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Anti-angiogenic activity of julibroside J8, a natural product isolated from Albizia julibrissin
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Phytomedicine 16 (2009) 703–711 www.elsevier.de/phymed
Anti-angiogenic activity of julibroside J8, a natural product isolated from Albizia julibrissin Hui Huaa, Lei Fenga, Xiao-ping Zhanga, Lian-fen Zhangb, Jian Jinb, a
Laboratory of Natural Medicine, School of Medicine and Pharmaceutics, Jiangnan University, 1800 Lihu RD, Wuxi, Jiangsu 214122, PR China b The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Medicine and Pharmaceutics, Jiangnan University, 1800 Lihu RD, Wuxi, Jiangsu 214122, PR China
Abstract Purpose: The purpose of this study was to investigate the anti-angiogenic properties of julibroside J8, a triterpenoid saponin isolated from Albizia julibrissin. Methods: In the presence of juliborside J8, the growth of human microvascular endothelial cells (HMEC-1), four human tumor cell lines, and a normal cell line (MRC-5) was evaluated by MTT assay. The in vivo anti-angiogenic effect of julibroside J8 was evaluated on a chorioallantoic membrane (CAM) and in transplanted colon carcinoma cells in a nude mice neovascularisation model. Results: Treatment with 0.5–4 mg/ml julibroside J8 resulted in dose-dependent inhibition of growth, migration, and tube formation in HMEC-1 cells; julibroside J8 also inhibited the formation of microvessels on CAM at a concentration of 10–50 mg/egg and reduced vessel density within tumor at a concentration of 0.5–3 mg/kg. Conclusions: Julibroside J8 may be a potent anti-angiogenetic and cytotoxic drug; further investigation is warranted. r 2009 Elsevier GmbH. All rights reserved. Keywords: Julibroside J8; Ginsenoside Rg3; Human microvascular endothelial cell; Angiogenesis
Introduction Angiogenesis is a fundamental method by which new capillaries are formed from pre-existing vasculature. This complex process involves degradation of extracellular matrix, migration and proliferation of endothelial cells, tube formation, and sprouting of new capillary branches (Folkman and Shing 1992). Under normal conditions, such as wound healing, embryonic development, and tissue or organ regeneration, the angiogenic Corresponding author at: Laboratory of Natural Medicine, School of Medicine and Pharmaceutics, Jiangnan University, 1800 Lihu RD, Wuxi, Jiangsu 214122, PR China. Tel./fax: +86 510 85918219. E-mail address: [email protected] (J. Jin).
0944-7113/$ - see front matter r 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2009.01.002
process switches on and off at the appropriate times, indicating tight regulation by stimulatory and inhibitory factors (Hanahan and Folkman 1996). With certain pathological conditions, such as the growth of solid tumors, tumor metastasis, rheumatoid arthritis, and diabetic retinopathy, angiogenesis occurs in a poorly controlled manner (Van Hinsbergh et al. 1999; O’Reilly 1997). Angiogenesis is not only needed for the growth of cancer but is also required for tumor transplantation and metastasis. Since inhibition of angiogenesis could suppress tumor growth and metastases, inhibition of tumor angiogenesis is one of the promising strategies in the development of novel anticancer therapy and in the treatment of other human diseases associated with angiogenesis.
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Albizia julibrissin Durazz is a plant widely distributed in China. Traditionally, the stem bark of Albizia julibrissin was dried and boiled with water. Asians administered this Albizia julibrissin soup as a folk medicine to treat insomnia, diuresis, sthenia, and confusion (Zhu 1998). Julibroside J8 was isolated from Albizia julibrissin and has been reported to have varying degrees of antiproliferation activity in six cancer cell lines (BGC-823, Bel-7402, HeLa, PC-3MIE8, MDAMB-435, and LH-60) in vitro (Zou, et al. 2005; Zheng et al. 2006). However, no reports on its anti-angiogenic activity have been published. In this study, bioassay-guided separation was used to isolate and identify the active component julibroside J8 from the traditional Chinese medicinal herb Albizia julibrissin. We then evaluated and confirmed the anti-angiogenic effect of julibroside J8 in vitro and in vivo. In vitro effect was assessed by growth, migration, and tube formation of human microvascular endothelial cells (HMEC-1). In vivo effect of julibroside J8 was tested in an angiogenesis model of chicken chorioallantoic membrane (CAM) and in transplanted colon carcinoma cells in a nude mice neovascularisation model.
Materials and methods Materials The dried stem bark of Ablizia julibrissin were collected from Zhejiang Province of China in September 2005, and were identified by Professor Jian-wei Chen, Chief of the Chinese Herbal Pharmacy in Nanjing University of Traditional Chinese Medicine. A voucher specimen (No. 20050909-1) is deposited in the Department of Natural Medicines, Jiangnan University. Ginsenoside Rg3 (Fig. 1B) was obtained from from Jilin yatai Pharmaceutical Co. Ltd. CD31 monoclonal antibody was purchased from BD PharMingen. MTT, MCDB-131, EGF, FBS, RPMI-1640, and trypsin were obtained from Sigma (St. Louis, MO, USA). HeLa, Bel-7402, MCF-7, B16F10, and C51 were purchased from Shanghai Institute of Cell Biology in the Chinese Academy of Sciences. HMEC-1 and MRC-5 were obtained from the French National Institute for Health and Medicine Research. HMEC-1 were grown in MCDB-131 medium supplemented with 10% FBS, 2 mM L-glutamine, 10 ng/ml epidermal growth factor, and 1 mg/ml hydrocortisone. HeLa, Bel-7402, MCF-7, B16F10, C51, MRC-5 cells were grown in RPMI1640 containing 10% FBS. All cells were maintained at 37 1C in a humidified 5% CO2 incubator.
