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Suppression of tumor-induced angiogenesis by Brazilian propolis: Major component artepillin C inhibits in vitro tube formation and endothelial cell proliferation
Cancer Letters 252 (2007) 235–243 www.elsevier.com/locate/canlet
Suppression of tumor-induced angiogenesis by Brazilian propolis: Major component artepillin C inhibits in vitro tube formation and endothelial cell proliferation Mok-Ryeon Ahn a, Kazuhiro Kunimasa b, Toshiro Ohta b,*, Shigenori Kumazawa a, Miya Kamihira a, Kazuhiko Kaji b, Yoshihiro Uto c, Hitoshi Hori c, Hideko Nagasawa d, Tsutomu Nakayama a a
Laboratory of Functional Food Science and COE Program in the 21st Century, School of Food and Nutritional Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan b Laboratory of Cell and Molecular Biology of Aging and COE Program in the 21st Century, School of Food and Nutritional Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan c Department of Life System, Institute of Technology and Science, The University of Tokushima Graduate School, Minamijosanjimacho-2, Tokushima 770-8506, Japan d Gifu Pharmaceutical University, 5-6-1 Mitahora-higashi, Gifu 502-8585, Japan Received 11 October 2006; accepted 27 December 2006
Abstract Propolis, a resinous substance collected by honeybees from various plant sources, possesses various physiological activities such as antitumor effects. We have previously shown that propolis of Brazilian origin was composed mainly of artepillin C and that its constituents were quite different from those of propolis of European origin. In this report, we examined an antiangiogenic effects of Brazilian propolis and investigated whether artepillin C was responsible for such effects. In an in vivo angiogenesis assay using ICR mice, we found that the ethanol extract of Brazilian propolis (EEBP) significantly reduced the number of newly formed vessels. EEBP also showed antiangiogenic effects in an in vitro tube formation assay. When compared with other constituents of EEBP, only artepillin C was found to significantly inhibit the tube formation of HUVECs in a concentration-dependent manner (3.13–50 lg/ml). In addition, artepillin C significantly suppressed the proliferation of HUVECs in a concentration-dependent manner (3.13–50 lg/ml). Furthermore, artepillin C significantly reduced the number of newly formed vessels in an in vivo angiogenesis assay. Judging from its antiangiogenic activity in vitro and in vivo, we concluded that artepillin C at least in part is responsible for the antiangiogenic activity of EEBP in vivo. Artepillin C may prove useful in the development of agents and foods with therapeutic or preventive activity against tumor angiogenesis. 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Angiogenesis; Artepillin C; Human umbilical vein endothelial cells; Propolis; Tube formation; Proliferation
*
Corresponding author. Tel./fax: +81 54 264 5571. E-mail address: [email protected] (T. Ohta).
0304-3835/$ - see front matter 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2006.12.039
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1. Introduction Angiogenesis, or new blood vessel growth, is defined as a process in which a network of new blood vessels emerges from preexisting vessels. Judah Folkman first observed in the early 1970s that such new blood vessel growth was required for a tumor to grow over a few mm3 in size, supplying the tumor with nutrition and oxygen for its exponential growth [1]. Since then many investigators have pursued studies for preventing or delaying cancer growth, or even completely eliminating cancer from a patient’s body, by suppressing such neovascularization [2,3]. Antiangiogenic treatment may be useful in the treatment and prevention of cancer progression [4]. Food factors capable of inhibiting angiogenesis, if found, would be useful to stop the progression of small cancers. Propolis, a folk medicine employed in treating various ailments, is a resinous substance collected by honeybees from the bud and bark of certain trees and plants, and stored inside their hives. It has been used in folk medicine from ancient times in many countries and has been extensively studied in Eastern European countries [5,6]. Recently, it has been reported to possess various biological activities such as antibacterial [7,8], antiviral [7,9], anti-inflammatory [10,11], anticancer [12,13], antifungal [7,14], and antitumoral [15] properties. For this reason, propolis is extensively used in foods and beverages to improve health and prevent diseases such as inflammation, diabetes, heart disease, and cancer [16,17]. Propolis has over 150 constituents such as polyphenols (flavonoids, phenolic acids and their esters), terpenoids, steroids, and amino acids, but its composition varies qualitatively and quantitatively with the geographical and botanical origins [18,19]. Because of the geographical differences, propolis samples from Europe, South America, and Asia have different chemical compositions [20–24]. Artepillin C (3,5-diprenyl-4-hydroxycinnamic acid) is one of the principal phenolic acids found specifically in Brazilian propolis. It has various biological activities, such as antibacterial [25], antiviral [26], apoptosis-inducing [27], antioxidant [28], and anticarcinogenic [29,30] properties. Konishi et al. reported that artepillin C has an extremely low absorption efficiency and bioavailability in vivo, in comparison to those of caffeic acid, which is absorbed and distributed by the monocarboxylic acid transporter (MCT)-mediated transport system
