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. 2020 Nov;9(11):6820-6832.
doi: 10.21037/tcr-20-1992.

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

Jing Chen #  1   2 , Bo-Xia Liu #  1   2 , Qin Shen  1   2 , Na Li  1   2 , Jun Ling  1   2 , Min Xiao  3 , Hai-Yan Jiao  1   2 , Tao Li  4
Affiliations

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

Jing Chen et al. Transl Cancer Res. 2020 Nov.

Abstract

Background: 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.

Methods: 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.

Results: 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.

Conclusions: Limonin is a promising anti-angiogenic and anti-metastatic candidate compound that can be further optimized as a therapeutic agent for breast cancer.

Keywords: Limonin; STAT3; angiogenesis; breast cancer; metastasis.

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Conflict of interest statement

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/tcr-20-1992). The authors have no conflicts of interest to declare.

Figures

Figure 1
Figure 1
The interaction of limonin with ATP-binding sites of VEGFR2 kinase domain. (A) Limonin’s molecular structure. (B) 2-dimensional maps of the interaction between limonin and related amino acids of VEGR2 protein were obtained by using LigPlot software. The types of closely related interactions and bonds. (C) The ribbon structure of green VEGFR2 protein was created by Chimera program (D) ATP binding site of VEGFR2 crystal. (E) Western blotting analysis showed that limonin dose- dependently inhibited the activation of both VEGFR2 (Tyr1175) and downstream STAT3 (Tyr705) triggered by VEGF in endothelial cells.
Figure 2
Figure 2
VEGF-induced angiogenesis in vitro and in vivo were inhibited by limonin. (A) VEGF-induced endothelial cells migration was significantly inhibited by limonin in Wound Healing assays, Scale bar=100 µm. (B) Quantification of the data from the Wound Healing assays. Scale bar=100 µm (n=3). *, P<0.05; **, P<0.01; ***, P<0.001. (C) Limonin inhibited HUVECs cell invasive ability using gelatin-coat Boyden inserts. (D) The formation of endothelial cell tubes was inhibited by limonin. Representative fields were displayed in each group (0.5% crystal violet staining, magnification at 100×). Columns, mean from three independent experiments with triplicate. *, P<0.05; **, P<0.01 versus VEGF control. (E,F) Representative matrigel plug images in each group (n=3). (G) Immunohistochemistry was used to analyze the anti-angiogenic effects of limonin on VEGFR2, CD31 and STAT3 on matrigel plug sections (magnification, 400×).
Figure 3
Figure 3
The IGFR1/STAT3 signaling pathway is involved in anti-metastatic effects in limonin treated breast cancer MDA-MB-231 cells. (A) Path enrichment of differentially expressed genes in limonin treated MDA-MB-231 cells. The down-regulated gene subpopulation regulated by the STAT family was used for enrichment analysis. The bar plot showed the highest enrichment score (_log (P value)) for the significant enrichment pathway, the P value cutoff is 0.05. (B) Limonin and Regorafenib dose-dependently inhibited the phosphorylation of both IGFR1 (Tyr1316) and STAT3 (Tyr705) in breast cancer cells. (C) Effects of limonin and Regorafenib on SHP-1 in MDA-MB-231 cells were analyzed by Western blotting assay. (D) Immunoblotting assay was used to detect the knockdown efficiency of SHP-1. (E) Transwell migration assay was used to test the inhibitory effect of limonin on MDA-MB-231 cell migration when silencing SHP-1, 0.5% crystal violet staining, Scale bar =100 µm. (F) RT-PCR and (G) Western blotting assays used to test the STAT3-dependent transcriptional activity of MMP9 and VEGF by suppressed of limonin and regorafenib. (H) Wound healing assay was used to examine the inhibition effect of limonin on breast cancer cells migration, Scale bar=100 µm.
Figure 4
Figure 4
Effect of prevention and treatment of limonin on mouse breast tumor metastasis by tail vein injection. (A) Bioluminescence analysis was used to determine tumor metastasis over a 45-day period. (B) Quantitative analysis of metastatic cells for use in lung bioluminescence analysis, ***, P<0.001. P values were calculated using a two sided Student’s t test. p/sec/cm2/sr = photons/second/cm2/steradian. (C) Quantitative analysis of incidence of lung metastasis. (D) Histological analyses of breast cancer cell lung metastases with administration of limonin or vehicle control (magnification, 400×).
Figure 5
Figure 5
Preventive effects of limonin on mouse breast tumor metastasis by left ventricle injection. (A) Bioluminescence analysis was used to determine the 28-day period tumor metastasis. (B) Limonin prevents metastasis in multiple organs in vivo.
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