Isolation and characterization of the anti-angiogenic component (julibroside J8) from Albizia julibrissin Durazz The powder of dried stem barks (15 kg) from Albizia julibrissin was extracted with 70% ethanol. The ethanol extract was evaporated, dissolved in water, and then sequentially extracted with chloroform, ethyl acetate, and n-butanol. The n-butanol fraction (214.5 g) containing the most cytotoxicity was dissolved in water and fractionated through a Diaion HP-20 resin column with gradient elution (100% water-100% ethanol). The fraction (65 g) eluted with 75% ethanol was then subjected to silica gel column chromatography using a solvent system of chloroform–methanol–water (9:1:0.1–6:4:1) to collect into 60 fractions (500 ml/ fraction). Analysis of individual fractions was performed by thin layer chromatography (TLC) plate with a solvent system butanol:acetic acid:water ratio of 4:1:5, and the separated spots on TLC were visualized with UV irradiation and 50% sulfuric acid. The fractions having the same pattern of separated spots on the TLC were collected together, and the cytotoxic activity of each fraction against HMEC-1 was determined by MTT assay. The active fraction (24.5 g) was applied on RP C18 column chromatography (eluted with 55–75% methanol), and subsequently on a preparative HPLC OBD-C18 column chromatography (eluted with methanol 65%, 3.0 ml/min, 215 nm); this produced 45 mg of cytotoxic compound with an IC50 value of 1.2 mg/ml against HMEC-1. The cytotoxic compound isolated by this procedure was over 99% pure, as determined by high-pressure liquid chromatography (HPLC). The chromatographic conditions were as follows: a Nova-pack C18 column (150 mm 4.6 mm; I.D. 5 mm) was used. The mobile phase was methanol/water (65:35, v/v) with a flow rate of 1.0 ml/min at a column temperature of 25 1C, and the detection wavelength was set at 215 nm. The structure of the cytotoxic compound was identified by comparing the chemical and spectral data (m.p., UV, IR, MS, 1H NMR, and 13C NMR) with those reported in the literature (Zou et al. 2005) and was identified as julibroside J8 (Fig. 1A). Julibroside J8 was dissolved in water and diluted to the desired concentrations immediately prior to use.
Cell growth inhibition assay Growth inhibitory effect of julibroside J8 on cells was measured by the MTT assay. Briefly, adherent cells in 0.1 ml media were plated in each well of 96-well plates and treated with different concentrations of julibroside J8 (0, 1.0, 2.0, 5.0, 10.0, and 20.0 mg/ml) after 24 h. Cells were incubated at 37 1C for another 48 h. The culture medium
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18 16 14
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Fig. 1. Chemical structures of julibroside J8 and ginsenoside Rg3, and effect of julibroside J8 on various cell lines. (A) Chemical structure of julibroside J8. (B) Chemical structure of ginsenoside Rg3. (C) Growth inhibition effects of julibroside J8 on various cell lines. Viable cell number was detected using MTT reduction. Values are mean7S.D. from triplicate cultures, and the experiments were repeated five times with similar results.
was removed and 20 ml of 5 mg/ml MTT was added. Four hours later, the supernatant was discarded and 100 ml of DMSO was added to each well. The mixture was shaken and measured at 570 nm using a Universal Microplate Reader (EL800, BIO-TEKINSTRUMENTS INC). The inhibition ratio (I%) was calculated by the following equation: I% ¼ (AcontrolAtreated)/Acontrol 100%, where Atreated and Acontrol are the average absorbance of three parallel experiments from treated and control groups, respectively. The IC50 was taken as the concentration causing 50% inhibition of cell proliferation and was calculated by SAS statistical software.
Cell migration assay Migration activity was tested using a previously described method (Valster et al. 2005). Briefly, 1 105 cells were seeded in each of 24-well plates and cultured overnight. After cells had attached completely, the cells were scraped with a line in the middle of the plate by a yellow tip; the width of the line gap is about 250–270 mm. After scraping the cells, the medium was changed with fresh medium with or without julibroside J8 (1.2 mg/ml). Cells were incubation for different time intervals (6, 12, and 24 h), and each culture was
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photographed at a magnification of 100 with a microscope video system (Olympus, IX70, Japan). The widths of each gap in three places were measured and averaged.
Capillary tube formation assay The tube formation assay was performed to determine the effect of julibroside J8 on angiogenesis in vitro. Formation of capillary tube-like structures by HMEC-1 was assessed in a matrigel-based assay as previously described (Kubota et al. 1988). Briefly, a 96-well plate coated with 60 ml matrigel per well was allowed to solidify at 37 1C for 1 h. Each well was seeded with 4 104 HMEC-1 and cultured in MCDB containing various concentrations of julibroside J8 (0.5, 1.0, 2.0, and 4.0 mg/ml) for 24 h; at the same time one negative control group was established. The enclosed networks of tubes were photographed from five randomly chosen fields under a microscope (Olympus, IX70, Japan). The total length of the tube structures in each photograph was measured using Adobe Photoshop software (Soeda et al. 2000). Inhibition of tube formation was calculated as [1(tube length treated/tube length control)] 100%.
Chicken chorioallantoic membrane (CAM) assay Anti-angiogenic activity was measured using CAM assay as previously described (Song et al. 2003). Fertilized, domestic chick embryos were incubated for 4 days and then windowed with slight modification. Briefly, a small hole (approximately 2 cm in diameter) was formed by removing the shell and inner shell membrane from the air space site and then the exposed area was sealed with cellophane tape. The eggs were returned to the incubator at 37.8 1C (humidity 55–60%) and incubated with the window upright for 3 days. On day 9, sterile filter paper disks saturated with julibroside J8 (10, 30, 50 mg), ginsenoside Rg3 (100 mg), or saline solution (control) were placed on the surface of the growing CAM vessels. After 48 h, the results of the samples relative to the controls were photographed and assayed.
julibroside J8 at doses of 0.5, 1.5, or 3 mg/kg in a volume of 200 ml vehicle. The control group was treated with vehicle mixture only and the positive group was treated with ginsenoside Rg3 at a dose of 10 mg/kg. Four weeks later, the mice were killed by an excess of ketamin and rompun, and the solid tumors were removed and weighed. During the session, signs of possible toxicity were recorded. Handling of animals in all animal experiments met the standards required by the National Institutes of Health Guide for Care and Use of Laboratory Animals.
