Longan and cancer

This article appeared in a journal published by Elsevier. The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues. Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited. In most cases authors are permitted to post their version of the article (e.g. in Word or Tex form) to their personal website or institutional repository. Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/authorsrights Author's personal copy JOURNAL OF FUNCTIONAL FOODS 5 ( 2 0 1 3 ) 1 0 8 8 –1 0 9 6 Available at www.sciencedirect.com journal homepage: www.elsevier.com/locate/jff Effects of dried longan seed (Euphoria longana Lam.) extract on VEGF secretion and expression in colon cancer cells and angiogenesis in human umbilical vein endothelial cells Atita Panyathepa, Teera Chewonarina, Khanittha Taneyhillb, Young-Joon Surhc, Usanee Vinitketkumnuena,* a Department of Biochemistry, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand Division of Clinical Chemistry, Department of Medical Technology, Faculty of Associated Medical Sciences, Chiang Mai University, Chiang Mai 50200, Thailand c Tumor Microenvironment Global Core Research Center, College of Pharmacy, Seoul National University, Seoul 151-724, South Korea b A R T I C L E I N F O A B S T R A C T Article history: Angiogenesis is a critical event in cancer metastasis, via delivery of needed oxygen and Received 18 January 2013 nutrients to tumor cells. Anti-angiogenesis is one strategy for controlling cancer progres- Received in revised form sion. We herein report anti-angiogenesis activity of dried longan seeds using colon adeno- 28 February 2013 carcinoma cells (SW480 cells) and human umbilical vein endothelial cells (HUVECs). Accepted 4 March 2013 Sephadex LH-20 column chromatography was used for separate three dried longan seed Available online 28 March 2013 fractions. We firstly evaluated vascular endothelial cell growth factor (VEGF) secretion, expression and colony formation of SW480 cells, using enzyme-linked immunosorbent Keywords: Dried longan seed Fraction atinase activity and tube formation of HUVECs were determined via proliferation assay, gelatin zymography and in vitro tube formation assay, respectively. The results suggest that VEGF SW480 cell Angiogenesis HUVEC 1. assay (ELISA), Western blot analysis and soft agar assays. Meanwhile cell proliferation, gel- dried longan seed fractions could be potential angiogenic inhibitors not only interruption of VEGF secretion and expression in SW480 cells but also abrogation of cell proliferation, the activity of gelatinase and tube formation of HUVECs.  2013 Elsevier Ltd. All rights reserved. Introduction Colorectal cancer (CRC) is one of the most common cancers and is the second leading cause of cancer-related deaths worldwide (Ferlay et al., 2011; Markowitz & Bertagnolli, 2009). Metastasis is considered to be the critical cause of CRC patient mortality (Carpizo & D’Angelica, 2009; Mayo & Pawlik, 2009). In metastasis, cancer cells spread to distant organs through blood or lymphatic vessels, circulate through the intravascular stream, and then proliferate at another site (Folkman, 1971). To sustain tumor growth metastasis, growth of a vascular network is necessary in tumor masses beyond 2 mm3 in order to provide oxygen and nutrients. The process of angiogenesis involves a complex interplay between cells, soluble factors and extracellular matrix (ECM) components. This process is initiated by release of angiogenic factors from tumors and binding with their specific receptors on endothelial cells (ECs), leading to ECs activation. Thereafter, four steps in the formation of new blood vessels include: (1) degradation of basement membrane and extracellular matrix by proteases such as matrix metalloproteinases (MMPs), which are produced by activated ECs (2) directional migration of ECs into * Corresponding author. Tel.: +66 53 945325; fax: +66 53 894031. E-mail address: usaneecmu@yahoo.com (U. Vinitketkumnuen). 