Anthraquinone derivative emodin inhibits tumor-associated angiogenesis through inhibition of extracellular signal-regulated kinase 1/2 phosphorylation

Abstract

An anthraquinone derivative, emodin, suppresses tumor development both in vitro and in vivo. In this study, we examined the anti-angiogenic activity of emodin and its modifying effect on the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. In cell cultures, emodin inhibited endothelial cell proliferation, migration, and tube formation in a dose-dependent manner. In addition, the mouse dorsal air sac assay revealed the vivo anti-angiogenic potential of emodin. Matrix metalloproteinase-9 (MMP-9) expression, which is critical for the angiogenic process, including migration and tube formation, decreased after exposure to emodin, as determined by polymerase chain reaction with reverse transcription (RT-PCR) and gelatin zymography. Moreover, the phosphorylation of ERK 1/2 decreased after exposure to emodin in a dose-dependent manner. These observations suggest that emodin has the potential to inhibit several angiogenic processes and that these effects may be related to suppression of the phosphorylation of ERK 1/2.

Introduction

Tumor cell proliferation and tumor progression are considered to largely depend on angiogenesis (Folkman et al., 1971). In angiogenesis, endothelial cells proliferate, lyse the basement membrane and the extracellular matrix, migrate into the surrounding stroma, and finally mature as new tubular vessels (Folkman and Shing, 1992, Carmeliet, 2000). In a healthy adult, new vessel formation is strictly restricted to the local the environment, including wound healing, acute inflammation, and menstruation. In contrast, angiogenesis in tumors is not controlled. Tumor cells continuously form abnormal vessels that are irregular, extensive, and circuitous. Expansion of a microtumor beyond 1–2 mm in size requires a continuous blood supply (Seno et al., 2002). Once tumor cells acquire the ability to induce angiogenesis, the tumor grows aggressively, which promotes invasion and metastasis. Angiogenesis involves many angiogenic molecules, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and interleukin-8 (IL-8), and anti-angiogenic molecules, including angiostatin, thrombospondin-1, and endostatin.

Because the angiogenic response is restricted in adults, although with a few exceptions, and normal endothelial cells are quiescent, resistance to anti-angiogenic therapy rarely develops, in contrast to tumor-targeted therapy. Therefore, inhibition of angiogenesis is considered a promising approach for cancer therapy or prevention.

Emodin (1,3,8-trihydroxy-6-methylanthraquinone) is derived from the rhizome of Rheum palmatum L. (Tsai and Chen, 1992, Liang et al., 1995). R. palmatum L. has been used since ancient times, to treat inflammatory diseases such as peptic ulcers and as a strong laxative. It has been documented that emodin, which suppresses bacterial and tumor growth, has a vasorelaxant effect (Koyama et al., 1988, Huang et al., 1991a, Huang et al., 1991b). Although, inhibitory effects of emodin on NF-κB, protein tyrosine kinase, protein kinase C, and activator protein-1 (AP-1) have been reported (Zhang and Hung, 1996, Huang et al., 2004b, Yang et al., 2004), its anti-angiogenic effect has not been well studied. Recently, it was reported that emodin suppressed angiogenesis induced by VEGF-A by blocking VEGFR2 phosphorylation (Kwak et al., 2006). However, whether emodin inhibits tumor-associated angiogenesis remains unclear. Therefore, the focus of this article was on the effect of emodin on angiogenesis. When searching for an inhibitor of tumor-associated angiogenesis, we found that emodin had strong anti-angiogenic activity. In the present study, we examined the in vitro effect of emodin on four important angiogenic processes: endothelial cell proliferation, migration, differentiation, and matrix degradation. Furthermore, we demonstrated the anti-angiogenic effect of emodin in vivo.