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Quercetin inhibits angiogenesis mediated human prostate tumor growth by targeting VEGFR- 2 regulated AKT/mTOR/P70S6K signaling pathways.

Pratheeshkumar P, Budhraja A, Son YO, Wang X, Zhang Z, Ding S, Wang L, Hitron A, Lee JC, Xu M, Chen G, Luo J, Shi X - PLoS ONE (2012)

Bottom Line: Quercetin (20 mg/kg/d) significantly reduced the volume and the weight of solid tumors in prostate xenograft mouse model, indicating that quercetin inhibited tumorigenesis by targeting angiogenesis.Furthermore, quercetin reduced the cell viability and induced apoptosis in prostate cancer cells, which were correlated with the downregulation of AKT, mTOR and P70S6K expressions.Collectively the findings in the present study suggest that quercetin inhibits tumor growth and angiogenesis by targeting VEGF-R2 regulated AKT/mTOR/P70S6K signaling pathway, and could be used as a potential drug candidate for cancer therapy.

View Article: PubMed Central - PubMed

Affiliation: Graduate Center for Toxicology, College of Medicine, University of Kentucky, Lexington, Kentucky, United States of America.

ABSTRACT
Angiogenesis is a crucial step in the growth and metastasis of cancers, since it enables the growing tumor to receive oxygen and nutrients. Cancer prevention using natural products has become an integral part of cancer control. We studied the antiangiogenic activity of quercetin using ex vivo, in vivo and in vitro models. Rat aortic ring assay showed that quercetin at non-toxic concentrations significantly inhibited microvessel sprouting and exhibited a significant inhibition in the proliferation, migration, invasion and tube formation of endothelial cells, which are key events in the process of angiogenesis. Most importantly, quercetin treatment inhibited ex vivo angiogenesis as revealed by chicken egg chorioallantoic membrane assay (CAM) and matrigel plug assay. Western blot analysis showed that quercetin suppressed VEGF induced phosphorylation of VEGF receptor 2 and their downstream protein kinases AKT, mTOR, and ribosomal protein S6 kinase in HUVECs. Quercetin (20 mg/kg/d) significantly reduced the volume and the weight of solid tumors in prostate xenograft mouse model, indicating that quercetin inhibited tumorigenesis by targeting angiogenesis. Furthermore, quercetin reduced the cell viability and induced apoptosis in prostate cancer cells, which were correlated with the downregulation of AKT, mTOR and P70S6K expressions. Collectively the findings in the present study suggest that quercetin inhibits tumor growth and angiogenesis by targeting VEGF-R2 regulated AKT/mTOR/P70S6K signaling pathway, and could be used as a potential drug candidate for cancer therapy.

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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.
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pone-0047516-g003: 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.

Mentions: CAM assay was used to determine the antiangiogenic effect of quercetin ex vivo. CAM revealed highly vascularized structure in the control group (Fig. 3a). Exposure to quercetin (20 and 40 µmol/egg) drastically reduced the vascular density. These results confirmed the anti-angiogenic potential of quercetin through an ex vivo assay.


Quercetin inhibits angiogenesis mediated human prostate tumor growth by targeting VEGFR- 2 regulated AKT/mTOR/P70S6K signaling pathways.

Pratheeshkumar P, Budhraja A, Son YO, Wang X, Zhang Z, Ding S, Wang L, Hitron A, Lee JC, Xu M, Chen G, Luo J, Shi X - PLoS ONE (2012)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3475699&req=5

pone-0047516-g003: 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.
Mentions: CAM assay was used to determine the antiangiogenic effect of quercetin ex vivo. CAM revealed highly vascularized structure in the control group (Fig. 3a). Exposure to quercetin (20 and 40 µmol/egg) drastically reduced the vascular density. These results confirmed the anti-angiogenic potential of quercetin through an ex vivo assay.

Bottom Line: Quercetin (20 mg/kg/d) significantly reduced the volume and the weight of solid tumors in prostate xenograft mouse model, indicating that quercetin inhibited tumorigenesis by targeting angiogenesis.Furthermore, quercetin reduced the cell viability and induced apoptosis in prostate cancer cells, which were correlated with the downregulation of AKT, mTOR and P70S6K expressions.Collectively the findings in the present study suggest that quercetin inhibits tumor growth and angiogenesis by targeting VEGF-R2 regulated AKT/mTOR/P70S6K signaling pathway, and could be used as a potential drug candidate for cancer therapy.

View Article: PubMed Central - PubMed

Affiliation: Graduate Center for Toxicology, College of Medicine, University of Kentucky, Lexington, Kentucky, United States of America.

ABSTRACT
Angiogenesis is a crucial step in the growth and metastasis of cancers, since it enables the growing tumor to receive oxygen and nutrients. Cancer prevention using natural products has become an integral part of cancer control. We studied the antiangiogenic activity of quercetin using ex vivo, in vivo and in vitro models. Rat aortic ring assay showed that quercetin at non-toxic concentrations significantly inhibited microvessel sprouting and exhibited a significant inhibition in the proliferation, migration, invasion and tube formation of endothelial cells, which are key events in the process of angiogenesis. Most importantly, quercetin treatment inhibited ex vivo angiogenesis as revealed by chicken egg chorioallantoic membrane assay (CAM) and matrigel plug assay. Western blot analysis showed that quercetin suppressed VEGF induced phosphorylation of VEGF receptor 2 and their downstream protein kinases AKT, mTOR, and ribosomal protein S6 kinase in HUVECs. Quercetin (20 mg/kg/d) significantly reduced the volume and the weight of solid tumors in prostate xenograft mouse model, indicating that quercetin inhibited tumorigenesis by targeting angiogenesis. Furthermore, quercetin reduced the cell viability and induced apoptosis in prostate cancer cells, which were correlated with the downregulation of AKT, mTOR and P70S6K expressions. Collectively the findings in the present study suggest that quercetin inhibits tumor growth and angiogenesis by targeting VEGF-R2 regulated AKT/mTOR/P70S6K signaling pathway, and could be used as a potential drug candidate for cancer therapy.

Show MeSH
Related in: MedlinePlus