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Extract of Artemisia lavandulaefolia Inhibits In Vitro Angiogenesis in Human Umbilical Vein Endothelial Cells

Angiogenesis is important processes for tumor growth and metastasis. Anti-angiogenesis target therapy has recently been known to be new anti-cancer therapeutic strategies. Natural products such as traditional medicine comprise a major source of angiogenesis inhibitors. Artemisia lavandulaefolia has been known to use in the traditional medical practices. However, its molecular mechanism on the tumor protection and therapy was not clearly elucidated. In this study, we investigated the possibility that extract of A. lavandulaefolia inhibits in vitro angiogenesis. Therefore, we examined the effect of extract of A. lavandulaefolia on the vascular network formation of human umbilical vein endothelial cells (HUVECs). We found that the treatment of A. lavandulaefolia extract suppressed the tube formation of HUVECs without any influence on the viability of HUVECs. In addition, extract of A. lavandulaefolia inhibited the migration and invasion of HUVECs. These results suggest that extract of A. lavandulaefolia could be act for an angiogenic inhibitor.


INTRODUCTION
Angiogenesis is a process of remodeling such as sprouting and growth of blood vessels. 1 It is regulated under the control of balance between stimulators and inhibitors. 2 In addition, angiogenesis involve complex and diverse cellular actions such as degradation of extracellular matrix, proliferation and migration of endothelial cells, and morphological differentiation of endothelial cells to form tubes. 3,4 Many traditional oriental medicines consist of medicinal products from plants and animals that are used for treatments. 5 It has been practiced in China, Korea, Japan, and other Asian countries for many centuries. 6 In many cases, traditional medicinal products showed anti-tumor activities and they have been considered as candidates for novel cancer therapeutics. 7,8 Artemisia lavandulaefolia is the Chrysanthemum family plant and is used in traditional medicine as a perennial plant that is widely distributed in Korea. 9 As well as being used as food material, A. lavandulaefolia has been used for the treatment of various diseases in traditional medicine in Korea. 10 A.
lavandulaefolia has been used as a digestive, anthelmintic, and effective odor remover. In addition, A. lavandulaefolia has known effects about gastrointestinal diseases, constipation, pain, belly pain, asthma, and gynecological problems. 10 Recently, it has been reported that A. lavandulaefolia has anti-bacterial and anti-fungal activity against many kinds of the pathogenic bacterium and fungi in the Chinese medicine. [11][12][13][14] Although many studies on the effects of A. lavandulaefolia have been conducted, no exists the information concerning relationship with angiogenesis and its molecular mechanisms.
Therefore, we examined the anti-angiogenic effects by A. lavandulaefolia in human umbilical vein endothelial cells (HUVECs).

A. lavandulaefolia was purchased from Kyeongdong Medicinal
Herb Market (Seoul, Korea). The biomass was dried root and leaf of A. lavandulaefolia. It was converted to a powdered form by cold-extraction using grain alcohol, and was sonicated at room temperature and ambient pressure. The A. lavandulaefolia extract was mixed with 70% grain alcoholic solution (30% pure water). The concentration of A. lavandulaefolia was 100 mg/mL, and the extract was diluted in distilled water. Finally, we used 50, 100, 500, and 1,000 μg/mL A. lavandulaefolia for cytotoxic tests and used 100 μg/mL for in vitro angiogenesis assays.

MTT assay for cell viability
The effect of extract of A. lavandulaefolia on the viability of HUVECs was determined using the MTT assay, which is based on the conversion of MTT (3-(4,5-dimethylthiazol-2-yl)-2,-5-diphenyltetrazoliumbromide) to insoluble MTT-formazan by cleavage of the tetrazolium ring by mitochondrial dehydrogenase enzymes in living cells. Briefly, HUVECs were grown in M199 with 20% FBS at a density of 2 × 10 4 cells on 24-well culture plates. After one night, the media was re-placed with M199 containing 1% FBS, and crude extract of A. lavandulaefolia and the cells were then incubated for 24 hours at 37 o C under a humidified atmosphere that was comprised of 5% CO2. Cells were treated with various concentrations of extract of A. lavandulaefolia (50, 100, 500 mg, and 1 mg). Next, MTT solution (5 mg/mL in H2O) was added to the well, followed by the addition of 0.3 mL of dimethyl sulfoxide to dissolve the MTT-formazan. The amount of MTT-formazan was then determined by measuring the absorbance at 540 nm. Each sample was assayed in triplicate, and the experiment was repeated three times.

