Evaluation of Antiangiogenic Efficacy of Emilia sonchifolia (L.) DC on Tumor-Specific Neovessel Formation by Regulating MMPs, VEGF, and Proinflammatory Cytokines

Abstract

Formation of new blood vessels from preexisting vasculature is an indispensable process in tumor initiation, invasion, and metastasis. Novel therapeutic approaches target endothelial cells involved in the process of angiogenesis, due to their genetic stability relative to the rapidly mutating drug-resistant cancer cells. In the present study, we investigated the effect of an active fraction from Emilia sonchifolia, belonging to the family Asteraceae, a plant well known for its anti-inflammatory and antitumor effects, on the inhibition of tumor-specific angiogenesis. Administration of the active fraction from E sonchifolia (AFES; 5 mg/kg, body weight, intraperitoneally) containing the major compound γ-humulene significantly inhibited B16F10 melanoma-induced capillary formation in C57BL/6 mice. The level of serum vascular endothelial growth factor and serum proinflammatory cytokines such as interleukin-1β, interleukin-6, tumor necrosis factor-α, and granulocyte-macrophage colony-stimulating factor were also reduced significantly. At the same time, administration of AFES significantly enhanced the production of antiangiogenic factors such as tissue inhibitor of matrix metalloproteinase-1. Dose-dependent reduction can be seen in the budding and expansion of microvessels from rat thoracic aorta by AFES treatment. Inhibition of the activation of proenzyme to active enzyme of matrix metalloproteinase along with a successful reduction of proliferation, invasion, and migration of human umbilical vein endothelial cells demonstrated the antiangiogenic effect of AFES in vitro. To date, no study has examined the antiangiogenic activity of this plant with already well-known anti-inflammatory and antitumor effects. Results obtained in the present study by using both in vivo and in vitro angiogenic models altogether proved the inhibitory effect of AFES on tumor-specific neovessel formation.

Keywords: Emilia sonchifolia; angiogenesis; endothelial cell migration; invasion; matrix metalloproteinase; vascular endothelial growth factor; γ-humulene.

Figure 1.

(A) Structure of γ-humulene. (B) MTT assay showing viability of HUVECs following treatment with the indicated concentrations of AFES.

Figure 2.

Effect of AFES on in vivo angiogenesis. Tumor angiogenesis was induced by subcutaneous injection of B16F10 melanoma cells (106 cells/mice) on shaven ventral side of C57BL/6 mice treated simultaneously with AFES or TNP-470: (A) Control; (B) AFES; (C) TNP-470.

Figure 3.

Effect of AFES on the serum cytokine levels: (A) IL-1β, (B) IL-6, (C) TNF-α, and (D) GM-CSF. The serum was collected from the caudal vein of C57BL/6 mice on the second and ninth days after induction of B16F10 melanoma cells (106 cells/mice). Values are mean ± SD. ^aP < .001 significantly different from untreated control.

Figure 4.

Effect of AFES on the serum (A) VEGF and (B) TIMP levels. The serum was collected from the caudal vein of C57BL/6 mice on the second and ninth days after induction of B16F10 melanoma cells (106 cells/mice). Values are mean ± SD. ^aP < .001 significantly different from untreated control.

Figure 5.

Effect of AFES on in vitro angiogenesis. The conditioned medium from normal semiconfluent bottles of B16F10 cells acts as the control: (A) Control with conditioned medium alone; (B) Conditioned medium + treatment with AFES (2.5 µg/mL); (C) Conditioned medium + treatment with AFES (1 µg/mL); (D) Conditioned medium + treatment with AFES (0.5 µg/mL).

Figure 6.

Inhibitory effect of AFES on HUVECs migration. The HUVECs (2 × 105 cells/well) were seeded on type I collagen coated 96-well titer plate and incubated overnight at 37°C. A clear area was made with a narrow tip in the monolayer and further incubated for 24 hours in the presence and absence of AFES (2.5, 1, and 0.5 µg/mL) along with VEGF (2 ng/mL). After incubation, the cells were fixed and stained using crystal violet and photographed. (A) Control “0” hour incubation; (B) Control after 24-hour incubation in medium without AFES; (C) AFES (2.5 µg/mL); (D) AFES (1 µg/mL); (E) AFES (0.5 µg/mL).

Figure 7.

Inhibitory effect of AFES on HUVECs invasion through collagen matrix. HUVECs (105 cells/150 µL medium 199) were seeded on to the upper chamber of Boyden chamber in the presence and absence of AFES (2.5, 1, and 0.5 µg/mL) along with 2 ng/mL VEGF and incubated at 37°C in 5% CO₂ atmosphere for 10 hours. After incubation, the cells migrating to the lower surface of the polycarbonate membrane were fixed with methanol and stained with crystal violet and photographed. (A) Untreated control; (B) Treatment with AFES (2.5 µg/mL); (C) Treatment with AFES (1 µg/mL); (D) Treatment with AFES (0.5 µg/mL).

Figure 8.

Effect of AFES on MMP-2 and MMP-9 production by HUVECs. (1) Condition medium from untreated HUVECs without trypsin activation; (2) Condition medium from untreated HUVECs after trypsin activation; (3) Condition medium from untreated HUVECs after trypsin activation + EDTA; (4) Condition medium from pretreated HUVECs (2.5 µg/mL AFES) after trypsin activation; (5) Condition medium from pretreated HUVECs (1 µg/mL AFES) after trypsin activation; (6) Condition medium from pretreated HUVECs (0.5 µg/mL AFES) after trypsin activation.