1756-4646/$ - see front matter  2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jff.2013.03.004 Author's personal copy JOURNAL OF FUNCTIONAL FOODS the newly created space (3) ECs division at the migration tip, and (4) lumen formation, generation of new basement membrane with the recruitment of pericytes for stabilization, formation of tubular structures and finally blood flow (Bussolino, Mantovani, & Persico, 1997). In general, angiogenesis is regulated through the stimulation of proangiogenic factors (e.g. VEGF, bFGF, and tumor necrosis factor-a) (Auerbach, Lewis, Shinners, Kubai, & Akhtar, 2003; Nicosia & Ottinetti, 1990; Risau, 1990). Among these, vascular endothelial growth factors (VEGFs) appear to be the most important angiogenic factor for sustaining tumor growth. They are commonly secreted by most types of human cancer cells, but usually not by endothelial cells. They function as mitogen, inducers for migration and release of MMP2, MMP-9, and MT1-MMP by ECs and vascular permeability factor (Beck & D’Amore, 1997; Dvorak, 2002; Velasco & Lange-Asschenfeldt, 2002). Overexpression of VEGF has been found in various types of tumor, including breast, colorectal, stomach, and lung cancers (Ishigami et al., 1998; Takahashi, Kitadai, Bucana, Cleary, & Ellis, 1995). Recently, VEGF has received attention as a target for therapeutic strategies against tumor angiogenesis. Anti-angiogenic agents derived from natural products are attractive, since natural products often contain mixtures of biological compounds that can act on the multistep angiogenesis process (Singh & Agarwal, 2003) and can synergistically combine with chemotherapy treatment for reduction of side effects (Sagar, Yance, & Wong, 2006). Longan fruit, Euphoria longana Lam. (Syn. Dimocarpus longan Lour.) is widely grown in Southern China, India, and Southeast Asia (Rangkadilok et al., 2007), and is used in Chinese traditional medicine. Dried longan seed is a source of polyphenol-rich antioxidant compounds such as corilagin, gallic acid, and ellagic acid (Rangkadilok, Worasuttayangkurn, Bennett, & Satayavivad, 2005). Moreover, longan seed extracts are capable of reducing proliferation and enhancing apoptosis in various colorectal cancer cells (Chung, Lin, Chou, & Hsu, 2010). Our previous research has shown that dried longan seed fractions obtained from Sephadex-LH-20 column chromatography exerted strong MMPs inhibitory activity. MMPs are the major factors leading to angiogenesis via facilitating the proliferation and migration of endothelial cells into new area and release of ECM-bound proangiogenic factors (e.g. bFGF, VEGF, and TGFb) (Rundhaug, 2003). Recent reports have shown that ellagic acid has potent anti-angiogenesis effects via reduced VEGF production in various cancer cell types (Losso, Bansode, Trappey, Bawadi, & Truax, 2004) and abolished the angiogenesis processes in HUVECs (Wang et al., 2012a, 2012b). The current study aimed to analyze the effect of dried longan seeds on VEGF secretion and expression in colon cancer cells, as well as and angiogenesis processes of endothelial cells. 2. Materials and methods 2.1. Plant material and extraction Fresh longan (E. longana Lam.) seeds were dried at 50 C for 2 days and then ground to powder. The powder was extracted 5 ( 2 01 3 ) 10 8 8–10 9 6 1089 with 80% acetone in a ratio 1:10 (w/v) at room temperature for 24 h. The resulting filtrate was collected through Whatman No.1 filter paper. The filtrate was evaporated and lyophilized to obtain a crude extract. The crude extract in 20 mg/mL ethanol was separated via Sephadex LH-20 column chromatography. Three fractions were collected according to the absorbance at 360 nm and labeled as fractions 1, 2 and 3. 2.2. Cell culture The human colon adenocarcinoma cell lines (SW480) (ATCC, Rockville, MD, USA) were selected as models. They were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Invitrogen Corporation, NY, USA) with 10% fetal bovine serum (Invitrogen Corporation, NY, USA) at 37 C under 5% CO2 humidified atmospheric air. Human Umbilical Vein Endothelial Cells (HUVECs) were obtained from American Type Culture Collection (ATCC, Rockville, MD, USA) and maintained in 0.1% (w/v) gelatin coated dishes with VascuLife Media supplemented growth factors (Lifeline Cell Technology, Frederick, MD, USA). Their growth condition was similar to the colon cancer cells. 