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. 2017 May;39(5):1255-1261.
doi: 10.3892/ijmm.2017.2940. Epub 2017 Mar 29.

Salinomycin exhibits anti-angiogenic activity against human glioma in vitro and in vivo by suppressing the VEGF-VEGFR2-AKT/FAK signaling axis

Yan-Ling Bi  1 , Pei-Yan Mi  1 , Shi-Jun Zhao  2 , Heng-Ming Pan  3 , Hui-Juan Li  1 , Fei Liu  4 , Lu-Rong Shao  2 , Hui-Fang Zhang  2 , Pu Zhang  1 , Shi-Liang Jiang  3
Affiliations

Salinomycin exhibits anti-angiogenic activity against human glioma in vitro and in vivo by suppressing the VEGF-VEGFR2-AKT/FAK signaling axis

Yan-Ling Bi et al. Int J Mol Med. 2017 May.

Abstract

Tumor angiogenesis plays a crucial role in tumor growth, progression and metastasis, and suppression of tumor angiogenesis has been considered as a promising anticancer strategy. Salinomycin (SAL), an antibiotic, displays novel anticancer potential against several human cancer cells in vitro and in vivo. However, little information concerning its anti-angiogenic properties is available. Therefore, the anti‑angiogenic effect of SAL and the underlying mechanism in human glioma were evaluated in the present study. The results indicated that SAL treatment significantly inhibited human umbilical vein endothelial cell (HUVEC) proliferation, migration, invasion and capillary-like tube formation. Further investigation on intracellular mechanisms showed that SAL markedly suppressed FAK and AKT phosphorylation, and downregulated vascular endothelial growth factor (VEGF) expression in HUVECs. Pretreatment of cells with a PI3K inhibitor (LY294002) and FAK inhibitor (PF562271) markedly enhanced SAL-induced inhibition of HUVEC proliferation and migration, respectively. Moreover, U251 human glioma xenograft growth was also effectively blocked by SAL treatment in vivo via inhibition of angiogenesis involving FAK and AKT depho-sphorylation. Taken together, our findings validated that SAL inhibits angiogenesis and human glioma growth through suppression of the VEGF-VEGFR2-AKT/FAK signaling axis, indicating the potential application of SAL for the treatment of human glioma.

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Figures

Figure 1
Figure 1
Salinomycin (SAL) inhibits the cell proliferation in human umbilical vein endothelial cells (HUVECs). (A) HUVECs seeded in 96-well plates (6,000 cells/well) were treated with the indicated concentrations of SAL for 48 h, and the cell viability was detected by MTT assay. Each value represents the mean ± SD of three independent experiments. *p<0.05; **p<0.01 vs. the control. (B) Changes in the morphology of HUVECs. Cells after treatment with the indicated concentrations of SAL for 48 h were examined by phase-contrast microscopy (magnification, ×200).
Figure 2
Figure 2
Salinomycin (SAL) blocks human umbilical vein endothelial cell (HUVEC) migration, invasion and capillary-like tube formation in vitro. Effects of SAL on (A) HUVEC migration, (C) invasion and (E) tube formation. Positive control [vascular endothelial growth factor (VEGF)] and negative control (cisplatin) were employed to evaluate the potential of SAL. The details of the experiments is provided in Materials and methods. (B) The migration ratio, (D) invasion rate and (F) the tube formation number were quantified by manual counting. All data are expressed as means ± SD of triplicate experiments. Bars with different characters (a, b and c) are statistically different at p<0.05 level.
Figure 3
Figure 3
Salinomycin (SAL) suppresses FAK-dependent cell migration in human umbilical vein endothelial cells (HUVECs). (A) Effects of SAL on the expression level of phosphorylated FAK (p-FAK). Cells were treated with the indicated concentrations of SAL for 48 h. (B) Western blotting of the time-dependent regulation of p-FAK in HUVECs. Cells were treated with 4 µM SAL for various periods of time. (C–E) Inhibitory effects of SAL and/or FAK inhibitor (PF562271) on the expression levels of p-FAK and cell migration in HUVECs. Cells were pretreated with or without 10 nM PF562271 for 1 h and co-treated with 4 µM of SAL for 48 h. All data are expressed as means ± SD of triplicate experiments. Bars with different characters (a, b and c) are statistically different at p<0.05 level.
Figure 4
Figure 4
Salinomycin (SAL) disturbs the vascular endothelial growth factor (VEGF)-VEGFR2-AKT signaling axis. (A) Western blotting of expression levels of VEGF, VEGFR2, phosphorylated VEGFR2 (p-VEGFR2), AKT and p-AKT in human umbilical vein endothelial cells (HUVECs) exposed to different concentrations of SAL for 48 h. (B) Inhibitory effects of SAL and/or LY294002 on HUVEC growth. Cells were pretreated with or without 10 µM LY294002 for 1 h and co-treated with 4 µM of SAL for 48 h. Cell viability was determined by MTT assay. All data are expressed as means ± SD of triplicate experiments. Bars with different characters (a, b and c) are statistically different at p<0.05 level.
Figure 5
Figure 5
Salinomycin (SAL) suppresses the U251 human glioma cell growth in vivo via inhibition of angiogenesis. (A and B) Tumor volume, (C) tumor weight and (D) body weight of the mice were all assessed at the end of the experiment. U251 tumor xenograft model was established as described in Materials and methods. (E) AKT and FAK expression. Homogenized cell lysate from each group were prepared for western blotting of expression levels of phosphorylated AKT (p-AKT) and p-FAK. (F) Immunohistochemical (IHC) analysis of the expression levels of Ki-67 and CD34 in SAL-treated U251 tumor xenografts.
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