[31]. Aga et al. found that artepillin C exhibits antibacterial and antiviral activity [25], but is also highly cytotoxic to a variety of malignant human and murine solid tumor cell lines in vitro [32]. Furthermore, artepillin C was found to inhibit the growth of transplanted solid human and mouse tumors including that of malignant melanoma, in athymic and thymic mice, respectively [32]. Konishi reported that artepillin C is mainly permeated across Caco-2 cells by transcellular passive diffusion [33]. However, there have been only a limited number of reports concerning the effects of propolis and no reports of the effects of artepillin C on angiogenesis [34–36]. In this study, we investigated in vivo anti-angiogenic activity of the ethanol extracts of Brazilian propolis in vivo. We further analyzed its effects on angiogenesis in vitro through inhibition of tube formation and endothelial cell proliferation and on angiogenesis in vivo by its major component artepillin C. 2. Materials and methods 2.1. Materials Medium 199 and all other chemicals were purchased from Sigma (St. Louis, MO, USA). Artepillin C was purchased from Wako Pure Chemicals Industries (Osaka, Japan) for in vitro studies and was synthesized according to the method of our previous report for in vivo studies [37,38]. Medium MCDB-104 was a product of Nihon Pharmaceutical (Tokyo, Japan). Fetal bovine serum (FBS) was purchased from Moregate (Brisbane, Australia). Cellgen was obtained from Koken (Tokyo, Japan). Epidermal growth factor (EGF) was purchased from BD Biosciences (Bedford, MA). Human basic FGF (Recombinant) was purchased from Austral Biologicals (San Ramon, CA, USA). 2.2. Experimental sample preparation Brazilian propolis was collected in Minas Gerais State, where Baccharis dracunculifolia DC. is the main botanical source of the propolis. Propolis sample was extracted with ethanol (30 ml of ethanol/g of propolis) at room temperature for 24 h. The ethanol suspension was separated by centrifugation at 470g for 15 min at 25 C, and the supernatant was concentrated in a rotary evaporator under reduced pressure at 40 C until reaching a constant weight, and then redissolved in ethanol. The preparation obtained was named ethanol extract of Brazilian propolis (EEBP), and the color of EEBP was brown. EEBP was stored under a dry condition at 4 C until analyzed.
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2.3. Cell culture Human umbilical vein endothelial cells (HUVECs) were grown in HUVEC growth medium (MCDB-104 medium supplemented with 10 ng/ml EGF, 100 lg/ml heparin, 100 ng/ml endothelial cell growth factor and 10% FBS) [39]. Incubation was carried out at 37 C under a humidified 95–5% (v/v) mixture of air and CO2. The cells were seeded on plates coated with 0.1% gelatin and allowed to grow to sub-confluence before experimental treatment. 2.4. Dorsal air sac assay The mouse dorsal air sac (DAS) assay was performed as described previously with slight modifications [40]. This study used female ICR mice (7 weeks old, Japan SLC, Japan) weighing 30 ± 5 g. The animals were kept on a 12 h-light/12 h-dark cycle environment with constant temperature and humidity. The body weight of mice was measured semi-daily. A chamber, which was prepared by covering both sides of a Millipore ring with Millipore filters of 0.45 lm pore size, was filled with a suspension of S180 tumor cells (3.0 · 106 cells) in 0.15 ml of calciumand magnesium-free phosphate buffered saline (PBS). This chamber was implanted into the subcutaneous dorsal air sac created in ICR mice by injecting an appropriate volume of air. For each experiment the mice were randomly divided into several groups as follows: (I): negative control mice (group I-1); S180-treated positive control mice (group I-2); animals with daily oral administration of 2.5% (group I-3) and 5% (group I-4) EEBP diet, (II): negative control mice (group II-1); S180-treated positive control mice (group II-2); animals with daily oral administration of 0.125% (group II-3), 0.25% (group II-4) and 0.5% (group II-5) artepillin C diet. The negative control mice were implanted only with the calcium- and magnesium-free PBS-containing chamber. The groups were administered once a day for 6 days starting from the day of implantation of the chamber. On day 7, the implanted chambers were removed from the subcutaneous area of the treated animals, and a white ring with the same inner diameter as that of the Millipore ring was placed at the site where the ring was located. The angiogenic response was assessed under a dissecting microscope by determining the number of newly formed blood vessels greater than 3 mm in length within the area encircled by the white ring. The blood vessels that were newly formed due to the malignant tumor cells were morphologically distinguishable from the preexisting background vessels by their zigzag character. 2.5. Tube formation assay Capillary tube-like structures formed by HUVECs in collagen gel were prepared as previously described with slight modifications [39]. Collagen gels were prepared with
