关键词: 黑色素瘤, 实验性； 木犀草素；肿瘤转移；受体, 血管内皮生长因子； 胶原酶类； 血管拟态

Abstract: 【Abstract】    Objective    To evaluate the effect of luteolin on the growth, migration and vasculogenic mimicry formation of a melanoma cell line B16. Methods    In vitro cultured  B16 melanoma cells were divided into 4 groups: low-, middle- and high-dose luteolin groups treated with 2.5, 5, 10 μmol/L luteolin respectively, and control group treated with 0.1% dimethyl sulfoxide （DMSO）. Scratch assay, Transwell invasion assay and vascular channel formation assay were performed to assess the migration, invasion of and vascular channel formation by melanoma cells. A model of subcutaneous transplanted B16 melanoma was established in 12 C57 mice, which were randomly and equally divided into 4 groups: control group gavaged with ultrapure water, low-, middle- and high-dose luteolin groups gavaged with 10, 20, 40 mg/kg luteolin respectively every day. The above treatment for the tumor-bearing mice lasted till day 28, and then these mice were sacrificed. Meanwhile, the lung and tumor tissues of the mice were excised, and the growth, metastasis and vasculogenic mimicry of transplanted melanoma were observed. Immunofluorescence and immunohistochemical studies were performed to evaluate the effects of luteolin on the expression of vascular endothelial cadherin （VE-cadherin）, vascular endothelial growth factor receptor 1 （VEGFR1）, VEGFR2, matrix metalloproteinase-2 （MMP-2） and MMP-9 in the transplanted melanoma. Means were compared among several groups by using one-way analysis of variation or rank sum test. Results    In vitro study showed that the relative scratch width at 48 hours significantly differed among the control group, low-, middle- and high-dose luteolin groups （0.47 ± 0.04, 0.64 ± 0.04, 0.73 ± 0.03, 0.84 ± 0.04 respectively; F = 34.51, P < 0.001）, and the migration ability of B16 cells was significantly lower in the low-, middle- and high-dose luteolin groups than in the control group （all P < 0.05）. At 24 hours, there were significant differences in the number of cells crossing the Transwell membrane among the control group, low-, middle- and high-dose luteolin groups （281.00 ± 8.79, 169.00 ± 15.35, 92.00 ± 14.79 and 57.00 ± 13.72 respectively; F = 275.30, P < 0.001）, and the invasive ability was significantly lower in the low-, middle- and high-dose luteolin groups than in the control group （P < 0.01）. Meanwhile, the number of formed vascular channels also differed among the above 4 groups （20.00 ± 2.77, 11.00 ± 1.28, 7.00 ± 1.86 and 2.00 ± 1.32 respectively; F = 48.61, P < 0.001）, and the number of vascular channels was significantly lower in the low-, middle- and high-dose luteolin groups than in the control group （all P < 0.01）. In vivo study showed that the tumor size significantly differed among the control group, low-, middle- and high-dose luteolin groups （5.10 ± 1.72, 4.02 ± 2.13, 2.98 ± 0.92, 1.49 ± 1.13 cm3 respectively; F = 28.76, P < 0.001）, and was significantly lower in the low-, middle- and high-dose luteolin groups than in the control group （t = 3.86, 7.11 and 13.06 respectively, all P < 0.01）. CD31-PAS double staining showed that the number of vasculogenic mimicry was significantly higher in the control group than in the low-, middle- and high-dose luteolin groups （all P < 0.01）. In vivo and in vitro studies both showed that the expression of vasculogenic mimicry-related markers in the cells or mouse tumor tissues was significantly lower in the high-dose luteolin group than in the control group （P < 0.05）. Conclusion    Luteolin can effectively inhibit the growth, metastasis and vasculogenic mimicry formation of melanoma.

Key words: Melanoma, experimental, Luteolin, Neoplasm metastasis, Receptors, vascular endothelial growth factor, Collagenases, Vasculogenic mimicry