Immunohistochemistry and microvessel counts Tumor weight was measured and data were statistically analyzed. The rate of inhibition (IR) was calculated according to the formula: IR ¼ [(mean tumor weight of the experimental groupmean tumor weight of the control group)/mean tumor weight of the control group] 100%. Expression of CD31 in tumor tissue was analyzed to evaluate the level of vessel formation during tumor development. The dissected tumors were fixed in 10% neutral buffered formalin and embedded in paraffin. To specifically immunostain the mouse CD31 using rat anti-mouse CD31 monoclonal antibody, zincfixed paraffin section preparation and immunohistochemistry analysis were performed according to the manufacturer’s instruction (Eno Gene). The vessel density was determined in tumor vessel ‘‘hot spots’’ by means of a Chalkley point-counting grid at high power (200 ) by two observers as described previously (Fox et al. 1995). The average of the vessel counts in five hot spots per section was recorded.
Statistical analysis Values were given as mean7S.D. Comparisons between two groups were performed by Student’s t-test; comparisons among multiple groups were performed by analysis of variance (ANOVA), or Dunnet t-test, with SPSS11.0 software (SPSS, Chicago, IL). po0.05 was considered statistically significant.
Constructions of animal model and treatment
Results
BALB/C-nu/nu mice, weighing 18–20 g, were obtained from Institute of Zoology, Chinese Academy of Sciences, and maintained in a standard animal room for 1 month prior to initiation of experiment. Mice were anaesthetized with 5 mg/kg sodium pentobarbital before inoculation. After the injection of 2 105 colon carcinoma cell (C51) subcutaneously into the right axilla of the mice, the mice were divided into five groups (six mice per group) at random and were treated with oral
Cell growth inhibition We first investigated the effect of julibroside J8 on growth of HMEC-1 and MRC-5. The results in Fig. 1C indicate that julibroside J8 caused a decrease in growth of HMEC-1. The IC50 was estimated to be 1.2 mg/ml. Julibroside J8 did not significantly inhibit growth of normal cells (MRC-5) at this concentration; the IC50 was at 15 mg/ml. We further investigated the effect of
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julibroside J8 on the growth of human cancer cells. Results showed that julibroside J8 had different cytotoxic potency against different tumor cells. The IC50 was estimated to be approximately 8–10 mg/ml.
4 mg/ml clearly inhibited the gel-induced tube formation of HMEC-1 in a dose-dependent manner.
Julibroside J8 inhibits HMEC-1 migration
In the absence of julibroside J8, the area of the CAM below the disks showed no alteration in vascular density, and a normal branching pattern of blood vessels was present (Fig. 4a). After 48 h treatment with julibroside J8, the branching pattern of the blood vessels below the disks was dramatically decreased (Figs. 4b and c) in a dose-dependent manner. Julibroside J8 at 10 mg/egg started to inhibit CAM angiogenesis and doses of 30 mg/egg and 50 mg/egg reduced the dense capillary network to approximately 32.6% and 64.4%, respectively, of the control and ginsenoside Rg3 (Fig. 4d); at 100 mg/egg capillary density was only 53.2% of the control value (Fig. 4B).
From Fig. 2A, we observed that endothelial cells migrated and narrowed down the width of the gap from 0 to 24 h. The width of the gap for the julibroside J8treated cells was broader than that of control cells as follows: 24078 mm vs. 238711 mm after 6 h; 180711 mm vs. 150719 mm after 12 h; 14074 mm vs. 75714 mm after 24 h (Fig. 2B).
Julibroside J8 capillary-like tube structures formation in matrigel In vitro angiogenesis test using gel-induced tube formation of HMEC-1 was carried out. As shown in Fig. 3, treatment with julibroside J8 ranging from 0.5 to
Control
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The width of the gap (um)
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Inhibition of CAM angiogenesis
Julibroside J8 inhibits tumor growth and angiogenesis in vivo When solid tumors in athymic mice were treated with the julibroside J8, suppression of tumor growth was observed. The rate of inhibition (IR) of tumor growth for 0.5, 1.5, and 3 mg/kg julibroside J8 treatment and 10 mg/kg ginsenoside Rg3 treatment in nude mice was 16.7%, 35.2%, 67.5%, and 54.3%, respectively (Fig. 5B). These data indicate significant inhibition of tumor growth by julibroside J8. During the treatment period, no evident changes in gross measures were observed, including weight loss, feeding, and behavior. Angiogenesis was evaluated by microvessel density assay of frozen sections stained with anti-CD31 monoclonal antibody, which has a highly specific affinity for vascular endothelial cells. The microvessel density (MVD) in tumor was markedly reduced in the julibroside J8-treated group compared to controls (Fig. 5A). The number of microvessels per vascular hot spot was 25.471.44 (0.5 mg/kg), 15.871.9 (1.5 mg/kg), and 10.471.8 (3 mg/kg) in the julibroside J8-treated group, 1271.9 (10 mg/kg) in the ginsenoside Rg3-treated group, and 31.573.4 in the control group, respectively. These results showed the MVD in julibroside J8 (3 mg/ kg) treatment group to be at least three times lower than that in control groups (Fig. 5C).
0 0
6
12 18 Time (h)
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Fig. 2. The effect of julibroside J8 on HMEC-1 cells migration. (A) The migration pattern of HMEC-1 cells after 24 h of julibroside J8 (1.2 mg/ml) treatment. (B) The migration ability was analyzed by averaging three widths of the line gap for three independent experiments. Julibroside J8 (~). **po0.01 vs. control (’) at 6, 12, and 24 h, n ¼ 3 per group.