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Cellgen (type I collagen). Aliquots (200 ll) of collagen solution (0.21% in M 199) were poured into the wells of 24-multiwell culture plates, and the plates were incubated at 37 C for 30 min to solidify gels. HUVECs (6.0 · 104 cells/cm2) in MCDB-104 with 0.5% FBS were seeded onto the collagen-coated wells, and left at 37 C in a 5% CO2 incubator for 1 h to become attached to the collagen gel. After removing the medium, 150 ll aliquots of the collagen solution was overlaid and subjected to gelation as described above. Subsequently, 650 ll aliquots of MCDB-104 with 0.5% FBS supplemented with 10 ng/ml bFGF, 8 nM/ml PMA, and 25 lg/ml ascorbic acid with various concentrations of either EEBP or artepillin C (0, 3.13, 12.5 and 50 lg/ml) were added to the wells and incubated for 36 h. The resulting web-like capillary structure was viewed with a microscope under 100· magnification and captured with a Olympus C4040ZOOM digital camera. The tube formation was quantified by determining the pixel number of tubes in each image using NIH Image program (developed at the U.S. National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/). 2.6. Measurement of proliferation inhibition of cells HUVECs were seeded onto gelatin-coated 24-multiwell plates at a density of 1.0 · 104 cells/cm2 in HUVEC growth media. After a 24-h incubation at 37 C in a 5% CO2 incubator, artepillin C was added to the wells (3.13–50 lg/ml) and the cells were further cultured for 3 days. The number of cells was counted with a Coulter Counter (Coulter Electronics, Hialeah, USA). 2.7. Statistical analysis The significance of difference between different groups was analyzed by the multiple comparison test. Results were expressed as means ± SE. obtained from three experiments. Comparisons between control and treatments were performed using Student’s unpaired t-test (*P < 0.05; **P < 0.005). 3. Results We previously analyzed the chemical composition of propolis from various geographic origins [20,41,42]. Table 1 shows the main constituents of the ethanol extract of Brazilian propolis which we used in the current study. The composition of propolis primarily depends upon the vegetation of the area where it was collected and secondarily upon the methods for its extraction [20]. It has been shown that Baccharis dracunculifolia is an important plant source of Brazilian propolis collected in Minas Gerais [43]. A distinctive feature of Brazilian propolis is that it lacks flavonoids, such as quercetin and kaempferol, and phenolic acid esters, such
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Table 1 Qualitative and quantitative analysis of Brazilian propolis Main constituents
Contenta (mg/g of EEP)
Caffeic acid p-Coumaric acid Artepillin C
1.6 27.4 43.9
a Each value (mg/g of EEP) is expressed as a mean of triplicate analyses for each sample. EEP, ethanol extracts of propolis. Data in this table are derived from our previous report [20].
as caffeic acid phenethyl ester and cinnamyl caffeate, both of which are commonly contained in propolis from other origins. On the other hand, artepillin C (Fig. 1), a unique substance found only in propolis from Brazilian origin, is the major constituent of EEBP. In our previous study, we found over 40 mg of artepillin C per g of EEBP [20]. The next most abundant components were p-coumaric acid at under 30 mg per g and caffeic acid at under 2 mg per g of EEBP, which can also be found in propolis from other origins. 3.1. Suppression of tumor-induced angiogenesis in vivo by oral administration of Brazilian propolis
2 mm
Angiogenesis index
To evaluate the effect of Brazilian propolis on the angiogenesis in vivo, we first performed a DAS assay using ICR mice. Oral administration of Brazilian propolis significantly suppressed tumor-induced angiogenesis in a dose-dependent manner (Fig. 2). Angiogenesis indexes (the numbers of newly formed blood vessels) were 5.4, 3.4 and 2.8 by positive control (group I-2), 2.5% (group I-3) and 5% EEBP (group I-4), respectively, while that of negative control (group I-1) was about 0.5. The numbers of newly formed blood vessels in mice treated with foods containing 2.5% and 5% EEBP were suppressed to 59% and 46%, respectively, compared to the positive control group. In addition, no signs of toxicity were observed in any ICR mice (body weight change during the feeding period; data not shown).
