(-)-Epigallocatechin-3-gallate inhibits growth of gastric cancer by reducing VEGF production and angiogenesis

Abstract

Aim: To investigate the effect of (-)-epigallocatechin-3-gallate (EGCG) on growth of gastric cancer and its possible mechanism.

Methods: Heterotopic tumors were induced by subcutaneously injection of SGC-7901 cells in nude mice. Tumor growth was measured by calipers in two dimensions. Tumor angiogenesis was determined with tumor microvessel density (MVD) by immunohistology. Vascular endothelial growth factor (VEGF) protein level and activation of signal transducer and activator of transcription 3 (Stat3) were examined by Western blotting. VEGF mRNA expression was determined by RT-PCR and VEGF release in tumor culture medium by ELISA. VEGF-induced cell proliferation was studied by MTT assay, cell migration by gelatin modified Boyden chamber (Transwell) and in vitro angiogenesis by endothelial tube formation in Matrigel.

Results: Intraperitoneal injection of EGCG inhibited the growth of gastric cancer by 60.4%. MVD in tumor tissues treated with EGCG was markedly reduced. EGCG treatment reduced VEGF protein level in vitro and in vivo. Secretion and mRNA expression of VEGF in tumor cells were also suppressed by EGCG in a dose-dependent manner. This inhibitory effect was associated with reduced activation of Stat3, but EGCG treatment did not change the total Stat3 expression. EGCG also inhibited VEGF-induced endothelial cell proliferation, migration and tube formation.

Conclusion: EGCG inhibits the growth of gastric cancer by reducing VEGF production and angiogenesis, and is a promising candidate for anti-angiogenic treatment of gastric cancer.

Figure 1

Inhibition of growth of human gastric cancer xenografts in an athymic mouce model. A: tumor tissues treated with EGCG or PBS on d 28 after treatment; B: an average of 60.4% suppression of primary tumor growth in the EGCG treatment group compared with control group; C: tumor growth suppression curve: volumes of EGCG treatment group versus PBS treatment group on indicated days. Data are presented as mean ± SE (n = 6, ^aP < 0.05, ^bP < 0.01).

Figure 2

MVD in tumor tissues determined by a specific antibody against mouse endothelial CD34 in PBS group (A) and EGCG group (B) (200 ×) while quantitative analysis showing significant reduction of tumor MVD after EGCG treatment (C). Data are presented as mean ± SE (n = 6, ^bP < 0.01).

Figure 3

Suppression of VEGF expression and Stat3 activation in tumor cells and tissues by EGCG. Protein level of VEGF and phosphorylated Stat3 was dose-dependently reduced in tumor cells treated with EGCG and also markedly in EGCG treated tumor tissues. EGCG did not change expression of Stat3 in tumor cells or tumor tissues.

Figure 4

Decreased VEGF secretion (A) and inhibition of of VEGF mRNA expression (B) in tumor cells. SGC-7901 cells were treated with various concentrations of EGCG for 24 h and VEGF protein level in the supernatant was measured by ELISA and mRNA expression in tumor cells was examined by RT-PCR. Data are presented as mean ± SE (n = 3, ^aP < 0.05, ^bP < 0.01).

Figure 5

Inhibitory effects of EGCG on VEGF-induced proliferation of endothelial cells. HUVECs were treated with 20 ng/mL VEGF and EGCG at the indicated concentration and time. EGCG time- and dose-dependently inhibited HUVEC proliferation induced by VEGF. Values are expressed as percent of control (means ± SE, n = 3).

Figure 6

EGCG inhibits endothelial cell migration induced by VEGF. EGCG inhibited migration of HUVECs induced by VEGF in a dose-dependent manner. Data are presented as mean ± SE (n = 3). VEGF-induced migration without EGCG treatment was used as a control. Values significantly lower than control are indicated (^aP < 0.05, ^bP < 0.01).

Figure 7

Inhibition of EGCG on VEGF-induced tube formation of endothelial cells. EGCG dose-dependently inhibited tube formation induced by VEGF on Matrigel. Data are expressed as percent of control. VEGF-induced tube formation without EGCG treatment was used as a control. Values significantly lower than control are indicated (^bP < 0.01).