Limonin inhibits angiogenesis and metastasis of human breast cancer cells by suppressing the VEGFR2/IGFR1-mediated STAT3 signaling pathway

Limonin is one of the major active ingredients of citrus. In the present study, the anti-angiogenic and anti-metastatic effects of limonin were investigated.The Molecular docking assay was carried out to assess the binding ability of limonin with VEGFR2 receptor. MTS assay was used to detect the effect of limonin on the proliferation of breast cancer cells (MDA-MB-231, MCF-7). The Wound-healing and Transwell chamber invasion assays were used to detect the inhibition effect of limonin on migration and invasion of HUVECs cells or breast cancer cells. The capillary-like tube formation assay and Matrixgel plug experiment were used to further measure the in vivo anti-angiogenic activity of limonin. Western blot, RNA isolation, microarray data analysis and RT-PCR were used to explore the molecular mechanism of limonin in suppressing breast cancer angiogenesis and metastasis. Left ventricular tumor metastasis model and caudal vein tumor metastasis model of breast cancer were both applied to verify in vivo anti-metastatic effects.Limonin dose-dependently inhibited the vascular endothelial growth factor (VEGF)-mediated tyrosine phosphorylation of vascular endothelial growth factor receptor 2 (VEGFR2) by blocking VEGF binding to VEGFR2 and suppressing constitutive STAT3 activation in human umbilical vein endothelial cells. Limonin effectively inhibited VEGF-induced endothelial cell proliferation, migration and tubular-structure formation in vitro and markedly reduced VEGF-triggered neovascularization in mouse matrigel plugs in vivo. Moreover, limonin treatment led to a remarkable suppression of tumor metastasis by decreasing the phosphorylation of insulin growth factor receptor 1-mediated STAT3 and the expression levels of its downstream members MMP-9 and VEGF in breast cancer cells. The data further showed that limonin increased the levels of the negative STAT3 regulator SHP-1 in breast cancer cells.Limonin is a promising anti-angiogenic and anti-metastatic candidate compound that can be further optimized as a therapeutic agent for breast cancer.