Elsevier

Vascular Pharmacology

Volume 47, Issues 2–3, August–September 2007, Pages 131-138
Vascular Pharmacology

Artesunate inhibits angiogenesis and downregulates vascular endothelial growth factor expression in chronic myeloid leukemia K562 cells

https://doi.org/10.1016/j.vph.2007.05.002Get rights and content

Abstract

Artesunate (ART), a semi-synthetic derivative of artemisinin extracted from the Chinese herb Artemisia annua, is a safe and effective antimalarial drug. In the present investigation, we analyzed the inhibitory effects of ART on angiogenesis and on VEGF production in chronic myeloid leukemia (CML) K562 cells in vitro and in vivo. In order to analyze the effect of ART on VEGF secretion in K562 cells, we examined the level of VEGF secreted in conditioned media (CM) by ELISA assay. The result showed that ART could decrease the VEGF level in CM of K562 cells, even at a lower concentration (2 μmol/l, P < 0.01). The inhibitory effect of in vitro angiogenesis was tested on aortic sprouting in fibrin gel. ART could effectively suppress the stimulating angiogenic ability of CM by pretreated with K562 cells for 48 h in a time-dependent manner (days 3–14). The antiangiogenic effect of ART was further evaluated in vivo in chicken chorioallantoic membrane (CAM) neovascularization model. The result indicated that the stimulating angiogenic activity was decreased in response to the K562 cells treated with ART or the CM from K562 cells pretreated with ART in a dose-dependent manner (3–12 μmol/l). Furthermore, we analyzed the level of VEGF expression by western blot and detected the form of VEGF mRNA by RT-PCR in K562 cells. The experiments showed that ART could inhibit the VEGF expression, correlated well with the level of VEGF secreted in CM. These findings suggest that ART might present potential antileukemia effect as a treatment for CML therapy, or as an adjunct to standard chemotherapeutic regimens.

Introduction

The development of new blood vessels from preexisting ones is generally referred to as angiogenesis (Kumar et al., 1998). In the last three decades, considerable research has been conducted documenting that tumor growth and metastasis require angiogenesis (Holash et al., 1999). Angiogenesis can be divided into a series of temporally regulated responses, including protease induction, endothelial migration, proliferation and differentiation (Klagsbrum and Moses, 1999). This is a highly complex process, in which a number of cytokines and growth factors released by endothelial cells (ECs), tumor cells and matrix cells are involved. Among many kinds of activators of angiogenesis, vascular endothelial growth factor (VEGF) is considered as one of the most potent angiogenic factors. The expression of VEGF has been suggested to be related to some fundamental features of solid tumors, such as growth rate, microvessel density, and the development of tumor metastasis (Drake and Little, 1999). Recently, there are increasing evidences indicating that VEGF also plays an important role in the development and progression of chronic myeloid leukemia (CML) (Aguayo et al., 2000). In addition, the VEGF concentrations in the bone marrows were found to correlate inversely with the length of survival in patients having chronic phase CML (Aguayo et al., 2003). Therefore, VEGF has been expected as an emerging target for CML therapy and some corresponding strategies have been raised including decreasing the production of VEGF (He et al., 2003), blocking the binding of VEGF to its receptors (Mordenti et al., 1999), and inhibiting VEGF receptor tyrosine kinases (Dias et al., 2001). In the previous studies, we have reported (Chen et al., 2004a, Chen et al., 2004b) that artesunate inhibits the VEGF expression in solid tumor xenograft and exhibits the potent antiangiogenic effect in solid tumors and described (Wu et al., 2005) the apoptotic activity of artesunate in cultured human umbilical vein endothelial cell (HUVEC). In this paper, we investigate the effects of artesunate on angiogenesis and on VEGF production in CML.

Artemisinin, a sesquiterpene lactone isolated from the plant Artemisia annua L., and its derivatives are presently used in various countries as an antimalarial drug with little toxicity to human and have a potent effect on chloroquine-resistant malarial parasites (Klayman, 1985, Woerdenbag et al., 1994). Artesunate (ART) is the main artemisinin derivatives and is more water-soluble and effective antimalaria than artemisinin. In present study, we developed the antiangiogenic potential of ART on in vitro models of the aortic sprouting in fibrin gel and on in vivo models of the chicken chorioallantoic membrane (CAM) neovascularization. We also examined the effect of ART on the production of VEGF by K562 cell and assessed the inhibitory effect of ART on expression of VEGF in K562 cells. Evidences indicate that ART was a potent inhibitor of K562 cell-induced angiogenesis.

Section snippets

Materials and animals

Artesunate was purchased from Guiling Pharmaceutical Co. (Guangxi, China). Working solutions were prepared by dissolving the compound in dimethyl sulphoxide (DMSO) before the experiments. The final concentration of DMSO is less than 0.1% in all experiments. Rabbit anti-human VEGF polyclonal antibody (A-20), Actin polyclonal antibody (I-19) and western blotting luminol reagent (sc-2048) were bought from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). Nitrocellulose membranes were supplied

ART decreases the VEGF level in CM of K562 cells

ELISA analysis was performed to determine the amount of secreted VEGF protein in conditioned media (CM). K562 cells pretreated with ART were grown in serum-free medium for 24 h, and the secreted protein of VEGF in culture media was determined by ELISA. Compared to normal K562 cells, K562 cells pretreated with 2 μmol/l ART showed a 25.7% decrease of the secreted VEGF level (P < 0.01) (Fig. 1).

Effects of ART on aortic sprouting in fibrin gel

Microscopically, incubated with CM from normal K562 cells, the aortic rings formed microvascular-like

Discussion

Based on the evidence that VEGF plays an important role in tumor angiogenesis, many different angiogenic inhibitors have been tested (Siemeister et al., 1998). In the previous studies we have confirmed (Chen et al., 2004a, Chen et al., 2004b) the antiangiogenic potential of ART on angiogenesis and on expressions of VEGF and VEGF receptor KDR/flk-1 in nude mice by means of human ovarian cancer HO-8910 implantation. We have yet demonstrated (Wu et al., 2005) that ART could induce apoptosis of

Acknowledgements

This work was supported by the grants from the Zhejiang Provincial foundation of Natural Science (No. M303842).

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