2.3. VEGF quantification assay A Human Vascular Endothelial Growth Factor (VEGF) ELISA kit (Calbiochem, Darmstadt, Germany) was used to measure VEGF production in SW480 cells supernatant samples. In short, conditioned media was collected from SW480 cells treated with each fraction at different concentrations (0– 100 lg/mL) for 24 h. Each sample was measured for total protein concentration and then analyzed for VEGF secretion according to the manufacturer’s instructions. The colorimetric reaction was quantified with a spectrophotometer at 450/ 540 nm. The VEGF standard curve used known concentrations of human VEGF protein. 2.4. Western blot analysis The immunoblotting assay was performed to examine the effect of dried longan seed fractions on VEGF protein expression in SW480 cells. Briefly, SW480 cells treated with each fraction at various concentrations (0–100 lg/mL) or ellagic acid (0– 30 lM) for 24 h were lysed with lysis buffer. Then, equal amounts of protein lysates in sample buffer were denatured and subjected to 10% sodium dodecyl sulfate–polyacrylamide gels (SDS–PAGE) and the separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). After blocking with blocking solution (5% nonfat dry milk) at room temperature for 1 h, the membranes were incubated with specific primary antibodies against human VEGF protein (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and b-actin (Sigma–Aldrich, St. Louis, MO, USA). For detection, the membranes were incubated with specific secondary antibodies conjugated with horseradish peroxidase and the specific VEGF band was visualized using an enhanced chemiluminescence (ECL) reagent (Amersham Life Science, Amersham, UK). Author's personal copy 1090 2.5. JOURNAL OF FUNCTIONAL FOODS Soft agar assay The soft agar assay is an anchorage independent growth assay in soft agar and is used for tumorigenesis analysis. In first step, 1% (w/v) base agar was mixed with 2X DMEM in a ratio of 1:1 to give 0.5% (w/v) agar and 1X DMEM in the final concentration. The mixture (2.5 mL) was added into 60 mm dishes and the agar was allowed to form. For top agar preparation, equal volumes of mixture between 0.7% (w/v) melting agarose and 2X DMEM in a centrifuge tube were mixed with an SW480 cell suspension at 1x105 cells/mL (100 lL) together with each fraction at various concentrations (0–100 lg/mL). The mixture was poured into the base agar and then incubated at 37 C for 10–14 days. The colonies were analyzed for morphology and the number of colonies formed counted under a (40·) microscope (4 fields/well). 2.6. 5 ( 2 0 1 3 ) 1 0 8 8 –1 0 9 6 with Amicon Ultra-2 Pre-launch Centrifugal Filter 10 K Devices (Millipore Corporation, Darmstadt, Germany). Equal amounts of concentrated protein samples in sample buffer (with non-reducing conditions) were loaded into 10% SDS– PAGE containing 0.1% gelatin. After gel electrophoresis, the gel was cut and then each gel was soaked in 2.5% Triton X-100 (Thermo Scientific, Rockford, IL USA) at room temperature for 1 h and then incubated at 37 C for 24 h in the reaction buffer (50 mM Tris–HCl, 200 mM NaCl, 10 mM CaCl2, pH 7.4). After incubation, each gel was stained with 0.1% Coomassie brilliant blue R-250 (Sigma–Aldrich, St. Louis, MO, USA) and destained with 10% acetic acid in 30% methanol until a clear band appeared against the blue background. The clear bands represented active gelatinases (MMP-2 and MMP-9) activity following specific molecular weight and could be quantified using Gel-Pro Analyzer 32 program (Media Cybernetics, Maryland, USA). Gelatin zymography 2.7. The activity of gelatinases from HUVECs was assessed by gelatin zymography as described previously (Chu, Chiou, Chen, Yang, & Hsieh, 2004). The culture supernatant of HUVECs after 48 h incubation was collected and concentrated Cell