* *
Negative control
Fig. 1. Chemical structure of artepillin C.
Positive control
2.5% EEBP
5% EEBP
Fig. 2. Suppressive effects of Brazilian propolis on tumorinduced angiogenesis in DAS assay (in vivo angiogenesis assay). Chamber filled with 0.15 ml of S180 cell suspension (3.0 · 106 cells) were inoculated subcutaneously to ICR mice. Arrowheads point to newly formed vessels with a zigzagging character. S180treated positive control mice (b) induced strong angiogenic responses compared to negative control mice (a). Oral administration of 2.5% (c) and 5.0% (d) Brazilian propolis (EEBP) reduced the number of newly formed vessels. Angiogenesis index was defined as the number of newly formed blood vessels above 3 mm in length and 0.075 mm in diameter. Brazilian propolis significantly reduced Angiogenesis index in a dose-dependent manner (e). Scale bar, 2 mm. Values are expressed as means ± SE (n = 5). *P < 0.05, as compared to the positive control.
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3.2. Inhibition of HUVEC tube formation, an in vitro model of angiogenesis, by EEBP We examined the effect of EEBP on angiogenesis in vitro using a tube formation model of HUVECs cultured in a 2-D system. After induction of tube formation, the endothelial cells formed a network of capillary-like tubes, which were composed of multiple cells that gathered together and adhered to each other. Fig. 3 shows the inhibitory effects of EEBP on tube formation of endothelial cells. At concentrations of 3.13 and 12.5 lg/ml, EEBP slightly reduced the width of the tubes. At a concentration of 50 lg/ml, EEBP completely inhibited elongation of HUVECs. 3.3. Inhibition of HUVEC tube formation, an in vitro model of angiogenesis, by artepillin C We then examined the effects of three constituents of EEBP on angiogenesis in vitro using a tube formation model of HUVECs. First, the inhibitory effects of artepillin C, the most abundant constituents of EEBP, on tube formation of endothelial cells cultured in a 2-D system were tested (Fig. 4). At a concentration of 3.13 lg/ml, artepillin C slightly reduced the width of the tubes. At a concentration of 12.5 lg/ml, artepillin C reduced both the width and the length of the tubes. At a concentration of 50 lg/ml, artepillin C completely inhibited tube formation in a concentration-dependent manner. Area ratios of the tubes per pictured field were 49.1%, 35.6%, 17.8% and 5.3% at concentrations of 0, 3.13, 12.5 and 50 lg/ml, respectively, which were calculated to be 72.5%, 36.2% and 10.8% (% of control) at concentrations of 3.13, 12.5 and 50 lg/ml, respectively (Fig. 5). On the other hand, both p-coumaric acid and caffeic acid did not show any significant inhibitory effects on tube formation (data not shown). 3.4. Inhibition of HUVEC proliferation, another in vitro model of angiogenesis, by artepillin C We then examined the effects of artepillin C on endothelial cell proliferation, another in vitro model of angiogenesis (Fig. 6). Artepillin C significantly reduced cell proliferation to 94.6%, 71.0% and 36.9% (% of control) at concentrations of 3.13, 12.5 and 50 lg/ml, respectively. Thus, artepillin C was shown to inhibit cell proliferation in a concentration-dependent manner, with a projected IC50 of 37.2 lg/ml. 3.5. Suppression of tumor-induced angiogenesis in vivo by oral administration of artepillin C
Fig. 3. Inhibitory effects of Brazilian propolis on tube formation of endothelial cells. Cell were sandwiched between two layers of collagen gels at a density of 6.0 · 104 cells/cm2 and induced to form blood vessel-like tubes. The experiment was repeated three times and representative data are shown. HUVECs were seeded and cultured between two collagen gel layers in the presence of various concentrations of Brazilian propolis. (a) control, (b) 3.13 lg/ml, (c) 12.5 lg/ml, (d) 50 lg/ml. Scale bar, 100 lm.
Oral administration of artepillin C was tested for effects on the angiogenesis in vivo. Artepillin C significantly suppressed tumor-induced angiogenesis in a dosedependent manner (Fig. 7). Angiogenesis indexes (the
numbers of newly formed blood vessels) were 5.2, 2.4, 2.0 and 1.75 by positive control (group II-2), 0.125% (group II-3), 0.25% (group II-4) and 0.5% (group II-5)
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Tube area (%)
50 40
*
30
*
20 10
*
0 control
3.13
12.5
50
Concentration (μg/ml)
Fig. 5. Quantification of inhibitory effects of artepillin C on tube formation of endothelial cells. Cells were treated with artepillin C and the areas of formed tubes were measured as described in materials and methods. At least four different pictures were used for each measurement. Values are expressed as means ± SE (n = 3). *P < 0.05, as compared to the control.
