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, 13 (6), 701-711
eCollection

Eupafolin Exhibits Potent Anti-Angiogenic and Antitumor Activity in Hepatocellular Carcinoma

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Eupafolin Exhibits Potent Anti-Angiogenic and Antitumor Activity in Hepatocellular Carcinoma

Honglei Jiang et al. Int J Biol Sci.

Abstract

Eupafolin is a flavonoid extracted from the common sage herb which has been used in China as traditional medicine. Previous studies had reported that eupafolin had antioxidative, anti-inflammatory and antitumor effects. However, the function and the mechanism of eupafolin to exert its antitumor activity, especially its effect on tumor angiogenesis, have not been elucidated. Herein, we showed that eupafolin significantly inhibited vascular endothelial growth factor (VEGF)-induced cell proliferation, migration and tube formation of human umbilical vascular endothelial cells (HUVECs) in a dose-dependent manner. Meanwhile, the new blood microvessels induced by VEGF in the matrigel plug were also substantially suppressed by eupafolin. The results of HCC xenograft experiments demonstrated eupafolin remarkably inhibited tumor growth and tumor angiogenesis in vivo, suggesting the antitumor activity exerted by eupafolin was closely correlated with its potency on tumor angiogenesis. Mechanism investigations revealed that eupafolin significantly blocked VEGF-induced activation of VEGFR2 in HUVEC cells as well as its downstream signaling pathway. In addition to the effect on endothelial cells, through inhibiting Akt activity in tumor cells, VEGF secretion in HepG2 was dramatically decreased after eupafolin treatment. Our study was the first to report the activity of eupafolin against tumor angiogenesis as well as the underlying mechanism by which eupafolin to exert its anti-angiogenic activity.

Keywords: Eupafolin; tumor angiogenesis.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
Eupafolin inhibited cell viability in HUVECs and HCC cells. A, the chemical structure of eupafolin. B, eupafolin inhibited VEGF-induced HUVECs proliferation. HUVECs(2× 104/well) were seeded into 96-well plate and starved with 0.1% FBS medium overnight and then incubated with or without VEGF(20 ng/ml) and various concentrations of eupafolin for 24 h, cell viability was analyzed by MTS assay. C, inhibitory effect of eupafolin on HUVECs in normal conditions. HUVECs (2×104/well) were seeded into 96-well plate and treated with different concentrations of eupafolin for 24 h, cell viability was analyzed by MTS assay. D and E, eupafolin suppressed HCC cell lines HepG2(D) and Hep3B(E) proliferation (the conditions were the same as described in C). Columns, mean of three independent experiments ;bars, SD. The asterisk (*, p<0.05) indicated a significant decrease of cell viability after treated with eupafolin.
Figure 2
Figure 2
Eupafolin inhibited VEGF-induced agiogenesis in vitro and ex vivo. A, eupafolin inhibited HUVECs migration. HUVECs were grown into full confluence in six-well plate and treated with 10 μg/ml mitomycin C for 2 h, then cells were wounded with pipette and incubated with or without 20 ng/ml VEGF as well as various concentrations of eupafolin. 12 h later, the migrated cells were quantified by manual counting. B, eupafolin suppressed HUVECs invasion. Cells were seeded in the upper chamber of Transwell and incubated with various concentrations of eupafolin. The bottom chamber was added with culture medium with VEGF. About 8 to 10 h later, the invasive cells were fixed and stained with crystal violet solution, the invasive cells were quantified by manual counting. C, eupafolin inhibited VEGF-induced tube formation of HUVECs. HUVECs incubated with different concentrations of eupafolin were seeded into 96-well plate pre-coated with Matrigel. After 6 to 8 h, tubular structures were photographed and the number of the tubes was quantified. D, eupafolin inhibited VEGF-induced angiogenesis in Matrigel plug assay. The Matrigel plug assay was performed as described in “Materials and Methods”. The number of vessels was counted. Representative photographs of each experiment (left panels) were shown, and the graph (right panels) showed the data of at least three independent experiments. Columns, mean; bars, SE; The asterisk (*, p< 0.05; **, p< 0.01) indicated significant difference versus VEGF alone.
Figure 3
Figure 3
Eupafolin suppressed HCC cells growth and angiogenesis in vivo. Antitumor activity of eupafolin was investigated in HepG2 and Hep3B xenograft models, mice were randomly assigned into groups when tumor volume reached 100 mm3. The dosage of eupafolin was 60 mg/kg and was administrated three times per week by intraperitoneal injection. A, the growth curve of HepG2(left panel) and Hep3B (right panel) xenograft; B, the weight of tumors in vehicle and eupafolin-treated group, left: HepG2, right: Hep3B; C, tumor tissues were performed with immunohistochemistry analysis by staining with anti-Ki67, anti-CD31 antibody to detect the change of angiogenesis in tumor tissue. Left panel, representative photograph of tumor tissue per group (200×); right panel, the expression of indicated marker in per group was quantified, the asterisks (*, p<0.05, **, p<0.01, Student's t test) indicated significant difference.
Figure 4
Figure 4
Eupafolin inhibited VEGFR2 signaling pathway in HUVECs and HCC cells. A and C, eupafolin inhibited VEGFR2 phosphorylation in HUVECs and HepG2 cells. HUVECs(A) and HepG2 cells(C) were incubated with various concentrations of eupafolin for 24 h and the cell lysates were subjected to western blotting and probed with indicated antibodies, β-actin was used as loading control. B and D, eupafolin suppressed VEGF-induced VEGFR2 phosphorylation in HUVECs and HepG2 cells. HUVECs(B) and HepG2(D) cells were starved with 0.1% FBS overnight and then incubated with various eupafolin for 2 h, after stimulating with 20 ng/ml VEGF for 10 min, cell lysates were collected and western blotting was used to examine eupafolin's effect on the activity on VEGFR2, ERK1/2 and Akt, β-actin was used as loading control.
Figure 5
Figure 5
Eupafolin suppressed VEGF secretion in HepG2 cells via inhibiting Akt signaling pathway. A, eupafolin inhibited VEGF secretion in HepG2 cells. HepG2 cells was treated with various concentrations of eupafolin for 24 h, the supernatant of cell culture medium was collected and the content of VEGF was examined with ELISA kit. B, eupafolin suppressed Akt signaling pathway in HepG2 cells. HepG2 cells were exposed to various concentrations of eupafolin for 24 h, and the cell lysates were analyzed by western blotting with indicated antibodies. C and D, Akt signaling pathway mediated VEGF secretion in HepG2 cells. HepG2 cells were treated with selective PI3K inhibitor(LY294002) or MEK inhibitor(PD98059) for 24 h respectively, cell lysates was subjected to western blotting with indicated antibodies (C), and the amount of VEGF in cell culture medium was analyzed with ELISA kit (D). E and F, exogenous overexpression of Akt attenuated eupafolin's effect on VEGF secretion. HepG2 cells were transfected with Myr-Akt1 for 24 h, and then treated with 20 µM eupafolin for 24 h, cell lysates were analyzed by western blot with indicated antibodies (E), VEGF secretion in cell culture medium was analyzed with ELISA kit (F). The graph showed the data of least three independent experiments and expressed as Mean±SD, the asterisks (*, p<0.05, Student's t test) indicated significant difference.

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