A cassaine diterpene alkaloid, 3β-acetyl-nor-erythrophlamide, suppresses VEGF-induced angiogenesis and tumor growth via inhibiting eNOS activation.

Nara Tae,Byung-Sun Min,Okwha Kim,Sunghun Na,Jeong-Hyung Lee,Tran Manh Hung,Suhyun Lee,Namho Kim

doi:10.18632/oncotarget.21307

Abstract

Angiogenesis is one of the hallmarks of cancer, playing an essential role in tumor growth, invasion, and metastasis. 3β-Acetyl-nor-erythrophlamide (3-ANE), a cassaine diterpene alkaloid compound from Erythrophleum fordii, exerts various pharmacological effects, including antitumor activity. However, the effects of 3-ANE on tumor angiogenesis and its potential molecular mechanism are still unknown. Here, we demonstrated that 3-ANE inhibited the vascular endothelial growth factor (VEGF)-mediated proliferation, migration, invasion, and capillary-like tube formation of human umbilical vascular endothelial cells (HUVECs), without inducing apoptosis. We also found that 3-ANE blocked angiogenesis in vivo, and suppressed tumor angiogenesis and human lung adenocarcinoma growth in the xenograft tumor model. Furthermore, we showed that 3-ANE blocked VEGF-mediated endothelial nitric oxide synthase (eNOS) phosphorylation, vascular permeability and NO production in HUVECs, via disrupting the VEGF-induced association of eNOS and heat-shock protein 90 (HSP90). Our studies therefore provide the first evidence that 3-ANE inhibits tumor angiogenesis by inhibiting the VEGF-mediated eNOS activation and NO production, and 3-ANE could be a potential candidate in angiogenesis-related disease therapy.

Highlights

Angiogenesis is defined as the generation of new vascular growth sprouting from pre-existing vessels [1, 2]
Figure 2: 3-ANE inhibits vascular endothelial growth factor (VEGF)-induced proliferation of human umbilical vascular endothelial cells (HUVECs). (A) HUVECs were incubated with the indicated concentrations of 3-ANE for 24 h with or without VEGF (40 ng/ml), and cell proliferation was determined by direct cell counting
Mean of three independent experiments performed in triplicate; bars, S.D.; **, P

Summary

INTRODUCTION

Angiogenesis is defined as the generation of new vascular growth sprouting from pre-existing vessels [1, 2]. Heat-shock protein 90 (HSP90) plays an essential role in NO production through the regulation of eNOS activity in ECs. Exposure of ECs to diverse eNOS agonists such as VEGF, induces an increased association of HSP90 with eNOS and eNOS phosphorylation by Akt, leading to elevation of NO production [11, 12, 13]. We found that a series of the cassaine diterpene alkaloids from E. fordii inhibits tube formation of human umbilical vascular endothelial cells (HUVECs) on Matrigel [19]. We demonstrated that 3-ANE significantly inhibits the VEGF-induced endothelial cell proliferation, cell cycle progression, migration and tube formation. It suppresses the VEGF-induced neovascularization in mouse Matrigel implantation model, and tumor-associated angiogenesis in a xenograft tumor model. Our data suggest that 3-ANE may function as a novel and potent angiogenesis inhibitor that suppresses VEGF-induced eNOS activation

RESULTS

DISCUSSION

Findings

MATERIALS AND METHODS