Viable number (% of control)
12 120
**
100
**
80 60
**
40 20 0 control
3.13
12.5
50
Concentration (μg/ml)
Fig. 6. Inhibitory effects of artepillin C on growth of endothelial cells. Cells were seeded on gelatin-coated plates at a density of 1.0 · 104 cells/cm2 in 1.0 ml of MCDB-104 with 10% FBS. After 24 h incubation at 37 C in a 5% CO2 incubator, artepillin C was added to the wells at the indicated concentrations and the cells were cultured further for 3 days before conducting cell count. Values are expressed as means ± SE (n = 3). **P < 0.005, as compared to the control.
Fig. 4. Inhibitory effects of artepillin C on tube formation of endothelial cells. Cell were sandwiched between two layers of collagen gels at a density of 6.0 · 104 cells/cm2 and induced to form blood vessel-like tubes. The experiment was repeated three times and representative data are shown. HUVECs were seeded and cultured between two collagen gel layers in the presence of various concentrations of artepillin C. (a) control, (b) 3.13 lg/ml, (c) 12.5 lg/ml, (d) 50 lg/ml. Scale bar, 100 lm.
artepillin C, respectively, while that of negative control (group II-1) did not have any incidence of newly formed blood vessels.
Thus, the oral dose of 0.125%, 0.25% and 0.5% artepillin C significantly suppressed the numbers of newly formed blood vessels in mice to 46.2%, 38.5% and 33.7%, respectively, compared to the positive control. In addition, no signs of toxicity were observed in any ICR mice (body weight change during the feeding period; data not shown).
4. Discussion In this report, we showed for the first time that artepillin C, a major component of Brazilian propolis, possesses antiangiogenic activity. Although some investigators have reported that propolis can
M.-R. Ahn et al. / Cancer Letters 252 (2007) 235–243
a
b
c
d
e
2mm
f
7
Angiogenesis index
6 5 4
*
3 2
*
*
1 0
Negative control
Positive control
Artepillin C Artepillin C Artepillin C 0.125% 0.25% 0.5%
Fig. 7. Suppressive effects of artepillin C on tumor-induced angiogenesis in DAS assay (in vivo angiogenesis assay). Chamber filled with 0.15 ml of S180 cell suspension (3.0 · 106 cells) were inoculated subcutaneously to ICR mice. Arrowheads point to newly formed vessels with a zigzagging character. S180-treated positive control mice (b) induced strong angiogenic responses compared to negative control mice (a). Oral administration of 0.125% (c) 0.25% (d) and 0.5% (e) artepillin C reduced the number of newly formed vessels. Angiogenesis index was defined as the number of newly formed blood vessels above 3 mm in length and 0.1 mm in diameter. Artepillin C significantly reduced Angiogenesis index in a dose-dependent manner (f). Scale bar, 2 mm. Values are expressed as means ± SE (n = 5). *P < 0.05, as compared to the positive control.
