Cyperenoic acid, a sesquiterpene derivative from Croton crassifolius, inhibits tumor growth through anti-angiogenesis by attenuating VEGFR2 signal pathway in breast cancer

Weihuan Huang; Yeyin Liang; Hau Yin Chung; Guocai Wang; Jim Junhui Huang; Yaolan Li

doi:10.1016/j.phymed.2020.153253

Cyperenoic acid, a sesquiterpene derivative from Croton crassifolius, inhibits tumor growth through anti-angiogenesis by attenuating VEGFR2 signal pathway in breast cancer

Phytomedicine. 2020 May 30:76:153253. doi: 10.1016/j.phymed.2020.153253. Online ahead of print.

Authors

Weihuan Huang¹, Yeyin Liang², Hau Yin Chung³, Guocai Wang², Jim Junhui Huang⁴, Yaolan Li⁵

Affiliations

¹ Key Laboratory of Regenerative Medicine, Ministry of Education, Jinan University, Guangzhou, China; Department of Developmental & Regenerative Biology, Jinan University, Guangzhou, China.
² Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou, China.
³ Food and Nutritional Sciences Programme, School of Life Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong SAR, China.
⁴ Environmental Research Institute, National University of Singapore, Singapore 117411, Republic of Singapore. Electronic address: erihj@nus.edu.sg.
⁵ Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan University, Guangzhou, China. Electronic address: tliyl@jnu.edu.cn.

PMID: 32531699
DOI: 10.1016/j.phymed.2020.153253

Abstract

Background: Cyperenoic acid, one of the main chemical constituents of the root of Croton crassifolius, exhibited potent anti-angiogenic property on the zebrafish embryo model with little cytotoxicity. Nevertheless, its anti-angiogenic mechanism and anti-tumor effect have not been investigated.

Purpose: To investigate the anti-angiogenic mechanisms of cyperenoic acid and evaluate it whether could exert anti-tumor effect by inhibiting angiogenesis.

Study design: Targeting vascular endothelial growth factor receptor-2 (VEGFR2) pathway to inhibit tumor angiogenesis is a significant strategy for cancer treatment. Initially, the anti-angiogenic effect of cyperenoic acid as well as the mechanisms of the action was studied using both in-vitro and in-vivo methodologies. Then, its anti-tumor effect through anti-angiogenesis by attenuating VEGFR2 signaling pathway was evaluated.

Methods: The in-vitro inhibitory effect of cyperenoic acid on the vascular endothelial growth factor (VEGF)-induced angiogenesis was evaluated using human umbilical vein endothelial cells (HUVECs) model. Moreover, its ex-vivo and in-vivo effects were evaluated using the aortic ring assay and the matrigel plug assay. The influence of the cyperenoic acid on tyrosine phosphorylation of VEGFR2 was studied by western blotting assay and the influence on downstream signaling pathway of VEGFR2 also be detected. Computer-docking simulations were carried out to study the interaction between cyperenoic acid and VEGFR2. Finally, its inhibitory effect on tumor growth was studied using breast cancer xenograft model.

Results: Cyperenoic acid possessed little toxicity to HUVECs, but it significantly inhibited VEGF-induced proliferation, invasion, migration and tube formation of HUVECs. Moreover, it inhibited VEGF-induced sprout formation ex vivo and vessel formation in vivo. Further mechanistic study showed that cyperenoic acid could suppress VEGFR2 tyrosine kinase activity and alter its downstream signaling pathways in VEGF-induced HUVECs. In addition, it could form two hydrogen bonds with the ATP binding pocket of the VEGFR2 kinase domain by docking. For breast cancer xenograft model, cyperenoic acid suppressed tumor growth, but no obvious toxic pathologic changes were observed in mice. Besides, it suppressed the phosphorylation of VEGFR2 in tumor, demonstrating its anti-angiogenic ability in vivo partly targeting the VEGFR2.

Conlusion: Cyperenoic acid could exert anti-tumor effect in breast cancer by inhibiting angiogenesis via VEGFR2 signaling pathway.

Keywords: Anti-angiogenesis; Cyperenoic acid; VEGFR2; breast cancer.