Discussion Using natural medicines in the treatment of various diseases, including those with angiogenic properties, is a long-standing practice in China. Based on this, it is highly possible that there may be some natural medicines that can be used to treat angiogenesis-related
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100
∗∗
Inhibition %
80 60
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40 20 0 0.5
1 2 Concentration (μg/ml)
4
Fig. 3. Effect of julibroside J8 on the matrix-induced tube formation. (A) HMEC-1 were treated with different concentrations of julibroside J8 as indicated for 24 h. (a) control; (b) julibroside J8, 1 mg/ml; (c) julibroside J8, 2 mg/ml; (d) julibroside J8, 4 mg/ml. (B) Quantification of the dose-dependent relationship of julibroside J8 inhibition of tube formation in HMEC-1 is shown. Each treatment was repeated at least three times. *po0.05. **po0.01.
diseases. Our laboratory has focused on examining plants used in traditional Chinese medicine for the identification of novel anti-angiogenic compounds. Our strategy is to explore potential anti-angiogenic compounds from traditional Chinese medicine used to treat angiogenesis-related diseases. We previously established models of angiogenesis in vitro and in vivo in order to screen for potentially active compounds from traditional Chinese medicine herbal treatments. Our earlier results showed that Albizia julibrissin extract had significant
anti-angiogenic effects in vitro and in vivo (data not shown). In the present study, we isolated an antiangiogenic agent, julibroside J8, from Albizia julibrissin extract based on polarization and bioactivities test in HMEC-1. The structure of the isolated anti-angiogenic agent was in accordance with the findings reported by Zou et al. (2005). To investigate the effect of julibroside J8 on the tumor growth and to evaluate cytotoxicity in normal cell, we selected four tumor cell lines and the normal cell line
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Inhibition %
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60 50 *
40 30 20 10 0 10
30 julibroside J8
50
100 μg/egg ginsenoside Rg3
Fig. 4. Inhibition of julibroside J8 on angiogenesis in the chicken chorioallantoic membrane (CAM). (A) Membranes were treated with (a) control; (b) 30 mg/egg julibroside J8; (c) 50 mg/egg julibroside J8; (d) 100 mg/egg ginsenoside Rg3 served as a positive control. (B) Quantification of the dose-dependent relationship of julibroside J8 inhibition of microvessel formation in CAM is shown. Photographs are representative pictures from three independent experiments. *po0.05, **po0.01.
MRC-5. Notably, julibroside J8 had little or no effect on normal growth or growth factor-induced proliferation of non-endothelial cells (Fig. 1C). These results indicated that endothelial cells were more sensitive to julibroside J8 compared to non-endothelial cells. It is well known that the mechanism of growth inhibition of HMEC-1 cells is complex. Cytotoxicity was assessed first using trypan blue staining, and the results showed that the viability of HMEC-1 was more than 90% even at 10 mg/ml of julibroside J8 treatment for 48 h (data not shown). Based on this result, angiogenesis assays in vitro were performed at a concentration range of 0.5–10 mg/ml of julibroside J8, but no cytotoxicity was observed.
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Angiogenesis depends on complex cellular activities, such as extracellular matrix degradation, proliferation and migration of endothelial cells, and morphological differentiation of endothelial cells to form tubes (Bussolino et al. 1997). We have demonstrated the dose-dependent anti-angiogenic properties of julibroside J8 using a series of in vitro models that include the most salient features of angiogenesis, such as endothelial cell proliferation, migration, and tube formation. To confirm the anti-angiogenesis of julibroside J8 in vivo, we tested its anti-angiogenic activity using CAM models; results showed that julibroside J8 could significantly inhibit the formation of microvessels in a dosedependent manner. The multistage inhibition of angiogenesis by julibroside J8 was further confirmed using tumor neoangiogenesis in transplanted colon carcinoma models. The process of angiogenesis within the tumor mass is critically required for the growth of solid tumor to a few millimeters in size (Cao et al. 1998; Kamphaus et al. 2000). The inhibition of microvessel formation within the tumor is therefore an important contributory cause of reduced tumor size in the julibroside J8-treated group, which can be explained by the limited supply of nutrients and oxygen due to the decreased vessel density in the tumor secondary to administration of julibroside J8. In order to prevent our findings being confounded by the fact that the vessel density is normally decreased in smaller tumors, the experiments were carried out by comparing the vessel density in tumors of similar size from the control and the julibroside J8 groups. The results, as revealed by CD31 immunostaining, confirmed the decreased vessel density in the julibroside J8 group. In addition, no evident changes in gross measures of function, such as weight loss, feeding, or other signs of possible side effects, were observed during treatment, indicating that julibroside J8 treatment seems to lack toxicity and is well tolerated in vivo, at least at the dose used in our study. Additionally, we compared the anti-angiogenic effects of julibroside J8 with ginsenoside Rg3 in the in vivo model. Ginsenoside Rg3, a saponin isolated from Panax ginseng, has demonstrated potent anti-angiogenic activity and antitumor activity in clinical studies, and is one of the most important parts of ginseng. The similarity between julibroside J8 and ginsenoside Rg3 presents in their chemical structures (Fig. 1). Thus, we used it as a positive control for angiogenesis inhibition. As illustrated in Fig. 4, in the presence of julibroside J8 at doses of 10–50 mg/egg, angiogenesis in the CAM model was inhibited in a concentration-dependent fashion. The inhibitory rate of angiogenesis at the dose of 50 mg/egg was approximately 64%, while that of ginsenoside Rg3 100 mg/egg was only 53%, suggesting that julibroside J8 has stronger anti-angiogenic activity. When julibroside J8 and ginsenoside Rg3 were evaluated with the tumor
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Fig. 5. (A) Inhibition of angiogenesis by Julibroside j8 was assessed by microvessel density (MVD) in tumor tissues. The microvessel endothelial cells, with specific reaction to CD31 antibody, were brown-stained and visualized in microscopy. (a) control; (b) julibroside J8, 1.5 mg/kg; (c) julibroside J8, 3.0 mg/kg; (d) ginsenoside Rg3, 10 mg/kg. (B) The inhibition ratio of solid tumors in athymic mice by julibroside J8 and ginsenoside Rg3 (*po0.05). (C) MVD was determined by counting the number of microvessels, as described in the text. The number of microvessels was significantly lower in the julibroside J8-treated group compared to control groups. Values are mean7S.E.M (n ¼ 6). *po0.05.
neoangiogenesis in transplanted colon carcinoma models, the result also showed that julibroside J8 had greater anti-angiogenic activity than the control drug ginsenoside Rg3, thus providing further evidence that the natural compound we isolated possesses potent antiangiogenic properties. In conclusion, julibroside J8 significantly inhibited angiogenesis in vitro and in vivo at relatively low concentrations and compared favorably with ginsenoside Rg3; this effect may result from inhibition of endothelial cells directly. These results taken collectively suggest that julibroside J8 may be a potent antiangiogenetic and cytotoxic drug; further investigation is warranted.
