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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 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.
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pone-0047516-g002: 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.

Mentions: Effect of quercetin on the chemotactic motility of HUVECs is shown in Figure 2a. HUVECs migrated into the clear area when stimulated with chemoattractant, VEGF. Quercetin significantly inhibited the VEGF induced migration of endothelial cells in a dose dependent manner and maximum inhibition of endothelial cell migration was observed at 40 µmol/L and was almost similar to that of zero hour incubation. This concentration is non-toxic as is evident from MTT assay (Fig. 1b) and hence the inhibitory effect could not be attributed to cytotoxic activity. HUVECs showed a high invasive property through the collagen matrix when stimulated with VEGF (Fig. 2b). Treatment of quercetin produced a significant inhibition in the invasion of the collagen matrix by HUVECs in a dose dependent manner. The tubular formation of endothelial cells is also a key step of angiogenesis [27]. Treatment of HUVECs with quercetin significantly inhibited tube formation (Fig. 2c). Incubation of HUVECs on matrigel with VEGF resulted in the formation of elongated and tube like structures. Quercetin effectively reduced the width and length of endothelial tubes at 20 and 40 µmol/L.


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 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.
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pone-0047516-g002: 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.
Mentions: Effect of quercetin on the chemotactic motility of HUVECs is shown in Figure 2a. HUVECs migrated into the clear area when stimulated with chemoattractant, VEGF. Quercetin significantly inhibited the VEGF induced migration of endothelial cells in a dose dependent manner and maximum inhibition of endothelial cell migration was observed at 40 µmol/L and was almost similar to that of zero hour incubation. This concentration is non-toxic as is evident from MTT assay (Fig. 1b) and hence the inhibitory effect could not be attributed to cytotoxic activity. HUVECs showed a high invasive property through the collagen matrix when stimulated with VEGF (Fig. 2b). Treatment of quercetin produced a significant inhibition in the invasion of the collagen matrix by HUVECs in a dose dependent manner. The tubular formation of endothelial cells is also a key step of angiogenesis [27]. Treatment of HUVECs with quercetin significantly inhibited tube formation (Fig. 2c). Incubation of HUVECs on matrigel with VEGF resulted in the formation of elongated and tube like structures. Quercetin effectively reduced the width and length of endothelial tubes at 20 and 40 µmol/L.

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