UPLC chromatogram was applied to determine the amount of piceatannol in supernatant after biotinylated VEGF or VEGF (100 ng/mL) in an immunoprecipitation assay by streptavidin magnetic beads, n = 3 (right panel).

Figure 2

Piceatannol inhibits VEGF-mediated endothelial cell migration, cell invasion and tube formation. In cell migration assay, HUEVCs, at a density of 2 × 10⁵/well were seeded into each well of a sterile 12-well plate. A wound located in the middle of cell monolayer was made manually. Pictures representing wounds were taken separately at 0 h and 8 h by using a phase-contrast microscope. Cells were treated with VEGF in the presence or absence of piceatannol. In cell invasion assay, 100 μL of cell cultures suspended in fresh medium was plated onto the upper part of chamber of a piece of a 12-well Transwell plate. In the upper compartment, cells were treated by VEGF with or without piceatannol. In the lower chambers, 500 μL fresh medium was applied. Drug application lasted for 24 h, and cells invaded into the lower chambers were analyzed and quantified by counting cell numbers manually after fixation. In tube formation assay, each well of a 24-well plate was precoated with Matrigel and 1 × 10⁵ endothelial cells were seeded. Cell suspension was incubated with VEGF with or without piceatannol, and the drug treatment lasted for 8 h. Images representing cell tube-like structures were photographed under a phase-contrast microscope. To perform the quantification of pictures, three fields in one picture were randomly determined and formed branching points were analyzed and counted manually. In all cases, VEGF was used at 10 ng/mL. Avastin (200 μg/mL) served as a positive control. Data expressed as mean ± SEM of the percentage of control group, where n = 3; p < 0.05 (*); p < 0.01 (**); p < 0.001 (***) vs. VEGF-treated group. Bar = 40 μm.

Figure 3

Piceatannol suppresses angiogenesis in vivo. Healthy zebrafish embryos were selected and divided into groups randomly. On 1st day of development, embryos were incubated with phenylthiourea (PTU) water containing VEGF (10 ng/mL) in the presence or absence of piceatannol at different concentrations. Avastin (200 μg/mL) served as a positive control. For the negative control, immunoglobulin prepared at the same concentration as Avastin was used here. Drug treatment lasted for 48 h and on the 3rd day development of zebrafish embryos, fish embryo staining was performed. Pictures demonstrating the subintestinal vessels and formation of blood vessels were captured. The branches and area of subintestinal vessels in different groups of drugs at different concentrations were analyzed and quantified by applying Image J software. Data expressed as mean ± SEM of the percentage of control group, where n = 3; p < 0.01 (**); p < 0.001 (***) vs. VEGF-treated group. p < 0.01 (##); p < 0.001 (###) vs. Avastin-treated group, as shown. Bar = 40 μm.

Figure 4

Piceatannol attenuates VEGF-induced VEGFR2 phosphorylation. (A) Endothelial cells were seeded into each well of a 12-well plate with cell density set at 2 × 10⁵ per well and cells were treated by VEGF (10 ng/mL) with or without piceatannol; (B) HUVECs were plated into each well of a 12-well plate with the density set at 2 × 10⁵ cells per well. The cells were treated with VEGF (10 ng/mL) with or without piceatannol. An inhibitor for VEGFR2 (SU5416 at 50 μM) was used. The treatment without SU5416, serving as control, was from values in Figure 4A. After the treatment, cell lysates were collected at different time points. Expressions of phosphorylated and total VEGFR2 proteins at ~210 kDa and ~230 kDa were determined by western blotting analysis. Avastin (200 μg/mL) served as a positive control. Data shown as X Basal, where the control group was set as 1, mean ± SEM, where n = 3; p < 0.05 (*); p < 0.01 (**); p < 0.001 (***) vs. VEGF-treated group; p < 0.05 (#); p < 0.01 (##); p < 0.001 (###) vs. its corresponding control, as shown.

Figure 5

Piceatannol exerts no effects on VEGF-induced VEGFR1 phosphorylation. About 2 × 10⁵ endothelial cells were seeded into each well of a sterile 12-well plate. VEGF, at a concentration of 10 ng/mL, was used to treat cells in the presence or absence of piceatannol. After drug treatment, cell lysates were collected from each well. The phosphorylated VEGFR1 at ~180 kDa was analyzed by western blotting analysis. Avastin (200 μg/mL) served as a positive control. Results are expressed as the fold of change than control (X Basal), where the control group (no drug) was set as 1, mean ± SEM, where n = 3; p < 0.001 (***) vs. VEGF-treated group.

Figure 6

Piceatannol inhibits VEGF-induced phosphorylation of Akt and Erk. HUVECs were plated into each well of a 12-well plate at a cell density of 2 × 10⁵ cells per well. The cells were treated by VEGF (10 ng/mL) with or without piceatannol. Cell lysates were collected after drug treatment at different time points as shown. Phosphorylated and total proteins of (A) ~60 kDa Akt, (B) Erk at ~42 kDa and ~44 kDa, were separately determined by western blotting analysis. Avastin (200 μg/mL) served as a positive control. Data expressed as X Basal, where the control was set as 1, mean ± SEM, where n = 3; p < 0.01 (**); p < 0.001 (***) vs. VEGF-treated group.

Figure 7

Piceatannol attenuates VEGF-triggered reactive oxygen species (ROS) formation. HUVECs with cell density set at 2 × 10⁵ cells per well were seeded into each well of a 12-well plate. After 24 h, cells were treated with VEGF (10 ng/mL) in the presence or absence of piceatannol for 48 h. After the treatment, cells were incubated with DCFH-DA at 37 °C for 30 min and the level of intracellular ROS was further analyzed by laser confocal fluorescent microscopy. Avastin (200 μg/mL) served as a positive control. Data expressed as mean ± SEM of the percentage of change in comparison to control group, where n = 4; p < 0.05 (*); p < 0.01 (**); p < 0.001 (***) vs. VEGF-treated group. p < 0.05 (#); p < 0.01 (##) vs. Avastin-treated group, as shown. Bar = 40 μm.

Figure 8

Piceatannol modulates VEGF-mediated function in colon cancer cells. (A) HT-29 colon cancer cells were treated with VEGF (10 ng/mL) with or without piceatannol or Avastin (200 μg/mL) for 24 h. Transwell^® motility assay was performed. The migrated cancer cells were counted. One of the representative images of the migrated cells is shown. The quantification data are demonstrated in right panel. Bar = 20 μm; (B) HT-29 colon cancer cells were treated with VEGF (10 ng/mL) in the presence or absence of piceatannol for 24 h. Total protein was collected and revealed by western blotting analysis by specific antibodies. GAPDH was used as an internal control. Quantitation (right panel) was done from the band intensity in western blotting (left panel). Avastin (200 μg/mL) served as a positive control. Data expressed as the percentage of control or the fold of change than control (X Basal), where the control group (no drug) was set as 1, mean ± SEM, where n = 3; p < 0.05 (*); p < 0.01 (**); p < 0.001 (***) vs. VEGF-treated group.