Theaflavin-3, 3'-digallate decreases human ovarian carcinoma OVCAR-3 cell-induced angiogenesis via Akt and Notch-1 pathways, not via MAPK pathways - PubMed (original) (raw)

Theaflavin-3, 3'-digallate decreases human ovarian carcinoma OVCAR-3 cell-induced angiogenesis via Akt and Notch-1 pathways, not via MAPK pathways

Ying Gao et al. Int J Oncol. 2016 Jan.

Abstract

Theaflavin-3, 3'-digallate (TF3) is a black tea polyphenol produced from polymerization and oxidization of the green tea ployphenols epicatechin gallate and (-)-epigallocatechin-3-gallate (EGCG) during fermentation of fresh tea leaves. TF3 has been reported to have anticancer properties. However, the effect of TF3 on tumor angiogenesis and the underlying mechanisms are not clear. In the present study, TF3 was verified to inhibit tumor angiogenesis. Compared with EGCG, TF3 was more potent. TF3 inhibited human ovarian carcinoma OVCAR-3 cell-induced angiogenesis in human umbilical vein endothelial cell model and in chick chorioallantoic membrane model. TF3 reduced tumor angiogenesis by downregulating HIF-1α and VEGF. One of the mechanisms was TF3 inactivated Akt/mTOR/p70S6K/4E-BP1 pathway and Akt/c-Myc pathway. Besides, TF3 suppressed the cleavage of Notch-1, subsequently decreased the expression of c-Myc, HIF-1α and VEGF, and finally the impaired cancer cells induced angiogenesis. Nevertheless, TF3 did not have any influence on the MAPK pathways. Taken together, these findings suggest that TF3 might serve as a potential anti-angiogenic agent for cancer treatment.

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Figures

Figure 1

Figure 1

Theaflavin-3, 3′-digallate (TF3) inhibits OVCAR-3 cell-induced angiogenesis by targeting HIF-1α and VEGF. (A) Molecular structure of TF3. (B) Viability of OVCAR-3 cells was decreased after TF3 treatment for 24 h. (C) OVCAR-3 cells-induced HUVEC tube formation was inhibited by TF3 treatment. (D) OVCAR-3 cell-induced blood vessel development in the CAM model was reduced by TF3 treatment. (E) The protein level of VEGF in TF3-treated OVCAR-3 cell culture supernatant was reduced. (F) TF3 diminished the transcriptional activity of VEGF promoter, and, overexpression of HIF-1α reversed it. The data are presented as the mean ± standard error of mean. *P<0.05 compared with control or between specific groups.

Figure 2

Figure 2

Angiogenesis-related proteins are affected by TF3 treatment in OVCAR-3 cells. Western blot analysis revealed that TF3 decreased the protein level of p-Akt, p-mTOR, p-p70S6K, p-4E-BP1, Notch-1 (NICD), c-Myc and HIF-1α in OVCAR-3 cells. TF3 had no impact on the protein level of p-ERK1/2, ERK1/2, JNK, p38 and p-FoxO 1. GAPDH served as the loading control.

Figure 3

Figure 3

Akt/mTOR/p70S6k/4E-BP1 pathway and Akt/c-Myc pathway are involved in TF3-induced inhibition of HIF-1α and VEGF. (A) Western blot analysis showed that 100 nM wortmannin, 10 μM TF3 and 100 nM wortmannin+10 μM TF3 decreased the phosphorylation of Akt, mTOR, p70S6K and 4E-BP1, and expression of c-Myc and HIF-1α. TF3+wortmannin exhibited the strongest effect among them. GAPDH served as the loading control. (B) Luciferase reporter assay and (C) VEGF ELISA showed 100 nM wortmannin, 10 μM TF3 and 100 nM wortmannin+10 μM TF3 suppressed the transcriptional activity of VEGF promoter and VEGF secretion, respectively. TF3+wortmannin elicited strongest effect among them. (D) Overexpression of active Akt attenuated the 15 μM TF3-induced decrease of phosphorylation of Akt, mTOR, p70S6K and 4E-BP1, and expression of c-Myc and HIF-1α. Overexpression of Akt, mTOR, p70S6K or 4E-BP1 attenuated TF3-induced inhibition of transcriptional activity of VEGF promoter (E) and HIF-1α promoter (F). (G) Overexpression of active Akt reversed the 15 μM TF3-induced reduction of VEGF secretion. The data are presented as the mean ± standard error of mean. *P<0.05 compared with control or between specific groups.

Figure 3

Figure 3

Akt/mTOR/p70S6k/4E-BP1 pathway and Akt/c-Myc pathway are involved in TF3-induced inhibition of HIF-1α and VEGF. (A) Western blot analysis showed that 100 nM wortmannin, 10 μM TF3 and 100 nM wortmannin+10 μM TF3 decreased the phosphorylation of Akt, mTOR, p70S6K and 4E-BP1, and expression of c-Myc and HIF-1α. TF3+wortmannin exhibited the strongest effect among them. GAPDH served as the loading control. (B) Luciferase reporter assay and (C) VEGF ELISA showed 100 nM wortmannin, 10 μM TF3 and 100 nM wortmannin+10 μM TF3 suppressed the transcriptional activity of VEGF promoter and VEGF secretion, respectively. TF3+wortmannin elicited strongest effect among them. (D) Overexpression of active Akt attenuated the 15 μM TF3-induced decrease of phosphorylation of Akt, mTOR, p70S6K and 4E-BP1, and expression of c-Myc and HIF-1α. Overexpression of Akt, mTOR, p70S6K or 4E-BP1 attenuated TF3-induced inhibition of transcriptional activity of VEGF promoter (E) and HIF-1α promoter (F). (G) Overexpression of active Akt reversed the 15 μM TF3-induced reduction of VEGF secretion. The data are presented as the mean ± standard error of mean. *P<0.05 compared with control or between specific groups.

Figure 4

Figure 4

Notch-1/c-Myc pathway is related to TF3-induced inhibition of HIF-1α and VEGF. (A) Western blot analysis showed that 10 μM TF3, 75 μM DAPT and 10 μM TF3+75 μM DAPT decreased the expression of NICD, c-Myc and HIF-1α. The dose of 10 μM TF3+75 μM DAPT exhibited the strongest effect. GAPDH served as the loading control. (B) Luciferase reporter assay and (C) VEGF ELISA showed 10 μM TF3, 75 μM DAPT and 10 μM TF3+75 μM DAPT suppressed the transcriptional activity of VEGF promoter and VEGF secretion, respectively. (D) Overexpression of NICD attenuated 15 μM TF3-induced decrease of NICD, c-Myc and HIF-1α. (E) Overexpression of NICD or c-Myc attenuated TF3-induced inhibition of transcriptional activity of VEGF promoter and HIF-1α promoter. (F) Overexpression of NICD or active Akt attenuated TF3-induced inhibition of transcriptional activity of c-Myc promoter. (G) Overexpression of NICD reversed the 15 μM TF3-induced reduction of VEGF secretion. The data are presented as the mean ± standard error of mean. *P<0.05 compared with control or between specific groups.

Figure 5

Figure 5

EGCG decreases VEGF secretion of OVCAR-3 cells. (A) Chemical structure of epicatechin gallate. (B) Chemical structure of EGCG. (C) Viability and (D) VEGF secretion of EGCG-treated OVCAR-3 cells. All data are presented as the mean ± standard error of mean. *P<0.05 compared with the control group.

Figure 6

Figure 6

Proposed mechanism of inhibition of tumor angiogenesis via Akt and Notch-1 pathways.

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