proliferation assay 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma–Aldrich, St. Louis, MO, USA) was utilized to determine the potency of dried longan seed fractions on A B C Fig. 1 – Partial purification and characterization of dried longan seed extract. (A) Partial purification of dried longan seed extract by using Sephadex LH-20 column chromatography monitoring an absorbance at 360 nm. (B) Gallic acid and ellagic acid content (lg/mL) of each fraction. (C) Total phenolic and total flavonoid content of each fraction. ap 6 0.001, bp 6 0.005 compared to the other fractions. a**p 6 0.001 vs fraction 3 (Panyathep et al., 2013). Author's personal copy JOURNAL OF FUNCTIONAL FOODS 1091 5 ( 2 01 3 ) 10 8 8–10 9 6 HUVECs proliferation. Briefly, HUVECs suspension at a density of 5 · 103 cells/well in VascuLife media was seeded into a 0.1% (w/v) gelatin coated 96-well plate and incubated overnight. Each fraction at various concentrations (0–400 lg/mL) was dissolved in new media and then added into each well. After incubation at 37 C for 24 h and 48 h, a mixture of new media and 15 lL of MTT solution (5 mg/mL in PBS) were added. The solution was allowed to stand at 37 C for 3–4 h. The formazan (purple) crystals were dissolved with DMSO and the absorbance measured at 540 nm. Hoang & Senger, 2005). This method was minor modified with the previous (Wang et al., 2012b). A 96-well plate was precoated with Matrigel (BD Bioscience, California, USA) (50 lL/well) and then placed in an incubator at 37 C for 30 min. After incubation, the mixture of HUVECs (at a density of 2.4 · 105 cells/mL) with each fraction at various concentrations (0–100 lg/mL) was added into the Matrigel coated plate. Tube formation occurred after incubation at 37 C for 4–6 h and was analyzed for morphology of HUVECs under a light microscope (40·). 2.8. 2.9. Tube formation assay The tube formation assay has been widely used as a screening method for angiogenic factors or angiogenic inhibitors (Auerbach et al., 2003; Browning, Dua, & Amoaku, 2008; Statistical analysis The results are shown as means ± SD of three independent experiments. Statistical difference values were analyzed by a one-way analysis of variance (ANOVA), where ap 6 0.001, A ‡ ‡ ‡ B C β ‡ Fig. 2 – Effects of dried longan seed fractions on VEGF secretion and expression of SW480 cells. Conditioned media was collected from treated SW480 cells with each fraction at various concentrations (0–100 lg/mL) for 24 h for analysis of VEGF secretion by ELISA. The VEGF concentration was compared with total protein concentration of each sample and then expressed as percent remaining VEGF (A). Treated SW480 cell lysate with each fraction at various concentrations (0–100 lg/ mL) or EA (0–30 lM) at 24 h was also collected and examined for VEGF expression via Western blot. The bands (B) were expressed as relative intensity VEGF/b-actin (C). All results shown are mean ± SD, n = 3. ap 6 0.001, bp 6 0.005 and cp 6 0.05 vs untreated controls. à and * were compared with treated conditions at 25 and 50 lg/mL of each fraction, respectively. Author's personal copy 1092 JOURNAL OF FUNCTIONAL FOODS 5 ( 2 0 1 3 ) 1 0 8 8 –1 0 9 6 A B Fig. 3 – Effects of dried longan seed fractions on colony formation of SW480 cells. Top agar containing SW480 cells (1 · 105 cells/60 mm dish) and each fraction at different concentrations (0–100 lg/mL) was poured onto base agar and then placed at 37 C for 2 weeks (until colonies formed). The colonies were counted under a light microscope and are shown as mean ± SD, n = 3. VC: vehicle control (0.5% of DMSO). ap 6 0.001, bp 6 0.005 and cp 6 0.05 vs untreated controls. à and * were compared with treated conditions at 25 and 50 lg/mL of each fraction, respectively. b p 6 0.005 and difference. 