suppress tumor growth in vivo [44], the actual mechanisms of these effects are not fully understood. EEBP showed significant antiangiogenic effects in a DAS assay (in vivo angiogenesis assay), significantly reducing the number of newly formed vessels induced by S180 sarcoma cells. When the areas of all tubes constructed in the 2-D culture model were measured, EEBP was shown to significantly inhibit
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tube formation of HUVECs in a concentrationdependent manner (3.13–50 lg/ml) and only artepillin C among three major constituents of EEBP was found to inhibit tube formation in a similar manner. Artepillin C was also found to inhibit proliferation of HUVECs in a concentration-dependent manner (3.13–50 lg/ml). Furthermore, the phenolic acid showed significant antiangiogenic effects in a DAS assay. From these results, we concluded that artepillin C, a major component of EEBP, had antiangiogenic activity in vitro and in vivo, and might be responsible, at least in part, for EEBP’s antiangiogenic activity in vivo. Propolis, a sticky material that honeybees collect from living plants, has been used for its pharmaceutical properties since ancient times [45]. Constituents of propolis are known to vary with the season and the vegetal source available in the geographic region where each propolis is produced. Propolis from Europe and China contains many flavonoids and phenolic acid esters [46]. Flavonoids are present only in small quantities in Brazilian propolis [47– 49], which suggests that its biological activities are due to constituents other than flavonoids. Fujimoto et al. analyzed various propolis samples from all over the world by UV and HPLC and classified the propolis into two groups according to the difference of their components: one is a Brazilian-type (Baccharis-type), and the other is a European-type (poplar-type) [50]. Brazilian-type propolis is rich in p-coumaric acid derivatives and is found only in Brazil. Phenolic compounds are commonly found in both edible and non-edible plants, and they have been reported to have multiple biological effects, including antioxidant and anti-proliferative activity [51,52]. Propolis contains a wide variety of phenolic compounds, mainly flavonoids. Variation in the flavonoid content of propolis is mainly attributable to the difference in the preferred regional plants collected by honeybees. Flavonoids and other phenolic substances have been suggested to play a preventive role in the development of cancer and heart disease [51]. Grunberger et al. reported on the preferential cytotoxicity on tumor cells of caffeic acid phenethyl ester isolated from propolis [53]. Propolis and its components caffeic acid, caffeic acid phenethyl ester, artepillin C, quercetin, galangin, naringenin and others are also promising candidates as antimutor agents. The major components in propolis of Brazilian origin are terpenoids and prenylated derivatives of p-coumaric acids. Artepillin C, one of the principal
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phenolic acids found in Brazilian propolis extract, exhibits various biological activities. The importance of artepillin C has been increasingly recognized and it has recently been designated as a criterion of the quality of Brazilian propolis. Brazilian propolis specifically contains artepillin C, because the bees collect materials from the Brazilian plant, Baccharis dracunculifolia, which contains a large amount of antioxidative artepillin C [27,32,47]. Kimoto et al. reported on the apoptosis and suppression of tumor growth by artepillin C [32]. They found that artepillin C exhibits direct anti-proliferative effects on human lung, gastric and colon cancers, murine and human hepatoma and murine malignant melanoma cells in vitro. Shimizu et al. reported that artepillin C suppressed the formation of colonic aberrant crypt foci (ACF) through the activation of antioxidant-responsive element (ARE) and induction of phase II enzymes in liver [54]. Our results together with these reports indicate that artepillin C has a tumor suppressing effect not only by directly inhibiting tumor cell growth but also by inhibiting angiogenesis. Such effects strongly support the possibility that we can prevent cancer by daily intake of artepillin C. We are studying further the mechanism of inhibition of angiogenesis by artepillin C at cellular and molecular levels. Acknowledgments This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of the Japanese government, and by a Grant-in-Aid from Japan Society for the Promotion of Sciences (JSPS) (to T. Ohta, Grant No. 15700470). References [1] J. Folkman, Tumor angiogenesis: therapeutic implications, N. Engl. J. Med. 285 (1971) 1182–1186. [2] A.I. Holleb, J. Folkman, Tumor angiogenesis, CA Cancer J. Clin. 22 (1972) 226–229. [3] J. Folkman, Clinical application of research on angiogenesis, N. Engl. J. Med. 333 (1995) 1757–1763. [4] F. Tosetti, N. Ferrari, S. de Flora, A. Albini, ‘Angioprevention’: angiogenesis is a common and key target for cancer chemopreventive agents, FASEB J. 16 (2002) 2–14. [5] S. Castaldo, F. Capasso, Propolis, an old remedy used in modern medicine, Fitoterapia 73 (2002) S1–S6. [6] V.S. Bankova, S.L.D. Castro, M.C. Marcucci, Propolis: recent advances in chemistry and plant origin, Apidologie 31 (2000) 3–15.