In vitro tube formation assay
Before performing the test, 0.3 mL matrigel was transferred to 24-well plate and incubated for 30 minutes. HUVECs (2 × 10 4 cells) were plated on a layer of polymerized matrigel and treated with or without extract of A. lavandulaefolia at 37 o C for 24 hours.
Cell morphological changes were captured through a phase contrast microscope and photographed at 40× magnification. Each sample was assayed in duplicate, and independent experiments were repeated three times.

In vitro wounding migration assay
HUVECs were seeded onto 24-well culture plate until confluence and left overnight. Media was aspirated the next day, and cells were scratched with a 200 μL pipette tip along the diameter of the well. Cells were washed twice with PBS and incubated at 37 o C and 5% CO2. After wounding, the cells were incubated in M199 with 1% serum, 1 mM thymidine, and/or extract of A. lavandulaefolia. These culture conditions minimized proliferation of HUVECs. Wound diameters were photographed at 24 hours. Wound closure was determined with optical microscopy at 40× magnification. Each sample was assayed in duplicate, and independent experiments were repeated three times.

In vitro invasion assay
Invasion assay was performed using a trans-well chambers system (Corning Inc., Cambridge, MA, USA) with 8.0-μm pore polycarbonate filter inserts. The upper side of trans-well was coated with 10 μL of matrigel (0.5 mg/mL) at room temperature for 1 hour. Complete media was plated in the lower parts of the trans-well chamber filters, and HUVECs (2 × 10 4 cells) and extract of A. lavandulaefolia in serum-free media were placed in the upper part. Cells were incubated at 37 o C for 24 hours, fixed with methanol, and then stained with hematoxyline/eosin. Cells on the upper surface of the filter membrane were removed by wiping with a cotton swab. Invaded cells were determined with optical microscopy at 40× magnification. Each sample was assayed in duplicate, and independent experiments were repeated three times.

Data analysis and statistics
Data are presented as means ± standard deviation. P < 0.05 was considered significant.

Extract of Artemisia lavandulaefolia do not show any cytotoxic effect on the viability of human umbilical vein endothelial cells
The cytotoxic effect of the A. lavandulaefolia extract on the HUVECs was examined by treatment with various concentration of extract of A. lavandulaefolia (from 50 to 1,000 mg/mL) and determined by MTT assay. Treatment with the A. lavandulaefolia extract for 24 hours has no effect on the viability of HUVECs (Fig.   1). This result indicates that the A. lavandulaefolia has no cytotoxic effect in HUVECs.

Extract of Artemisia lavandulaefolia suppress tube formation of human umbilical vein endothelial cells
To examine the effect of A. lavandulaefolia on the differentiation of endothelial cells, we conducted the tube formation assay. The capillary tube formation was conducted on polymerized matrigel beds and tube formation with elongated tube networks was observed in 6 to 24 hours in HUVECs (Fig. 2).
Importantly, when HUVECs were treated with A. lavandulaefolia extract, tube network was completely abrogated (Fig. 2). This finding demonstrates that extract of A. lavandulaefolia suppressed the tube formation of HUVECs.

