Research paperAnlotinib inhibits angiogenesis via suppressing the activation of VEGFR2, PDGFRβ and FGFR1
Introduction
Angiogenesis is the sprouting and growth of new vessels from an existing vasculature. It occurs in normal development and disease such as embryogenesis and wound healing (Wang et al., 2015). Tumor angiogenesis plays an important role in tumor progress. To support their growth, tumors need to form new vessels to transport nutrients and oxygen (Kerbel, 2000). As such, inhibiting the formation of new vessels in tumor is a potent strategy for cancer treatment.
Previous studies indicated that tumor cells secreted pro-angiogenic cytokines to induce migration and tube formation in endothelial cells and then to generate neovascular. Vascular endothelial growth factor (VEGF), one of the most important pro-angiogenic factors, is expressed by in vast majority of cancers, especially VEGFA (Bergers and Benjamin, 2003). VEGFA has high affinity to VEGFR2 expressed in endothelial cells. The binding of VEGFA to VEGFR2 activates the tyrosine kinase receptor and the phosphorylation of VEGFR2 triggers a network of downstream pathways to promote proliferation, survival and migration of endothelial cells (Hicklin and Ellis, 2005; Claesson-Welsh and Welsh, 2013). Similar to VEGF, fibroblast grow factor 2 (FGF-2) and platelet-derived growth factor-BB (PDGF-BB) function as pro-angiogenic factors in tumor angiogenesis (Nissen et al., 2007b). FGF-2 triggers the autophosphorylation of FGF receptor 1 (FGFR1) and activates downstream signaling cascades to induce angiogenesis (Katoh and Nakagama, 2014). PDGF-BB binds to its receptor PDGF receptor β (PDGFRβ) to regulate tumor angiogenesis, growth and metastasis (Zhao and Adjei, 2015). Now clinical strategy to target angiogenesis has achieved a qualitative progress in cancer treatment. The small-molecule tyrosine kinase inhibitors, such as sunitinib, sorafenib and nintedanib, have shown clinical efficacy in diverse cancer types (Gotink and Verheul, 2010). In this study, we found a new chemical compound (Fig. 1A), named as anlotinib, has a better anti-angiogenic effect than sunitinib, sorafenib and nintedanib. The clinical trials of anlotinib has been completed recently and is going to come into the market soon. As a TKI, anlotinib targeted multiple angiogenic kinases including VEGFR2, PDGFRβ and FGFR1. Therefore, anlotinib inhibited VEGF/PDGF-BB/FGF-2-induced angiogenesis in vitro and in vivo, suggesting that anlotinib might become a potential angiogenesis inhibitor.
Section snippets
Reagent
Anlotinib, sunitinib malete, sorafenib tosylate, nintedanib esylate were provided by CTTQ Pharma (Lianyungang, China). The compounds were dissolved in dimethylsulfoxide (DMSO) to 10 mM as stock solution and stored at −20 °C for in vitro studies and diluted with medium before each experiment. Primary antibodies against β-actin and p-ERK were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Primary antibodies against VEGFR2, p-VEGFR2, FGFR1, p-FGFR1, PDGFRβ, p-PDGFRβ and ERK were
Anlotinib inhibited VEGF/PDGF-BB/FGF-2-induced migration in EA.hy 926 cells
In order to eliminate the inhibition effect of anlotinib on the proliferation of endothelial cells, MTT assay was conducted on EA.hy 926 cells and the IC50 was 30.26 μM. Anlotinib had little effect on cell viability of EA.hy 926 cells for 24 h at the concentration of 0.01, 0.1, 1 μM. Therefore, these dosage of anlotinib were applied to the following experiments.
We detected the inhibitory of anlotinib on kinase activity and found that anlotinib worked best in inhibiting VEGFR2, PDGFRβ and FGFR1 (
Discussion
VEGF is a critical growth factor which drives tumor angiogenesis to stimulate tumor growth. High VEGF concentration correlates with poor clinical prognosis of many cancers. Inhibition of tumor angiogenesis through pharmacological blockade of VEGF signaling is clinically approved but tumors can easily acquire drug resistance at the same time (Moens et al., 2014). VEGF stimulates the proliferation, migration, survival and new-vessel formation of endothelial cells in tumors, and blocking the
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These authors contributed equally to this work.