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Phytomedicine 16 (2009) 703–711 www.elsevier.de/phymed
Anti-angiogenic activity of julibroside J8, a natural product isolated from Albizia julibrissin Hui Huaa, Lei Fenga, Xiao-ping Zhanga, Lian-fen Zhangb, Jian Jinb, a
Laboratory of Natural Medicine, School of Medicine and Pharmaceutics, Jiangnan University, 1800 Lihu RD, Wuxi, Jiangsu 214122, PR China b The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Medicine and Pharmaceutics, Jiangnan University, 1800 Lihu RD, Wuxi, Jiangsu 214122, PR China
Abstract Purpose: The purpose of this study was to investigate the anti-angiogenic properties of julibroside J8, a triterpenoid saponin isolated from Albizia julibrissin. Methods: In the presence of juliborside J8, the growth of human microvascular endothelial cells (HMEC-1), four human tumor cell lines, and a normal cell line (MRC-5) was evaluated by MTT assay. The in vivo anti-angiogenic effect of julibroside J8 was evaluated on a chorioallantoic membrane (CAM) and in transplanted colon carcinoma cells in a nude mice neovascularisation model. Results: Treatment with 0.5–4 mg/ml julibroside J8 resulted in dose-dependent inhibition of growth, migration, and tube formation in HMEC-1 cells; julibroside J8 also inhibited the formation of microvessels on CAM at a concentration of 10–50 mg/egg and reduced vessel density within tumor at a concentration of 0.5–3 mg/kg. Conclusions: Julibroside J8 may be a potent anti-angiogenetic and cytotoxic drug; further investigation is warranted. r 2009 Elsevier GmbH. All rights reserved. Keywords: Julibroside J8; Ginsenoside Rg3; Human microvascular endothelial cell; Angiogenesis
Introduction Angiogenesis is a fundamental method by which new capillaries are formed from pre-existing vasculature. This complex process involves degradation of extracellular matrix, migration and proliferation of endothelial cells, tube formation, and sprouting of new capillary branches (Folkman and Shing 1992). Under normal conditions, such as wound healing, embryonic development, and tissue or organ regeneration, the angiogenic Corresponding author at: Laboratory of Natural Medicine, School of Medicine and Pharmaceutics, Jiangnan University, 1800 Lihu RD, Wuxi, Jiangsu 214122, PR China. Tel./fax: +86 510 85918219. E-mail address: [email protected] (J. Jin).
0944-7113/$ - see front matter r 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2009.01.002
process switches on and off at the appropriate times, indicating tight regulation by stimulatory and inhibitory factors (Hanahan and Folkman 1996). With certain pathological conditions, such as the growth of solid tumors, tumor metastasis, rheumatoid arthritis, and diabetic retinopathy, angiogenesis occurs in a poorly controlled manner (Van Hinsbergh et al. 1999; O’Reilly 1997). Angiogenesis is not only needed for the growth of cancer but is also required for tumor transplantation and metastasis. Since inhibition of angiogenesis could suppress tumor growth and metastases, inhibition of tumor angiogenesis is one of the promising strategies in the development of novel anticancer therapy and in the treatment of other human diseases associated with angiogenesis.
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Albizia julibrissin Durazz is a plant widely distributed in China. Traditionally, the stem bark of Albizia julibrissin was dried and boiled with water. Asians administered this Albizia julibrissin soup as a folk medicine to treat insomnia, diuresis, sthenia, and confusion (Zhu 1998). Julibroside J8 was isolated from Albizia julibrissin and has been reported to have varying degrees of antiproliferation activity in six cancer cell lines (BGC-823, Bel-7402, HeLa, PC-3MIE8, MDAMB-435, and LH-60) in vitro (Zou, et al. 2005; Zheng et al. 2006). However, no reports on its anti-angiogenic activity have been published. In this study, bioassay-guided separation was used to isolate and identify the active component julibroside J8 from the traditional Chinese medicinal herb Albizia julibrissin. We then evaluated and confirmed the anti-angiogenic effect of julibroside J8 in vitro and in vivo. In vitro effect was assessed by growth, migration, and tube formation of human microvascular endothelial cells (HMEC-1). In vivo effect of julibroside J8 was tested in an angiogenesis model of chicken chorioallantoic membrane (CAM) and in transplanted colon carcinoma cells in a nude mice neovascularisation model.
Materials and methods Materials The dried stem bark of Ablizia julibrissin were collected from Zhejiang Province of China in September 2005, and were identified by Professor Jian-wei Chen, Chief of the Chinese Herbal Pharmacy in Nanjing University of Traditional Chinese Medicine. A voucher specimen (No. 20050909-1) is deposited in the Department of Natural Medicines, Jiangnan University. Ginsenoside Rg3 (Fig. 1B) was obtained from from Jilin yatai Pharmaceutical Co. Ltd. CD31 monoclonal antibody was purchased from BD PharMingen. MTT, MCDB-131, EGF, FBS, RPMI-1640, and trypsin were obtained from Sigma (St. Louis, MO, USA). HeLa, Bel-7402, MCF-7, B16F10, and C51 were purchased from Shanghai Institute of Cell Biology in the Chinese Academy of Sciences. HMEC-1 and MRC-5 were obtained from the French National Institute for Health and Medicine Research. HMEC-1 were grown in MCDB-131 medium supplemented with 10% FBS, 2 mM L-glutamine, 10 ng/ml epidermal growth factor, and 1 mg/ml hydrocortisone. HeLa, Bel-7402, MCF-7, B16F10, C51, MRC-5 cells were grown in RPMI1640 containing 10% FBS. All cells were maintained at 37 1C in a humidified 5% CO2 incubator.
