Figure 1
Interactive effects of AKT inhibitor and MEK1/2 inhibitor on EGCG-induced apoptosis. Human umbilical vein endothelial cells (HUVECs) were pretreated with AKT inhibitor-IV (1 μM) or MEK1/2 inhibitor PD98059 (10 μM) for 4 h, followed by treatment with EGCG (40 μM) for 2 h. Cells were harvested and TUNEL assay was performed as per manufacturer's instructions (Promega). Data represent mean ± SD. * = significantly different from control, P < 0.05.
Figure 2
EGCG inhibits migration and capillary tube formation by HUVEC cells. (A) Migration of HUVEC cells was assessed using Transwell Boyden chamber containing a polycarbonated filter. HUVECs (4 × 104 cells) were pretreated with AKT inhibitor IV (1 μM) and/or MEK1/2 inhibitor PD98059 (10 μM) for 2 h, followed by treatment with EGCG (40 μM) or DMSO (control). Migration through the membrane was determined after 24 h of incubation at 37°C. Cells that had migrated to the lower chamber were fixed with 90% methanol, stained with giemsa, quantified by counting the number of cells under a microscope. Data represent mean ± SD. * = significantly different from control, P < 0.05. (B), HUVEC cells were treated as described in A. Cells that had migrated to the lower chamber were fixed with 90% methanol, and photographed with a digital camera attached to a microscope. (C), HUVECs (10 × 104) were seeded in 24-well plates containing matrigel, and pretreated with AKT inhibitor IV (1 μM) and/or MEK1/2 inhibitor PD98059 (10 μM) for 2 h, followed by treatment with EGCG (40 μM) or DMSO (control) for 24 h. Capillary tube structures were photographed with a digital camera attached to a microscope. (D), HUVECs cells were seeded and treated as described in C. Capillary tubes were counted under a microscope. Data represent mean ± SD. * = significantly different from control, P < 0.05.
Figure 3
Phosphorylation deficient mutants of FOXO enhance the inhibitory effects of EGCG on migration and capillary tube formation. (A) HUVEC (4 × 104) cells were transiently transfected with empty vector, FOXO1-TM or FOXO3A-TM along with pCMV-LacZ vector (as transfection control) and treated with or without EGCG (40 μM). Migration through the membrane was determined after 24 h of incubation at 37°C. Cells that had migrated to the lower chamber were fixed with 90% ethanol, stained with Geimsa, quantified by counting the number of cells under a microscope. Data represent mean ± SD. *, ** = significantly different from control, P < 0.05. (B), HUVEC (4 × 104) cells were transiently transfected with empty vector, FOXO1-TM or FOXO3A-TM along with pCMV-LacZ vector (as transfection control) and treated with or without EGCG (40 μM) for 24 h. Capillary tubes were counted under a microscope. Data represent mean ± SD. *, ** = significantly different from control, P < 0.05.
Figure 4
Inhibition of PI3K/AKT and MEK/ERK pathways synergistically enhanced EGCG-induced FOXO activity in HUVEC cells. HUVEC cells were transiently transfected with 6X DBE-luciferase and pRL-TK plasmids for 24 h [69]. After transfection, HUVEC cells were pretreated with AKT inhibitor IV (1 μM) and/or MEK1/2 inhibitor PD98059 (10 μM) for 2 h, followed by treatment with or without EGCG (40 μM) for 24 h. Cells were harvested for firefly/Renilla luciferase assays using the Dual-Luciferase Reporter Assay System (Promega). Luciferase counts were normalized using Renilla luciferase transfection control. Data represent the mean ± S.D. *, #, ** = significantly different from respective controls, P < 0.05.
Figure 5
Phosphorylation deficient mutants of FOXO enhance EGCG-induced FOXO transcriptional activity in HUVEC cells. HUVEC cells were transiently transfected with empty vector or constructs encoding FOXO1-TM, FOXO3a-TM, or FOXO4-TM together with 6X DBE-luciferase and pRL-TK plasmids for 24 h [69]. Luciferase counts were normalized using Renilla luciferase transfection control. After transfection, cells were washed, treated with EGCG (20 μM) for 24 h, and harvested for firefly/Renilla luciferase assays using the Dual-Luciferase Reporter Assay System (Promega). Data represent the mean ± S.D. *, #, ** = significantly different from respective controls, P < 0.05.