3. c p 6 0.05 were considered as a significant Results 3.1. Effect of dried longan seed fractions on VEGF secretion and expression of SW480 cells In a previous report (Panyathep, Chewonarin, Taneyhill, & Vinitketkumnuen, 2013), three dried longan seed fractions were purified by Sephadex LH-20 column chromatography (Fig. 1A). These fractions were characterized by high performance liquid chromatography (HPLC). Fraction 1 was determined as a gallic acid-rich fraction while fraction 3 was an ellagic acid-rich fraction. In addition, fraction 2 contained a low concentration of both compounds (Fig. 1B). In this study, SW480 cells were treated with each fraction for 24 h at nontoxic concentrations (0–100 lg/mL) which have no growth inhibition within 24 h by MTT assay (unpublished results). In each treatment, culture supernatant and cell lysates were collected for determining VEGF secretion by ELISA and VEGF expression by Western blot analysis, respectively. The results are shown in Fig. 2. VEGF secretion in treated SW480 cells at 50 and 100 lg/mL with fraction 1 and fraction 2 was signifi- cantly suppressed by approximately 40–50% as compared with the untreated controls (at 0 lg/mL) (Fig. 2A), while fraction 3 was effective only at 100 lg/mL (cp 6 0.05), by approximately 60% of untreated controls (ap 6 0.001) (Fig. 2B and C). On the other hand fraction 1 and fraction 2 had no effect on the expression of VEGF which was not correlated with the secretion manner. 3.2. Effect of dried longan seed fractions on colony formation of SW480 cells A colony formation or anchorage independent growth assay was used to determine cancer transformation ability and to confirm VEGF secretion from SW480 cells. VEGF secreted from colon cancer cells is able to autoactivate VEGF receptors (VEGFR-1) on the cell surface, leading to the colony growing in soft agar (Fan et al., 2005). Each fraction was mixed with SW480 cell suspension (at 1 · 104 cell/60 mm dish) and top agar. After 2 weeks of colony growth, the colony (>50 cells) was examined under a light microscope (40·). Each fraction clearly diminished colony formation by dose response; fraction 3 seemed to have the greatest effect, with the percentage inhibition at 100 lg/mL more than 90% compared to untreated controls (ap 6 0.001) (Fig. 3). Author's personal copy JOURNAL OF FUNCTIONAL FOODS 5 ( 2 01 3 ) 10 8 8–10 9 6 1093 A A B B * ‡ * ‡ Fig. 4 – Effect of dried longan seed fractions on MMP-2 activity of HUVECs. Culture supernatant of SW480 cells at 48 h was collected for MMP-2 activity assessment by gelatin zymography. Each gel was incubated with each fraction at distinct concentrations (0–100 lg/mL) for 24 h. The clear bands represented MMP-2 activity (A). The intensity of each band was read and is shown as mean ± SD, n = 3 (B). a p 6 0.001, bp 6 0.005 and cp 6 0.05 vs untreated controls. à and * were compared with treated conditions at 25 and 50 lg/mL of each fraction, respectively. 3.3. Effect of dried longan seed fractions on gelatinases activity from HUVECs The releasing of gelatinases from activated ECs contributes to not only dissolution of the extracellular matrix proteins and basement membrane, but also involving in endothelial cell migration and proliferation. This is the first event of the angiogenesis process of endothelial cells. Culture supernatant of HUVECs was collected and then gelatinases activity was analyzed by gelatin zymography. Each gel was co-incubated with each fraction at 0–100 lg/mL or ellagic acid (0–60 lM) for 24 h and then the clear bands were analyzed for MMP-2 and MMP9 activity. Fig. 4A and B shows the potential inhibitory effect of each fraction at 100 lg/mL on MMP-2 activity, approximately by 60–90% inhibition compared to untreated controls (ap 6 0.001), while anti-MMP-9 activity of each fraction was Fig. 5 – Effects of dried longan seed fractions on HUVECs proliferation. HUVECs were treated with each fraction at various concentrations (0–100 lg/mL) or EA and GA (0– 30 lM) for 24 h (A) and 48 h (B). The data are expressed as the average percentage of cell proliferation compared with untreated controls from three independent experiments ± SD. At the corner of each graph, the effects of EA and GA treatment on HUVECs proliferation are given. a p 6 0.001, bp 6 0.005 and cp 6 0.05 vs untreated controls. à and * were compared with treated conditions at 25 and 50 lg/mL of each fraction, respectively. not obviously apparent. The EA-rich fraction 3 showed the strongest potential anti-gelatinase activity, while the inhibition of single EA treatment was also effective to a lesser degree. 