Cancer Letters

Cancer Letters

Volume 239, Issue 2, 8 August 2006, Pages 271-280
Cancer Letters

Rosmarinic acid inhibits angiogenesis and its mechanism of action in vitro

https://doi.org/10.1016/j.canlet.2005.08.025Get rights and content

Abstract

Rosmarinic acid (RA), a water-soluble polyphenolic compound with anti-oxidative and anti-inflammatory activities, inhibited several important steps of angiogenesis including proliferation, migration, adhesion and tube formation of human umbilical vein endothelial cells (HUVEC) in a concentration-dependent manner. RA also reduced intracellular reactive oxygen species (ROS) level, H2O2-dependent VEGF expression and IL-8 release of endothelial cells. These findings suggested that the anti-angiogenic potential of RA might be related to its anti-oxidative activity, which further resulted in the inhibition of ROS-associated VEGF expression and IL-8 release.

Introduction

Angiogenesis is characterized by the formation of new capillary from pre-existing vessels. The process consists of the following steps, beginning with activation of endothelial cells by growth factors, followed by enzymatic degradation of basement membrane, detachment of endothelial cells from adhesion proteins, endothelial cell migration into the perivascular spaces and proliferation, and final new vessels formation. The event is highly regulated by various growth factors and cytokines. Vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), tumor necrosis factor alpha (TNF-α) and interleukin-8 (IL-8) are the potent angiogenic factors [1]. Angiogenesis plays a crucial role in the growth and metastasis of tumor and several chronic inflammatory diseases such as rheumatoid arthritis and proliferative diabetic retinopathy. Inhibition of angiogenesis has been recognized as a promising therapeutic approach for the control of tumor growth and metastasis and chronic inflammatory diseases [2]. While many ischemic diseases such as ischemic coronary artery disease, critical limb ischemia and brain infarction may benefit from the induction of angiogenesis [3].

A growing body of evidence has demonstrated that reactive oxygen species (ROS) may act as fundamental signaling molecules during angiogenesis. It is reported that hydrogen peroxide (H2O2) stimulated angiogenesis in vitro [4], [5] and many antioxidants, such as ascorbic acid [6], green tea catechins, vitamin E [7], [8] and resveratrol [9], exhibited anti-angiogenic activity. Rosmarinic acid (RA, Fig. 1), a water-soluble polyphenolic component isolated from many medicinal plants including Salviae miltiorrhizae, a traditional Chinese herb with blood vessel dilation activity, has been reported to have anti-oxidative [10], [11], [12], anti-inflammatory [13], [14], [15] and anti-depressive activities, [16] and protective activity on lipopolysaccharide-induced liver injury [17]. However, the potential effect of RA on angiogenesis and its mechanism had not yet been found. The aim of the present study is to explore the effect of RA on angiogenesis and its mechanism.

Section snippets

Chemicals

Rosmarinic acid (RA), fibrinogen, thrombin from human plasma, 6-aminohexanoic acid, sulforhodamine B (SRB), type I collagen, fibronectin, 2′,7′-dichlorofluorescin diacetate (DCFH-DA) were purchased from Sigma (St Louis, MO, USA). Cell culture medium RPMI 1640 was purchased from Gibco (CA, USA). Calf serum was the product of Si-Ji-Qing Co. (Hangzhou, China). Anti-VEGF antibodies and S–P immunochemical staining kit were purchased from Fuzhou Maixin Biological Technology Ltd (Fuzhou, China). IL-8

Inhibition of tube formation

Endothelial cells seeded on fibrin gel developed tube-like structures spontaneously in the control group (Fig. 2A). Within the tested concentration (12.5–200 μM), RA significantly inhibited tube formation in a concentration-dependent manner. Treatment with 12.5, 25, 50, 100, and 200 μM RA resulted in the inhibition of tube formation by 12.7, 24.8, 39.1, 55.0 and 62.1%, respectively, as compared with the control group (Fig. 2B and C).

Inhibition of cell growth

The proliferation of endothelial cells was examined with SRB

Discussion

Angiogenesis is a complex process that includes degradation of basement membrane, endothelial cell proliferation, migration and adhesion and tube formation. In the present study, although the inhibitory effects of rosmarinic acid (RA) on endothelial cell proliferation and adhesion were less potent than on tube formation and cell migration, RA did inhibit all four important steps of angiogenesis process including proliferation, migration, adhesion and tube formation of endothelial cells.

Reactive

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