Extract of Artemisia lavandulaefolia inhibit migration and invasion of human umbilical vein endothelial cells
Endothelial cell migration and invasion are one of the critical steps in the formation of new blood vessels. 15 Therefore, we investigated the effect of extract of A. lavandulaefolia on the movement of HUVECs from a wounded edge to an open area by wound healing assay. Treatment with A. lavandulaefolia extract for 24 hours markedly decreased the migration of HUVECs compared with that of control (Fig. 3A). Migration of endothelial cells was decreased by about 63% by A. lavandulaefolia extract treatment compared with that of control (Fig. 3B).
To examine the effect of extract of A. lavandulaefolia on the invasiveness of HUVECs, we performed invasion assay with a trans-well system. Trans-wells were prepared such that the upper sides of the filter were coated with matrigel, and used for invasion assay. Extract of A. lavandulaefolia inhibited the invasiveness of HUVECs compared with that of control after 24 hours of incubation (Fig. 4A). The invasiveness of endothelial cells was inhibited by about 62% by A. lavandulaefolia extract treatment compared with that of control (Fig. 4B). Taken together, extract of A. lavandulaefolia strongly suppressed the migration and invasion of HUVECs.

DISCUSSION
Angiogenesis is the developmental process of new capillaries by sprouting from pre-existing vasculature, 16,17 and is required as a significant component of a wide variety of physiological process and pathological conditions. 18 In 1971, Folkman 15 was firstly built up that angiogenesis is essential process in tumor growth. Since then, angiogenesis have been recognized as important strategies for cancer therapy. 19,20 Angiogenesis are initiated by the secreted growth factors, chemokines/cytokines and other mediators. 3 Tumor initiation and progression are also closely linked to angiogenesis. Thus, angiogenic responses in tumor could be targets for development of anti-cancer therapeutic drugs. 5 The studies for novel anti-cancer drugs from natural products have been continued through the research of scientists worldwide in looking for new bio-active compounds. 20 A. lavandulaefolia is one of the traditional medicinal products and is usually used for food materials. 11 Effects of A. lavandulaefolia have been known to use for treatment in the traditional medicinal field. However, scientific studies on the effects of A. lavandulaefolia were not clearly elucidated. Recently, A. lavandulaefolia has been known to show anti-bacterial and anti-fungal effects. A. lavandulaefolia has been also reported that it has induced apoptosis and necrosis of hela cells. 21 In this study, we investigated whether extract of A. lavandulaefolia has anti-angiogenic activities in HUVECs. The MTT assay was first carried out. Because the MTT assay is one of the most widely used in cell viability assay, which measures the cytotoxicity of molecules. The result is that extract of A. lavandulaefolia did not affect the viability of HUVECs (Fig. 1). This result was expected because extract of A. lavandulaefolia widely using as food materials. To determine the effect on the anti-angiogenesis of extract of A. lavandulaefolia, we examined in vitro tube formation assay. It is important step of angiogenesis which promote morphological differentiation into capillary-like structure. In the absence of extract of A. lavandulaefolia, HUVECs formed capillary-like networks. However, tube-like structure was suppressed in the presence of extract of A. lavandulaefolia in HUVECs (Fig. 2). Endothelial cell migration and invasion are fundamental step during angiogenesis. 4 Therefore, we determined the effect of extract of A. lavandulaefolia on the migration and invasion of HUVECs. As shown in Figures 3 and 4, migration and invasion were remarkably reduced by treatment extract of A. lavandulaefolia in HUVECs.
Research of the functional ingredients from the traditional medicinal products is very important for therapy development.
Chemical composition of A. lavandulaefolia essentioal oil has been previously reported in several studies. 22,23 The main components of A. lavandulaefolia essentioal oil was reported caryophyllene, b-thujone, eucalytol and b-farnesene. 24 However, the numerous activities of these components for medical therapy were not clearly elucidated. Thus, a functional study of the chemical composition of traditional medicine material will be added for effective disease treatment. In summary, the major findings reported here are that extract of A. lavandulaefolia inhibited angiogenesis in HUVECs. Therefore, extract of A. lavandulaefolia may have potential to be a useful angiogenesis inhibitor. Further study is required to elucidate the mechanism of action on angiogenesis by extract of A. lavandulaefolia.