Isolation and characterization of the anti-angiogenic component (julibroside J8) from Albizia julibrissin Durazz The powder of dried stem barks (15 kg) from Albizia julibrissin was extracted with 70% ethanol. The ethanol extract was evaporated, dissolved in water, and then sequentially extracted with chloroform, ethyl acetate, and n-butanol. The n-butanol fraction (214.5 g) containing the most cytotoxicity was dissolved in water and fractionated through a Diaion HP-20 resin column with gradient elution (100% water-100% ethanol). The fraction (65 g) eluted with 75% ethanol was then subjected to silica gel column chromatography using a solvent system of chloroform–methanol–water (9:1:0.1–6:4:1) to collect into 60 fractions (500 ml/ fraction). Analysis of individual fractions was performed by thin layer chromatography (TLC) plate with a solvent system butanol:acetic acid:water ratio of 4:1:5, and the separated spots on TLC were visualized with UV irradiation and 50% sulfuric acid. The fractions having the same pattern of separated spots on the TLC were collected together, and the cytotoxic activity of each fraction against HMEC-1 was determined by MTT assay. The active fraction (24.5 g) was applied on RP C18 column chromatography (eluted with 55–75% methanol), and subsequently on a preparative HPLC OBD-C18 column chromatography (eluted with methanol 65%, 3.0 ml/min, 215 nm); this produced 45 mg of cytotoxic compound with an IC50 value of 1.2 mg/ml against HMEC-1. The cytotoxic compound isolated by this procedure was over 99% pure, as determined by high-pressure liquid chromatography (HPLC). The chromatographic conditions were as follows: a Nova-pack C18 column (150 mm 4.6 mm; I.D. 5 mm) was used. The mobile phase was methanol/water (65:35, v/v) with a flow rate of 1.0 ml/min at a column temperature of 25 1C, and the detection wavelength was set at 215 nm. The structure of the cytotoxic compound was identified by comparing the chemical and spectral data (m.p., UV, IR, MS, 1H NMR, and 13C NMR) with those reported in the literature (Zou et al. 2005) and was identified as julibroside J8 (Fig. 1A). Julibroside J8 was dissolved in water and diluted to the desired concentrations immediately prior to use.
Cell growth inhibition assay Growth inhibitory effect of julibroside J8 on cells was measured by the MTT assay. Briefly, adherent cells in 0.1 ml media were plated in each well of 96-well plates and treated with different concentrations of julibroside J8 (0, 1.0, 2.0, 5.0, 10.0, and 20.0 mg/ml) after 24 h. Cells were incubated at 37 1C for another 48 h. The culture medium
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Fig. 1. Chemical structures of julibroside J8 and ginsenoside Rg3, and effect of julibroside J8 on various cell lines. (A) Chemical structure of julibroside J8. (B) Chemical structure of ginsenoside Rg3. (C) Growth inhibition effects of julibroside J8 on various cell lines. Viable cell number was detected using MTT reduction. Values are mean7S.D. from triplicate cultures, and the experiments were repeated five times with similar results.
was removed and 20 ml of 5 mg/ml MTT was added. Four hours later, the supernatant was discarded and 100 ml of DMSO was added to each well. The mixture was shaken and measured at 570 nm using a Universal Microplate Reader (EL800, BIO-TEKINSTRUMENTS INC). The inhibition ratio (I%) was calculated by the following equation: I% ¼ (AcontrolAtreated)/Acontrol 100%, where Atreated and Acontrol are the average absorbance of three parallel experiments from treated and control groups, respectively. The IC50 was taken as the concentration causing 50% inhibition of cell proliferation and was calculated by SAS statistical software.
Cell migration assay Migration activity was tested using a previously described method (Valster et al. 2005). Briefly, 1 105 cells were seeded in each of 24-well plates and cultured overnight. After cells had attached completely, the cells were scraped with a line in the middle of the plate by a yellow tip; the width of the line gap is about 250–270 mm. After scraping the cells, the medium was changed with fresh medium with or without julibroside J8 (1.2 mg/ml). Cells were incubation for different time intervals (6, 12, and 24 h), and each culture was
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photographed at a magnification of 100 with a microscope video system (Olympus, IX70, Japan). The widths of each gap in three places were measured and averaged.
Capillary tube formation assay The tube formation assay was performed to determine the effect of julibroside J8 on angiogenesis in vitro. Formation of capillary tube-like structures by HMEC-1 was assessed in a matrigel-based assay as previously described (Kubota et al. 1988). Briefly, a 96-well plate coated with 60 ml matrigel per well was allowed to solidify at 37 1C for 1 h. Each well was seeded with 4 104 HMEC-1 and cultured in MCDB containing various concentrations of julibroside J8 (0.5, 1.0, 2.0, and 4.0 mg/ml) for 24 h; at the same time one negative control group was established. The enclosed networks of tubes were photographed from five randomly chosen fields under a microscope (Olympus, IX70, Japan). The total length of the tube structures in each photograph was measured using Adobe Photoshop software (Soeda et al. 2000). Inhibition of tube formation was calculated as [1(tube length treated/tube length control)] 100%.
Chicken chorioallantoic membrane (CAM) assay Anti-angiogenic activity was measured using CAM assay as previously described (Song et al. 2003). Fertilized, domestic chick embryos were incubated for 4 days and then windowed with slight modification. Briefly, a small hole (approximately 2 cm in diameter) was formed by removing the shell and inner shell membrane from the air space site and then the exposed area was sealed with cellophane tape. The eggs were returned to the incubator at 37.8 1C (humidity 55–60%) and incubated with the window upright for 3 days. On day 9, sterile filter paper disks saturated with julibroside J8 (10, 30, 50 mg), ginsenoside Rg3 (100 mg), or saline solution (control) were placed on the surface of the growing CAM vessels. After 48 h, the results of the samples relative to the controls were photographed and assayed.
julibroside J8 at doses of 0.5, 1.5, or 3 mg/kg in a volume of 200 ml vehicle. The control group was treated with vehicle mixture only and the positive group was treated with ginsenoside Rg3 at a dose of 10 mg/kg. Four weeks later, the mice were killed by an excess of ketamin and rompun, and the solid tumors were removed and weighed. During the session, signs of possible toxicity were recorded. Handling of animals in all animal experiments met the standards required by the National Institutes of Health Guide for Care and Use of Laboratory Animals.
