A novel angiogenesis inhibitor impairs lovo cell survival via targeting against human VEGFR and its signaling pathway of phosphorylation

B L Dai,X L Wang,W W Smith,Y Z Zhan,L Zheng,Y N Chen,J Zhang,Y M Zhang,L C He

doi:10.1038/cddis.2012.145

Abstract

Colorectal cancer represents the fourth commonest malignancy, and constitutes a major cause of significant morbidity and mortality among other diseases. However, the chemical therapy is still under development. Angiogenesis plays an important role in colon cancer development. We developed HMQ18–22 (a novel analog of taspine) with the aim to target angiogenesis. We found that HMQ18–22 significantly reduced angiogenesis of chicken chorioallantoic membrane (CAM) and mouse colon tissue, and inhibited cell migration and tube formation as well. Then, we verified the interaction between HMQ18–22 and VEGFR2 by AlphaScreen P-VEGFR assay, screened the targets on angiogenesis by VEGF Phospho Antibody Array, validated the target by western blot and RNAi in lovo cells. We found HMQ18–22 could decrease phosphorylation of VEGFR2(Tyr1214), VEGFR1(Tyr1333), Akt(Tyr326), protein kinase Cα (PKCα) (Tyr657) and phospholipase-Cγ-1 (PLCγ-1) (Tyr771). Most importantly, HMQ18–22 inhibited proliferation of lovo cell and tumor growth in a human colon tumor xenografted model of athymic mice. Compared with normal lovo cells proliferation, the inhibition on proliferation of knockdown cells (VEGFR2, VEGFR1, Akt, PKCα and PLCγ-1) by HMQ18–22 decreased. These results suggested that HMQ18–22 is a novel angiogenesis inhibitor and can be a useful therapeutic candidate for colon cancer intervention.

Highlights

Vascular endothelial growth factor (VEGF) is one of the most important regulators of angiogenesis and a key drug target in anticancer treatment
To test the effects of HMQ18–22 on chorioallantoic membrane (CAM) blood vessels, we treated them with HMQ18–22 (0, 2.5, 5, 10 mg/per egg) for 72 h, and found HMQ18–22 inhibited the CAM angiogenesis in a dosedependent manner (Figures 2a and b)
Many new microvessels grew after 5 days, and the density and area of the blood vessels tended to increase after 7 days in the untreated control group (Figure 2d), whereas microvessels were less in the HMQ18–22-treated group (Figure 2c6–c8)

Summary

Introduction

Vascular endothelial growth factor (VEGF) is one of the most important regulators of angiogenesis and a key drug target in anticancer treatment. Clinical trials have shown inhibitors to this pathway are effective in reducing tumor size, metastasis and blood vessel formation.[14] There are various molecular players and signaling cascades involved in the VEGF/VEGFR pathway, such as the phosphatidylinositol 3kinase (PI3K)/AKT, Ras/Raf/mitogen-activated protein kinase (MAPK) and phospholipase-Cg/protein kinase C (PLCg/PKC) pathway. These signaling pathways regulate important cellular functions including cellular proliferation, migration, angiogenesis and apoptosis.[15,16,17,18]. Taspine was originally identified from a screen of Radix et Rhizoma leonticis (Hong Mao Qi in Chinese) using cell

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Results

Conclusion