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Suppression of tumor-induced angiogenesis by Brazilian propolis: Major component artepillin C inhibits in vitro tube formation and endothelial cell proliferation Mok-Ryeon Ahn a, Kazuhiro Kunimasa b, Toshiro Ohta b,*, Shigenori Kumazawa a, Miya Kamihira a, Kazuhiko Kaji b, Yoshihiro Uto c, Hitoshi Hori c, Hideko Nagasawa d, Tsutomu Nakayama a a
Laboratory of Functional Food Science and COE Program in the 21st Century, School of Food and Nutritional Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan b Laboratory of Cell and Molecular Biology of Aging and COE Program in the 21st Century, School of Food and Nutritional Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan c Department of Life System, Institute of Technology and Science, The University of Tokushima Graduate School, Minamijosanjimacho-2, Tokushima 770-8506, Japan d Gifu Pharmaceutical University, 5-6-1 Mitahora-higashi, Gifu 502-8585, Japan Received 11 October 2006; accepted 27 December 2006
Abstract Propolis, a resinous substance collected by honeybees from various plant sources, possesses various physiological activities such as antitumor effects. We have previously shown that propolis of Brazilian origin was composed mainly of artepillin C and that its constituents were quite different from those of propolis of European origin. In this report, we examined an antiangiogenic effects of Brazilian propolis and investigated whether artepillin C was responsible for such effects. In an in vivo angiogenesis assay using ICR mice, we found that the ethanol extract of Brazilian propolis (EEBP) significantly reduced the number of newly formed vessels. EEBP also showed antiangiogenic effects in an in vitro tube formation assay. When compared with other constituents of EEBP, only artepillin C was found to significantly inhibit the tube formation of HUVECs in a concentration-dependent manner (3.13–50 lg/ml). In addition, artepillin C significantly suppressed the proliferation of HUVECs in a concentration-dependent manner (3.13–50 lg/ml). Furthermore, artepillin C significantly reduced the number of newly formed vessels in an in vivo angiogenesis assay. Judging from its antiangiogenic activity in vitro and in vivo, we concluded that artepillin C at least in part is responsible for the antiangiogenic activity of EEBP in vivo. Artepillin C may prove useful in the development of agents and foods with therapeutic or preventive activity against tumor angiogenesis. 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Angiogenesis; Artepillin C; Human umbilical vein endothelial cells; Propolis; Tube formation; Proliferation
*
Corresponding author. Tel./fax: +81 54 264 5571. E-mail address: [email protected] (T. Ohta).
0304-3835/$ - see front matter 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2006.12.039
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1. Introduction Angiogenesis, or new blood vessel growth, is defined as a process in which a network of new blood vessels emerges from preexisting vessels. Judah Folkman first observed in the early 1970s that such new blood vessel growth was required for a tumor to grow over a few mm3 in size, supplying the tumor with nutrition and oxygen for its exponential growth [1]. Since then many investigators have pursued studies for preventing or delaying cancer growth, or even completely eliminating cancer from a patient’s body, by suppressing such neovascularization [2,3]. Antiangiogenic treatment may be useful in the treatment and prevention of cancer progression [4]. Food factors capable of inhibiting angiogenesis, if found, would be useful to stop the progression of small cancers. Propolis, a folk medicine employed in treating various ailments, is a resinous substance collected by honeybees from the bud and bark of certain trees and plants, and stored inside their hives. It has been used in folk medicine from ancient times in many countries and has been extensively studied in Eastern European countries [5,6]. Recently, it has been reported to possess various biological activities such as antibacterial [7,8], antiviral [7,9], anti-inflammatory [10,11], anticancer [12,13], antifungal [7,14], and antitumoral [15] properties. For this reason, propolis is extensively used in foods and beverages to improve health and prevent diseases such as inflammation, diabetes, heart disease, and cancer [16,17]. Propolis has over 150 constituents such as polyphenols (flavonoids, phenolic acids and their esters), terpenoids, steroids, and amino acids, but its composition varies qualitatively and quantitatively with the geographical and botanical origins [18,19]. Because of the geographical differences, propolis samples from Europe, South America, and Asia have different chemical compositions [20–24]. Artepillin C (3,5-diprenyl-4-hydroxycinnamic acid) is one of the principal phenolic acids found specifically in Brazilian propolis. It has various biological activities, such as antibacterial [25], antiviral [26], apoptosis-inducing [27], antioxidant [28], and anticarcinogenic [29,30] properties. Konishi et al. reported that artepillin C has an extremely low absorption efficiency and bioavailability in vivo, in comparison to those of caffeic acid, which is absorbed and distributed by the monocarboxylic acid transporter (MCT)-mediated transport system