Immunohistochemistry and microvessel counts Tumor weight was measured and data were statistically analyzed. The rate of inhibition (IR) was calculated according to the formula: IR ¼ [(mean tumor weight of the experimental groupmean tumor weight of the control group)/mean tumor weight of the control group] 100%. Expression of CD31 in tumor tissue was analyzed to evaluate the level of vessel formation during tumor development. The dissected tumors were fixed in 10% neutral buffered formalin and embedded in paraffin. To specifically immunostain the mouse CD31 using rat anti-mouse CD31 monoclonal antibody, zincfixed paraffin section preparation and immunohistochemistry analysis were performed according to the manufacturer’s instruction (Eno Gene). The vessel density was determined in tumor vessel ‘‘hot spots’’ by means of a Chalkley point-counting grid at high power (200 ) by two observers as described previously (Fox et al. 1995). The average of the vessel counts in five hot spots per section was recorded.
Statistical analysis Values were given as mean7S.D. Comparisons between two groups were performed by Student’s t-test; comparisons among multiple groups were performed by analysis of variance (ANOVA), or Dunnet t-test, with SPSS11.0 software (SPSS, Chicago, IL). po0.05 was considered statistically significant.
Constructions of animal model and treatment
Results
BALB/C-nu/nu mice, weighing 18–20 g, were obtained from Institute of Zoology, Chinese Academy of Sciences, and maintained in a standard animal room for 1 month prior to initiation of experiment. Mice were anaesthetized with 5 mg/kg sodium pentobarbital before inoculation. After the injection of 2 105 colon carcinoma cell (C51) subcutaneously into the right axilla of the mice, the mice were divided into five groups (six mice per group) at random and were treated with oral
Cell growth inhibition We first investigated the effect of julibroside J8 on growth of HMEC-1 and MRC-5. The results in Fig. 1C indicate that julibroside J8 caused a decrease in growth of HMEC-1. The IC50 was estimated to be 1.2 mg/ml. Julibroside J8 did not significantly inhibit growth of normal cells (MRC-5) at this concentration; the IC50 was at 15 mg/ml. We further investigated the effect of
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julibroside J8 on the growth of human cancer cells. Results showed that julibroside J8 had different cytotoxic potency against different tumor cells. The IC50 was estimated to be approximately 8–10 mg/ml.
4 mg/ml clearly inhibited the gel-induced tube formation of HMEC-1 in a dose-dependent manner.
Julibroside J8 inhibits HMEC-1 migration
In the absence of julibroside J8, the area of the CAM below the disks showed no alteration in vascular density, and a normal branching pattern of blood vessels was present (Fig. 4a). After 48 h treatment with julibroside J8, the branching pattern of the blood vessels below the disks was dramatically decreased (Figs. 4b and c) in a dose-dependent manner. Julibroside J8 at 10 mg/egg started to inhibit CAM angiogenesis and doses of 30 mg/egg and 50 mg/egg reduced the dense capillary network to approximately 32.6% and 64.4%, respectively, of the control and ginsenoside Rg3 (Fig. 4d); at 100 mg/egg capillary density was only 53.2% of the control value (Fig. 4B).
From Fig. 2A, we observed that endothelial cells migrated and narrowed down the width of the gap from 0 to 24 h. The width of the gap for the julibroside J8treated cells was broader than that of control cells as follows: 24078 mm vs. 238711 mm after 6 h; 180711 mm vs. 150719 mm after 12 h; 14074 mm vs. 75714 mm after 24 h (Fig. 2B).
Julibroside J8 capillary-like tube structures formation in matrigel In vitro angiogenesis test using gel-induced tube formation of HMEC-1 was carried out. As shown in Fig. 3, treatment with julibroside J8 ranging from 0.5 to
Control
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Inhibition of CAM angiogenesis
Julibroside J8 inhibits tumor growth and angiogenesis in vivo When solid tumors in athymic mice were treated with the julibroside J8, suppression of tumor growth was observed. The rate of inhibition (IR) of tumor growth for 0.5, 1.5, and 3 mg/kg julibroside J8 treatment and 10 mg/kg ginsenoside Rg3 treatment in nude mice was 16.7%, 35.2%, 67.5%, and 54.3%, respectively (Fig. 5B). These data indicate significant inhibition of tumor growth by julibroside J8. During the treatment period, no evident changes in gross measures were observed, including weight loss, feeding, and behavior. Angiogenesis was evaluated by microvessel density assay of frozen sections stained with anti-CD31 monoclonal antibody, which has a highly specific affinity for vascular endothelial cells. The microvessel density (MVD) in tumor was markedly reduced in the julibroside J8-treated group compared to controls (Fig. 5A). The number of microvessels per vascular hot spot was 25.471.44 (0.5 mg/kg), 15.871.9 (1.5 mg/kg), and 10.471.8 (3 mg/kg) in the julibroside J8-treated group, 1271.9 (10 mg/kg) in the ginsenoside Rg3-treated group, and 31.573.4 in the control group, respectively. These results showed the MVD in julibroside J8 (3 mg/ kg) treatment group to be at least three times lower than that in control groups (Fig. 5C).
0 0
6
12 18 Time (h)
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30
Fig. 2. The effect of julibroside J8 on HMEC-1 cells migration. (A) The migration pattern of HMEC-1 cells after 24 h of julibroside J8 (1.2 mg/ml) treatment. (B) The migration ability was analyzed by averaging three widths of the line gap for three independent experiments. Julibroside J8 (~). **po0.01 vs. control (’) at 6, 12, and 24 h, n ¼ 3 per group.
