Ethyl cinnamate suppresses tumor growth through anti-angiogenesis by attenuating VEGFR2 signal pathway in colorectal cancer

Ethnopharmacological relevance: Kaempferia galanga Linn. is an aromatic medicinal herb with extensively applied in India, China, Malaysia and Other South Asia countries for thousands of years. It has been mentioned to treat abdominal tumors. Ethyl cinnamate (EC), one of the main chemical constituents of the rhizome of K. galanga, exhibited nematocidal, sedative and vasorelaxant activities. However, its anti-angiogenic activity, and anti-tumor effect have not been investigated.

Aim of the study: To investigate the anti-angiogenic mechanism of EC and its anti-tumor effect by suppressing angiogenesis.

Materials and methods: The in vitro anti-angiogenic effect was evaluated using HUVECs model induced by VEGF and zebrafish model in vivo. The influence of the EC on phosphorylation of VEGFR2/KDR/Flk-1 and its downstream signaling pathways were evaluated by western blotting assay. Molecule docking technology was conducted to explore the interaction between EC and VEGFR2/KDR/Flk-1. SPR assay was used for detecting the binding affinity between EC and VEGFR2/KDR/Flk-1. To further investigate the molecular mechanism of EC on anti-angiogenesis, VEGFR2/KDR/Flk-1 knockdown in HUVECs and examined the influence of the EC. Anti-tumor activity of EC was evaluated using colony formation assay and Apoptosis assay. The inhibitory effect of EC on tumor growth was explored using HT29 colon Cancer xenograft model.

Results: EC obviously inhibited proliferation, migration, invasion and tube formation of VEGF-induced HUVECs. EC also induced Apoptosis of HUVECs. Moreover, it inhibited the development of vessel formation in zebrafish. Further investigations demonstrated that EC could suppress the phosphorylation of VEGFR2/KDR/Flk-1, and its downstream signaling pathways were altered in VEGF-induced HUVECs. EC formed a hydrogen bond to bind with the ATP binding site of the VEGFR2/KDR/Flk-1, and EC-VEGFR2 interaction was shown in SPR assay. The suppressive effect of EC on angiogenesis was abrogated after VEGFR2/KDR/Flk-1 knockdown in HUVECs. EC inhibited the colon Cancer cells colony formation and induced Apoptosis. In addition, EC suppressed tumor growth in colon Cancer xenograft model, and no detectable hepatotoxicity and nephrotoxicity. In addition, it inhibited the phosphorylation of VEGFR2/KDR/Flk-1, and its downstream signal pathways in tumor.

Conclusions: EC could inhibit tumor growth in colon Cancer by suppressing angiogenesis via VEGFR2/KDR/Flk-1 signaling pathway, and suggested EC as a promising candidate for colon Cancer treatment.

Anti-angiogenic; Ethyl cinnamate; Kaempferia galanga Linn; VEGFR2.