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, 117 (3), 505-15

Green Tea Catechins Inhibit Angiogenesis Through Suppression of STAT3 Activation

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Green Tea Catechins Inhibit Angiogenesis Through Suppression of STAT3 Activation

Hoyee Leong et al. Breast Cancer Res Treat.

Abstract

Previous studies indicate that green tea extract may inhibit breast cancer progression by blocking angiogenesis, although the molecular mechanisms are not well defined. We demonstrate that administration of Polyphenon E (Poly E), a standardized green tea extract, inhibited MDA-MB231 breast cancer and human dermal microvascular endothelial (HMVEC) cell migration and the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinase 9 (MMP9). In addition, Poly E inhibited VEGF-induced neovascularization in vivo. We also demonstrate that Poly E blocked signal transducers and activators of transcription (STAT) signaling by suppressing interferon-gamma (IFN-gamma)-induced gene transcription via IFN-gamma-activating sequence (GAS) elements and downstream STAT3 activation by inhibiting STAT1 and STAT3 dimerization in MDA-MB231 cells. Transient expression of constitutively active STAT3 significantly reduced the inhibitory effect of Poly E on cell migration and VEGF and MMP9 expression. Taken together, these observations indicate that green tea extract inhibits angiogenesis partly through the disruption of STAT3-mediated transcription of genes, including VEGF.

Figures

Fig. 1
Fig. 1
Poly E inhibits invasive behavior and capillary-like structure formation. a Treatment of MDA-MB231 and HMVEC cells by Poly E (>10 ng/µl) and 50 µM EGCG significantly reduced cell invasive behavior by Boyden chamber assay. Microscopic images reflect representative crystal violet staining of migrated MDA-MB231 cells 24 h after catechin treatment. Data points indicate an average of triplicate samples. Bars represent SD. * P < 0.05 by Student’s t test compared to the control group. b Poly E treatment (20–50 ng/µl) inhibited morphologic differentiation of HMVEC cells into capillary-like structures. Microscopic images reflect representative reorganization of endothelial cells into vascular networks 6 h after plating and catechin treatment
Fig. 2
Fig. 2
Poly E inhibits neovascularization in vivo. A matrigel plug assay was performed in C57Bl6 mice treated with Poly E (0.75, 1.5, or 2.5 mg) five times weekly for a duration of 2 weeks. a Cell migration was significantly reduced by all doses of Poly E treatment as indicated by H&E staining of matrigel plug sections. b Poly E treatment significantly inhibited MVD as indicated by reduced CD31 staining of plug sections. Images reflect representative staining of all sections. c The hemoglobin content of matrigel plugs, assayed by Drabkin’s reagent, was reduced by all doses of Poly E. Data points indicate an average of five samples with bars representing SD. *P < 0.05 by Student’s t test compared to the control group
Fig. 3
Fig. 3
Poly E modulates VEGF expression and secretion. MCF-7 and MDA-MB231 cells were treated with Poly E for 24 h. a The release of VEGF into cell culture media, as determined by ELISA assay, was significantly inhibited by Poly E in MCF-7 (10–50 ng/µl) and MDA-MB231 cells (50 ng/µl). b Poly E significantly inhibited VEGF mRNA expression by quantitative RTPCR analysis in a concentration-dependent manner in MDA-MB231 but not in MCF-7 cells. c A similar decrease in VEGF promoter activity was observed by luciferase assay. Bars represent SD. *P < 0.05 by Student’s t test compared to the respective control group.
Fig. 4
Fig. 4
Poly E downregulates the expression and secretion of MMP9. MDA-MB231 and MCF-7 cells were treated with Poly E for 48 h. a MMP9 activity was significantly decreased by Poly E (20–50 ng/µl) as determined by zymography analysis of conditioned media of MDA-MB231-treated cells. MMP9 was loaded in the last lane as an internal positive control. b Poly E inhibited MMP9 mRNA expression by quantitative RTPCR analysis. Bars represent SD. *P < 0.05 by Student‘s t test compared control.
Fig. 5
Fig. 5
Inhibition of STAT3 activation by Poly E. MDA-MB231 and MCF-7 cells were treated with Poly E or EGCG in the presence or absence of 1,000 U/ml IFN-α or IFN-γ. Immunoblotting for total and phosphorylated forms of STAT3 were performed after 24 h of treatment. Actin was detected as an internal loading control.
Fig. 6
Fig. 6
Poly E inhibits interferon-induced STAT1/STAT3. MDA-MB231 and MCF-7 cells were treated for 24 h with Poly E (20 ng/µl) in the presence or absence of IFN-α or IFN-γ. Cell extracts were coimmunoprecipitated with STAT1 or STAT3 antibody and analyzed for STAT1 and STAT3 association by western analysis.
Fig. 7
Fig. 7
Poly E suppresses interferon-induced GAS activation. a MCF-7, and b MDA-MB231, cells were transfected with pGAS-Luc and control β-galactosidase expression reporter plasmids. Cells were treated with vehicle or Poly E in the presence or absence of 1,000 U/ml IFN-α or IFN-γ. Luciferase activity was assayed and reported as the fold induction relative to vehicle-treated control. Bars indicate SD. *P < 0.05 by Student’s t test compared to the respective control group.
Fig. 8
Fig. 8
Poly E mediates antiangiogenic effects through STAT-3 dependent mechanisms. a MDA-MB231 cells transiently transfected with pSTAT3C or control pCMV-Tag1 expression vector were treated with Poly E (20 ng/µl) for 24 h. Quantitative RTPCR analysis demonstrated that constitutive activation of STAT3 blocked the inhibitory effects of Poly E on VEGF and MMP9 gene expression. Results are expressed as fold induction relative to control cells transfected with pCMV-Tag1 in the absence of Poly E treatment. Bars represent SD. *P < 0.05 by Student’s t test compared to the respective control group. b MDA-MB231 cells were plated in Boyden chambers following transient transfection with pSTAT3C or control pCMV-Tag1 expression vector. The suppressive effect of 24 h Poly E (20 ng/µl) treatment on cell migration was inhibited by constitutive STAT3 activation. Data points indicate the percent change in migration relative to control cells transfected with pCMV-Tag1. Bars represent SD. *P < 0.05 by Student’s t test compared to the respective control group.

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