Discussion Using natural medicines in the treatment of various diseases, including those with angiogenic properties, is a long-standing practice in China. Based on this, it is highly possible that there may be some natural medicines that can be used to treat angiogenesis-related
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100
∗∗
Inhibition %
80 60
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1 2 Concentration (μg/ml)
4
Fig. 3. Effect of julibroside J8 on the matrix-induced tube formation. (A) HMEC-1 were treated with different concentrations of julibroside J8 as indicated for 24 h. (a) control; (b) julibroside J8, 1 mg/ml; (c) julibroside J8, 2 mg/ml; (d) julibroside J8, 4 mg/ml. (B) Quantification of the dose-dependent relationship of julibroside J8 inhibition of tube formation in HMEC-1 is shown. Each treatment was repeated at least three times. *po0.05. **po0.01.
diseases. Our laboratory has focused on examining plants used in traditional Chinese medicine for the identification of novel anti-angiogenic compounds. Our strategy is to explore potential anti-angiogenic compounds from traditional Chinese medicine used to treat angiogenesis-related diseases. We previously established models of angiogenesis in vitro and in vivo in order to screen for potentially active compounds from traditional Chinese medicine herbal treatments. Our earlier results showed that Albizia julibrissin extract had significant
anti-angiogenic effects in vitro and in vivo (data not shown). In the present study, we isolated an antiangiogenic agent, julibroside J8, from Albizia julibrissin extract based on polarization and bioactivities test in HMEC-1. The structure of the isolated anti-angiogenic agent was in accordance with the findings reported by Zou et al. (2005). To investigate the effect of julibroside J8 on the tumor growth and to evaluate cytotoxicity in normal cell, we selected four tumor cell lines and the normal cell line
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Inhibition %
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30 julibroside J8
50
100 μg/egg ginsenoside Rg3
Fig. 4. Inhibition of julibroside J8 on angiogenesis in the chicken chorioallantoic membrane (CAM). (A) Membranes were treated with (a) control; (b) 30 mg/egg julibroside J8; (c) 50 mg/egg julibroside J8; (d) 100 mg/egg ginsenoside Rg3 served as a positive control. (B) Quantification of the dose-dependent relationship of julibroside J8 inhibition of microvessel formation in CAM is shown. Photographs are representative pictures from three independent experiments. *po0.05, **po0.01.
MRC-5. Notably, julibroside J8 had little or no effect on normal growth or growth factor-induced proliferation of non-endothelial cells (Fig. 1C). These results indicated that endothelial cells were more sensitive to julibroside J8 compared to non-endothelial cells. It is well known that the mechanism of growth inhibition of HMEC-1 cells is complex. Cytotoxicity was assessed first using trypan blue staining, and the results showed that the viability of HMEC-1 was more than 90% even at 10 mg/ml of julibroside J8 treatment for 48 h (data not shown). Based on this result, angiogenesis assays in vitro were performed at a concentration range of 0.5–10 mg/ml of julibroside J8, but no cytotoxicity was observed.
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Angiogenesis depends on complex cellular activities, such as extracellular matrix degradation, proliferation and migration of endothelial cells, and morphological differentiation of endothelial cells to form tubes (Bussolino et al. 1997). We have demonstrated the dose-dependent anti-angiogenic properties of julibroside J8 using a series of in vitro models that include the most salient features of angiogenesis, such as endothelial cell proliferation, migration, and tube formation. To confirm the anti-angiogenesis of julibroside J8 in vivo, we tested its anti-angiogenic activity using CAM models; results showed that julibroside J8 could significantly inhibit the formation of microvessels in a dosedependent manner. The multistage inhibition of angiogenesis by julibroside J8 was further confirmed using tumor neoangiogenesis in transplanted colon carcinoma models. The process of angiogenesis within the tumor mass is critically required for the growth of solid tumor to a few millimeters in size (Cao et al. 1998; Kamphaus et al. 2000). The inhibition of microvessel formation within the tumor is therefore an important contributory cause of reduced tumor size in the julibroside J8-treated group, which can be explained by the limited supply of nutrients and oxygen due to the decreased vessel density in the tumor secondary to administration of julibroside J8. In order to prevent our findings being confounded by the fact that the vessel density is normally decreased in smaller tumors, the experiments were carried out by comparing the vessel density in tumors of similar size from the control and the julibroside J8 groups. The results, as revealed by CD31 immunostaining, confirmed the decreased vessel density in the julibroside J8 group. In addition, no evident changes in gross measures of function, such as weight loss, feeding, or other signs of possible side effects, were observed during treatment, indicating that julibroside J8 treatment seems to lack toxicity and is well tolerated in vivo, at least at the dose used in our study. Additionally, we compared the anti-angiogenic effects of julibroside J8 with ginsenoside Rg3 in the in vivo model. Ginsenoside Rg3, a saponin isolated from Panax ginseng, has demonstrated potent anti-angiogenic activity and antitumor activity in clinical studies, and is one of the most important parts of ginseng. The similarity between julibroside J8 and ginsenoside Rg3 presents in their chemical structures (Fig. 1). Thus, we used it as a positive control for angiogenesis inhibition. As illustrated in Fig. 4, in the presence of julibroside J8 at doses of 10–50 mg/egg, angiogenesis in the CAM model was inhibited in a concentration-dependent fashion. The inhibitory rate of angiogenesis at the dose of 50 mg/egg was approximately 64%, while that of ginsenoside Rg3 100 mg/egg was only 53%, suggesting that julibroside J8 has stronger anti-angiogenic activity. When julibroside J8 and ginsenoside Rg3 were evaluated with the tumor
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Fig. 5. (A) Inhibition of angiogenesis by Julibroside j8 was assessed by microvessel density (MVD) in tumor tissues. The microvessel endothelial cells, with specific reaction to CD31 antibody, were brown-stained and visualized in microscopy. (a) control; (b) julibroside J8, 1.5 mg/kg; (c) julibroside J8, 3.0 mg/kg; (d) ginsenoside Rg3, 10 mg/kg. (B) The inhibition ratio of solid tumors in athymic mice by julibroside J8 and ginsenoside Rg3 (*po0.05). (C) MVD was determined by counting the number of microvessels, as described in the text. The number of microvessels was significantly lower in the julibroside J8-treated group compared to control groups. Values are mean7S.E.M (n ¼ 6). *po0.05.
neoangiogenesis in transplanted colon carcinoma models, the result also showed that julibroside J8 had greater anti-angiogenic activity than the control drug ginsenoside Rg3, thus providing further evidence that the natural compound we isolated possesses potent antiangiogenic properties. In conclusion, julibroside J8 significantly inhibited angiogenesis in vitro and in vivo at relatively low concentrations and compared favorably with ginsenoside Rg3; this effect may result from inhibition of endothelial cells directly. These results taken collectively suggest that julibroside J8 may be a potent antiangiogenetic and cytotoxic drug; further investigation is warranted.
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