Combinative effects of β-elemene and propranolol on the proliferation, migration, and angiogenesis of hemangioma

Abstract

Hemangioma (HA) is one of the most common benign vascular tumors among children. Propranolol is used as the first-line treatment for hemangioma and is a non-selective blocker of the β-adrenergic receptor. β-elemene is a compound extracted from Rhizoma zedoariae and has been approved for the treatment of tumors in clinical practice. However, the combinatorial effects of β-elemene and propranolol in the treatment of HA remains unclear. This study explored the combinative effects and mechanisms of β-elemene and propranolol using hemangioma-derived endothelial cells (HemECs). Cytotoxic assays showed that the combinatorial treatment of β-elemene and propranolol did not increase the cytotoxic effects of HemECs. Furthermore, functional analysis showed that the combinatorial treatment with β-elemene and propranolol significantly inhibited the proliferation, migration, and tube formation of the HemECs compared to the single treatment regimens. Mechanistic analysis showed that combinative treatment with β-elemene and propranolol synergistically down-regulated the hypoxia-inducible factor-1 alpha/vascular endothelial growth factor-A (HIF-1-α/VEGFA) signaling pathway. Additionally, in a xenograft tumor model, angiogenesis in the combinatorial treatment group was significantly lower than in the control, propranolol, and β-elemene treatment alone groups. Our results suggest that β-elemene combined with propranolol can significantly inhibit the proliferation, migration, and tube formation of HemECs via synergistically down-regulating the HIF-1-α/VEGFA signaling pathway without increasing any cytotoxic side effects.

Keywords: Angiogenesis; Hemangioma; Proliferation; Propranolol; β-elemene.

Figure 1. Cytotoxic effect of propranolol and β-elemene on HemECs.

(A) A concentrations of β-elemene lower than or equal to 75 µg/ml did not produce a cytotoxic effect on HemECs. (B) There was no apparent effect in the cytotoxicity of propranolol on HemECs at concentrations lower than or equal to 25 µM. (C) Compared with the control group, proliferation of the HemECs decreased at concentrations of β-elemene more than 75 µg/ml, the data was evaluated via a cell counting kit-8 assay. (D) Compared to the control group, proliferation of the HemECs decreased at concentrations of propranolol more than 25 µM, the data was evaluated via a Cell counting kit-8 assay. (E) Representative photomicrographs for Calcein-AM/PI double staining of the HemECs in five different groups: control, β-elemene (75 µg/ml), propranolol (50 µM), combinatorial treatment with β-elemene (75 µg/ml) and propranolol (50 µM), and the propranolol groups (75 µM) (scale bar = 100 µm). (F) Quantification of Calcein-AM/PI double staining. The experiment was repeated in triplicate and results are presented as mean ± SD; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant.

Figure 2. Combinatorial treatment of β-elemene and propranolol inhibited the migration and proliferation and tube formation of HemECs.

(A) Representative photomicrographs of the HemECs scratch test of five different groups: control, β-elemene (75 µg/ml), propranolol (50 µM), and combinatorial treatment with β-elemene (75 µg/ml) and propranolol (50 µM), and propranolol groups (75 µM) (scale bar = 250 µm). (B) Quantitative analyses of the relative migration rate. (C) HemECs treated with β-elemene and propranolol for 24 h, 48 h, and 72 h. The proliferation of the HemECs were evaluated using the cell counting kit-8 assay. (D) Quantification of proliferation in HemECs treated with β-elemene and propranolol for 72 h. (E, F) Representative photomicrographs of the tube formation assay in HemECs within five different groups. The corresponding quantitative analysis of the tube number of the capillary tubes formed by the HemECs (scale bar = 200 µm). The experiment was repeated in triplicate and results are presented as mean ± SD; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant.

Figure 3. HIF-1- α/VEGFA signaling pathway was activiated in hemangioma.

(A) The expression of HIF-1- α and VEGFA observed by RT-PCR. (B) The expression of HIF-1- α, phospho-Akt, Akt, phospho-Erk, Erk, and VEGFA observed by western blotting. (C) Quantified protein expression levels of HIF-1- α, VEGFA, p-Akt/Akt, and p-Erk/Erk. (D) Representative photomicrographs of the HUVECs and HemECs scratch test (scale bar = 250 µm). (E) Quantitative analyses of the relative migration rate. (F) Compared with HUVECs, proliferation of the HemECs were increased, data was evaluated via a cell counting kit-8 assay. (G) Representative photomicrographs of the tube formation assay in HUVECs and HemECs (scale bar = 200 µm). (H) Quantitative analysis of the tube number of the capillary tubes . The experiment was repeated in triplicate and results are presented as mean ± SD; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant.

Figure 4. Combinatorial treatment of β-elemene and propranolol inhibits the expression of HIF-1- α and VEGFA in HemECs.

HemECs treated with β-elemene and propranolol for 24 h, (A and B) The expression of HIF-1- α, and VEGFA observed by RT-PCR. (C) The expression of HIF-1- α, VEGFA, p-Akt/Akt, and p-Erk/Erk observed by western blotting. (D) Quantified protein expression levels of HIF-1- α, VEGFA, p-Akt/Akt, and p-Erk/Erk. The experiment was repeated in triplicate and results are presented as mean ± SD; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant.

Figure 5. Combinatorial treatment of β-elemene and propranolol inhibited the cytokines in HemECs.

(A) The expression of IL-1 α, IL-1 β, IL-6, IL-8, IL-10, TNF- α in HUVECs and HemECs observed by RT-PCR. (B) HemECs treated with β-elemene and propranolol for 24 h, the expression of IL-6, IL-8 and TNF- α observed by RT-PCR. The experiment was repeated in triplicate and results are presented as mean ± SD; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant.

Figure 6. Combinatorial treatment of β-elemene and propranolol inhibited growth and angiogenesis of hemangioma.

The HemECs were injected subcutaneously into the left flank of the nude mice. After 30 days of β-elemene (75 mg/kg d) and propranolol (50 mg/kg d) treatment the primary hemangioma tumor was carefully removed from the left flank for analysis. (A) Hemangioma images (scale bar 1 cm). (B) The size of the hemangioma before resection. (C) Statistical analysis of tumor volume after tumors separated. (D) Statistical analysis of tumor weight. (E) Immunofluorescence used to detect the expression of CD31 on hemangioma (scale bar 200 µm). (F) Quantification of microvessel density (MVD). The experiment was repeated in triplicate and results are presented as mean ± SD; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant.

Figure 7. Schematic diagram of the possible mechanisms of action of the combinatorial treatment using β-elemene and propranolol on hemangioma.

β-elemene in combination with propranolol inhibits the expression of VEGFA by synergistically down-regulating HIF-1- α expression. Decreased VEGFA expression inhibits VEGFR2 activation, which in turn affects the phosphorylation of downstream signaling molecules, including Akt and Erk; therefore, ultimately inhibiting hemangioma growth and angiogenesis.