3.4. Effect of dried longan seed fractions on HUVECs proliferation Activated ECs proliferation was analyzed by the MTT assay. The proliferation of HUVECs was markedly reduced after Author's personal copy 1094 JOURNAL OF FUNCTIONAL FOODS 5 ( 2 0 1 3 ) 1 0 8 8 –1 0 9 6 A B G H C D I J E F K L Fig. 6 – Effects of dried longan seed fractions on tube formation of HUVECs. HUVECs (1.2 · 105 cells/well) were added into Matrigel coated 96 well plates together with each fraction at distinct concentrations (50 and 100 lg/mL) or EA and GA (15 and 30 lM) for 4–6 h. The morphology of capillary like tubes was captured by phase contrast microscopy. Production of a fragmented network of cells or reduction in capillary like formation of HUVEC was shown with the arrows. The data are representative of for independent experiments. VC: vehicle control (0.5% of DMSO). treatment with each fraction. The results showed percentages of growth inhibition of F1, F2 and F3 at 100 lg/mL were 40.8 (cp 6 0.05), 58.1 (bp 6 0.005) and 41.8 (bp 6 0.005) of untreated controls at 24 h, while at 48 h these were 57.2 (cp 6 0.05), 67.2 (ap 6 0.001) and 66.2 (ap 6 0.001) vs untreated controls, respectively (Fig. 5A and B). There was no effect on single GA treated HUVECs. Of all, fraction 3 even displayed the efficient anti-proliferation of endothelial cells. 3.5. Effect of dried longan seed fractions on tube formation of HUVECs In vitro tube formation is a commonly used method for analyzing angiogenesis (Arnaoutova, George, Kleinman, & Benton, 2009). HUVECs are activated with supplement growth factors contained in the media aligned themselves into a network of capillary-liked tubes after 4–6 h in the absence of treatment conditions (Fig. 6A and B). Co-incubation with each fraction at 50 and 100 lg/mL destroyed the tube networks into small fragments; the effect was most pronounced with the EA-rich fraction 3 (Fig. 6G and L). Single EA and GA treatments also produced such tube fragmentation to a lesser extent (Fig. 6C and F). 4. Discussion Many natural products contain active compounds that are able to interfere with tumor angiogenesis (Sagar et al., 2006; Singh & Agarwal, 2003). Longan (E. longana Lam.) seeds have been reported as a potential source of antioxidant polyphenols, including ellagic acid and gallic acid. Our previous study has collected three fractions (fraction 1, fraction 2 and fraction 3) from dried longan seed extract. In previous data, they were quantified in their total phenolic contents (202.7, 122.9 and 121.2 mg GAE/g DW) and the total flavonoid contents (89.1, 80.4 and 53.2 mg CAE/g DW) (Fig. 1C). Fraction 1 was obviously contained with gallic acid-rich, whereas the amount of ellagic acid was plentiful in fraction 3 (Fig. 1B) (Panyathep et al., 2013). However, anti-cancer properties of ellagic acid and gallic acid have also been studied with evidence of anti-angiogenesis effects in both cancer cells and endothelial cells (Losso et al., 2004; Lu et al., 2010; Wang et al., 2012a). In this study, we focused on the action of dried longan seed fractions on angiogenesis process at the cellular level of both colon cancer cells (SW480 cells) and human umbilical vein endothelial cells (HUVECs). First, the release and expression of VEGF from SW480 cells were indicated that all fractions showed the anti-VEGF secretion activity; however, the only EA-rich fraction 3 being the most potent VEGF expression inhibitor (Fig. 2). The single EA-treated SW480 cells did not suppress the protein levels of VEGF, indicating that the role of synergistic compounds in the EA-rich fraction 3 contributed to down-regulation of VEGF in SW480 cells. Nevertheless, there are numerous polyphenol compounds that can exert anti-angiogenetic effects in colon cancer cells, these evidences