Figure 1.
Quercetin inhibits the VEGF induced cell proliferation in HUVECs.
(a) Chemical structure of quercetin. (b) Effect of quercetin on HUVECs viability in culture. HUVECs (5000 cells/well) were plated in a 96 well titer plate with different concentrations of quercetin and incubated for 48 h. Relative cell viability was determined by MTT assay. Values are means ± SD (mean of triplicate). *p<0.05 denotes a statistically significant difference from untreated controls. (c) Quercetin inhibits the VEGF induced proliferation of endothelial cells. HUVECs (5000 cells/well) in 96-well flat bottomed titer plate with different concentrations of quercetin and VEGF and incubated for 24 h. Relative cell proliferation was determined by MTT assay. Values are means ± SD (mean of triplicate). *p<0.05 denotes a statistically significant difference from untreated controls; #p<0.05 denotes a statistically significant difference from VEGF control.
Figure 2.
Quercetin inhibits VEGF-induced migration, invasion, and tube formation of endothelial cells.
(a) Quercetin inhibited HUVECs migration. HUVECs were grown into wells of collagen coated 24 well plate dishes to 100% confluence. Cells were starved to inactivate cell proliferation and then wounded by pipette tips. EGM-2 containing 0.5% FBS was added with or without 10 ng/mL VEGF and different dilutions of quercetin. Migrated cells were quantified by manual counting. (b) Quercetin inhibited HUVECs invasion. HUVECs (105 cells/Transwell) along with the indicated concentrations of quercetin were seeded into the upper compartment of invasion chambers. The bottom chambers were filled with EGM-2 supplemented with VEGF. After 24 h incubation, migrated cells were fixed, stained and quantified. (c) Quercetin inhibited the tube formation of HUVECs. HUVECs in medium EGM-2 were seeded into the matrigel layer in 24–well plates with VEGF. Various dilutions of quercetin were added into the wells and incubated for 24 h, cells were fixed, and tubular structures were photographed. Values are means ± SD (mean of triplicate). *p<0.05 denotes a statistically significant difference from untreated controls; #p<0.05 denotes a statistically significant difference from VEGF control.
Figure 3.
Quercetin inhibits ex vivo angiogenesis by CAM and matrigel plug assay and in vitro angiogenesis by rat aortic ring assay.
(a) Quercetin inhibits ex vivo angiogenesis in CAM assay. Fertile leghorn chicken eggs were candled on embryonic day 8; a small opening was made at the top of the live eggs. Quercetin for treatment was mixed with 0.5% methyl cellulose in water and gently placed on the CAM. The eggs were incubated for 48 h and photographed. Blood vessels density was quantified by Image J software and represented as a bar diagram. Values are means ± SD (mean of triplicate). *p<0.05 denotes a statistically significant difference from untreated controls; #p<0.05 denotes a statistically significant difference from 20 and 40µmol/L quercetin. (b) Quercetin inhibits ex vivo angiogenesis in matrigel plug assay. Matrigel plug containg VEGF and quercetin were implanted into the CAM at day 9 of fertilized chicken eggs. After 96 h of incubation, the matrigel plugs were taken out and dispersed in PBS and incubated at 4°C overnight. Hemoglobin levels were determined using Drabkin’s reagent according to manufacturer instructions. Values are means ± SD (mean of triplicate). *p<0.05 denotes a statistically significant difference from untreated controls; #p<0.05 denotes a statistically significant difference from VEGF control. (c) Quercetin inhibits microvessel outgrowth from the rat aortic ring. Dorsal aorta from a freshly sacrificed Sprague–Dawley rat was taken out in a sterile manner and rinsed in ice cold PBS. It was then cut into ∼1 mm long pieces using surgical blade. Each ring was placed in a collagen pre-coated 96-well plate. VEGF, with or without different dilutions of quercetin, was added to the wells. On day 6, the rings were analyzed by phase-contrast microscopy and microvessel outgrowths were quantified and photographed. Values are means ± SD (mean of triplicate). *p<0.05 denotes a statistically significant difference from untreated controls.
Figure 4.
Quercetin inhibits the activation of VEGFR2-mediated signaling pathways in HUVECs.
Quercetin suppressed the activation of VEGFR2 and their down stream AKT/mTOR/p70S6K pathway triggered by VEGF in HUVECs. Proteins from different treatments was tested by western blotting and probed with specific antibodies. Experiments were repeated for three times.
Figure 5.
Quercetin induces cell apoptosis and inhibits the activation of AKT/mTOR/p70S6K pathway in prostate cancer cells.
(a) Quercetin inhibited VEGF secretion in prostate PC-3 cancer cells. VEGF level was estimated by ELISA method. Values are means ± SD (mean of triplicate). *p<0.05 denotes a statistically significant difference from untreated controls. (b) Quercetin inhibited cell viability of prostate PC-3 cancer cells. Cell viability was quantified by MTT assay. Values are means ± SD (mean of triplicate). *p<0.05 denotes a statistically significant difference from untreated controls. (c) Quercetin induced PC-3 cancer cell apoptosis by the cleaved-PARP analysis. PC-3 cells were treated with quercetin for 48 h, and whole cell proteins were analysed by Western blotting with antipoly (ADP-ribose) polymerase (PARP). (d) Quercetin inhibited the activation of AKT/mTOR/p70S6K pathway in PC-3 cells. Proteins from different treatments was tested by western blotting and probed with specific antibodies. Experiments were repeated for three times.
Figure 6.
Quercetin inhibits tumor growth in a xenograft mouse model.
PC-3 cells were injected into 6-week old BALB/cA nude mice (5×106 cells per mouse). After tumors grew to about 100 mm3, mice were treated intraperitoneally with or without quercetin (20 mg/kg/d). (a) Solid tumors in the quercetin treated mice were significantly smaller than those in the control mice. Quercetin significantly reduced (b) tumor volume, and (c) tumor weight, (d) but had no effect on the body weight of mice. Values are means ± SD (mean of triplicate). *p<0.05 denotes a statistically significant difference from untreated controls.
Figure 7.
Quercetin inhibits tumor angiogenesis in vivo by suppressing AKT/mTOR/p70S6K pathway.
(a) Quercetin inhibited the activation of AKT/mTOR/p70S6K pathway in vivo. Proteins from tumor tissue was tested by western blotting and probed with specific antibodies. Experiments were repeated for three times. Quercetin inhibited tumor angiogenesis as evident from (b) CD31 and (c) CD34 immunohistochemistry. Tumor sections (5 µm) were incubated with a rabbit anti-CD31 and mouse anti-CD34 antibodies and were subsequently incubated with biotinylated anti-rabbit/anti-mouse secondary antibody, followed by staining with Vectastain ABC Kit.