reveal the importance of combining these polyphenols and thus significant increasing the anti-carcinogenetic effects (Araujo, Goncalves, & Martel, 2011). The present result also showed the decreasing of colony formation with each fraction treatment was associated with anti-VEGF secretion effect in SW480 cells. Previous research indicated that VEGF production from colon cancer cells contributes to autoactivation of VEGFR-1 on the cell surface and mediates colon cancer cell invasion, migration and colony formation (Fan et al., 2005). Our results suggest that this inhibitory effects of dried longan seed fractions on VEGF Author's personal copy JOURNAL OF FUNCTIONAL FOODS secretion was associated not only the invasive ability of colon cancer cells (SW480 cells) (unpublished results), but also colony growth on soft agar (Fig. 3). Additionally, colony formation or anchorage-independent growth is a key prerequisite for cells to acquire metastatic ability (Wang, 2004). Our data also suggest that the anti-anchorage independent growth effect of the dried longan seed fractions was associated with inhibition of the initiation step of metastasis, which led to attenuation of cancer-mediated angiogenesis. In the processes of new blood vessel synthesis in solid tumors, endothelial cells lining the vessel are a key target (Davis & Senger, 2005; Folkman, 2006; Harris & Thorgeirsson, 1998), and thus treatment strategies involve with the inhibition of endothelial cell growth until the forming of capillary (Oklu, Walker, Wicky, & Hesketh, 2010). In present study human umbilical vein endothelial cells (HUVECs) were chosen as an in vitro angiogenesis model because they are able to create capillary-like tube structures in response to appropriate growth factors (Matsui, Wakabayashi, Asada, Yoshimatsu, & Okada, 2004). First, activated HUVECs release protease enzymes for facilitation of endothelial cell migration and proliferation particularly in MMP-2 and MMP-9 (Kraling et al., 1999; Puyraimond, Weitzman, Babiole, & Menashi, 1999). We observed anti-gelatinases activity of dried longan seed fractions not only in the colon cancer cells (unpublished results), but also in HUVECs (Fig. 4). Correlatively, dried longan seed fractions also belong to the efficient inhibitor in proliferation activity (Fig. 5) as well as tube formation ability in HUVECs (Fig. 6). Indeed the breakage of tube formation was also implied the inhibitory action of each fraction on multiple angiogenesis processes including adhesion, migration, proteases activity and cell alignment. Even though all fractions contribute to diminish in several processes of angiogenesis in HUVECs, EA-rich fraction 3 still manifested the most outstanding anti-angiogenesis activity. Overall the anti-angiogenetic properties of dried longan seeds no doubt lie in synergistic effects of multiple compounds. Their actions seem to interfere with multiple steps of angiogenesis, including VEGF secretion and expression and anchorage-independent growth in colon cancer cells as well as cell proliferation, gelatinase activity, and tube formation of endothelial cells. However, the inhibitory mechanism of dried longan seed fractions is still unclear; this study has provided preliminary data for further investigation. Their anti-angiogenesis and anti-cancer results are reasonable evidence for possible development and utilized in combination with a new cancer treatment approach to reduce cytotoxicity dose and adverse events of conventional chemotherapy drug in the future. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgement This study was supported by Grants of the Royal Golden Jubilee (RGJ) Ph.D. program and the Graduate School, Chiang Mai University. 5 ( 2 01 3 ) 10 8 8–10 9 6 1095 R E F E R E N C E S Araujo, J. R., Goncalves, P., & Martel, F. (2011). Chemopreventive effect of dietary polyphenols in colorectal cancer cell lines. Nutrition Research, 31(2), 77–87. Arnaoutova, I., George, J., Kleinman, H. K., & Benton, G. (2009). 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