[31]. Aga et al. found that artepillin C exhibits antibacterial and antiviral activity [25], but is also highly cytotoxic to a variety of malignant human and murine solid tumor cell lines in vitro [32]. Furthermore, artepillin C was found to inhibit the growth of transplanted solid human and mouse tumors including that of malignant melanoma, in athymic and thymic mice, respectively [32]. Konishi reported that artepillin C is mainly permeated across Caco-2 cells by transcellular passive diffusion [33]. However, there have been only a limited number of reports concerning the effects of propolis and no reports of the effects of artepillin C on angiogenesis [34–36]. In this study, we investigated in vivo anti-angiogenic activity of the ethanol extracts of Brazilian propolis in vivo. We further analyzed its effects on angiogenesis in vitro through inhibition of tube formation and endothelial cell proliferation and on angiogenesis in vivo by its major component artepillin C. 2. Materials and methods 2.1. Materials Medium 199 and all other chemicals were purchased from Sigma (St. Louis, MO, USA). Artepillin C was purchased from Wako Pure Chemicals Industries (Osaka, Japan) for in vitro studies and was synthesized according to the method of our previous report for in vivo studies [37,38]. Medium MCDB-104 was a product of Nihon Pharmaceutical (Tokyo, Japan). Fetal bovine serum (FBS) was purchased from Moregate (Brisbane, Australia). Cellgen was obtained from Koken (Tokyo, Japan). Epidermal growth factor (EGF) was purchased from BD Biosciences (Bedford, MA). Human basic FGF (Recombinant) was purchased from Austral Biologicals (San Ramon, CA, USA). 2.2. Experimental sample preparation Brazilian propolis was collected in Minas Gerais State, where Baccharis dracunculifolia DC. is the main botanical source of the propolis. Propolis sample was extracted with ethanol (30 ml of ethanol/g of propolis) at room temperature for 24 h. The ethanol suspension was separated by centrifugation at 470g for 15 min at 25 C, and the supernatant was concentrated in a rotary evaporator under reduced pressure at 40 C until reaching a constant weight, and then redissolved in ethanol. The preparation obtained was named ethanol extract of Brazilian propolis (EEBP), and the color of EEBP was brown. EEBP was stored under a dry condition at 4 C until analyzed.
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2.3. Cell culture Human umbilical vein endothelial cells (HUVECs) were grown in HUVEC growth medium (MCDB-104 medium supplemented with 10 ng/ml EGF, 100 lg/ml heparin, 100 ng/ml endothelial cell growth factor and 10% FBS) [39]. Incubation was carried out at 37 C under a humidified 95–5% (v/v) mixture of air and CO2. The cells were seeded on plates coated with 0.1% gelatin and allowed to grow to sub-confluence before experimental treatment. 2.4. Dorsal air sac assay The mouse dorsal air sac (DAS) assay was performed as described previously with slight modifications [40]. This study used female ICR mice (7 weeks old, Japan SLC, Japan) weighing 30 ± 5 g. The animals were kept on a 12 h-light/12 h-dark cycle environment with constant temperature and humidity. The body weight of mice was measured semi-daily. A chamber, which was prepared by covering both sides of a Millipore ring with Millipore filters of 0.45 lm pore size, was filled with a suspension of S180 tumor cells (3.0 · 106 cells) in 0.15 ml of calciumand magnesium-free phosphate buffered saline (PBS). This chamber was implanted into the subcutaneous dorsal air sac created in ICR mice by injecting an appropriate volume of air. For each experiment the mice were randomly divided into several groups as follows: (I): negative control mice (group I-1); S180-treated positive control mice (group I-2); animals with daily oral administration of 2.5% (group I-3) and 5% (group I-4) EEBP diet, (II): negative control mice (group II-1); S180-treated positive control mice (group II-2); animals with daily oral administration of 0.125% (group II-3), 0.25% (group II-4) and 0.5% (group II-5) artepillin C diet. The negative control mice were implanted only with the calcium- and magnesium-free PBS-containing chamber. The groups were administered once a day for 6 days starting from the day of implantation of the chamber. On day 7, the implanted chambers were removed from the subcutaneous area of the treated animals, and a white ring with the same inner diameter as that of the Millipore ring was placed at the site where the ring was located. The angiogenic response was assessed under a dissecting microscope by determining the number of newly formed blood vessels greater than 3 mm in length within the area encircled by the white ring. The blood vessels that were newly formed due to the malignant tumor cells were morphologically distinguishable from the preexisting background vessels by their zigzag character. 2.5. Tube formation assay Capillary tube-like structures formed by HUVECs in collagen gel were prepared as previously described with slight modifications [39]. Collagen gels were prepared with
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Cellgen (type I collagen). Aliquots (200 ll) of collagen solution (0.21% in M 199) were poured into the wells of 24-multiwell culture plates, and the plates were incubated at 37 C for 30 min to solidify gels. HUVECs (6.0 · 104 cells/cm2) in MCDB-104 with 0.5% FBS were seeded onto the collagen-coated wells, and left at 37 C in a 5% CO2 incubator for 1 h to become attached to the collagen gel. After removing the medium, 150 ll aliquots of the collagen solution was overlaid and subjected to gelation as described above. Subsequently, 650 ll aliquots of MCDB-104 with 0.5% FBS supplemented with 10 ng/ml bFGF, 8 nM/ml PMA, and 25 lg/ml ascorbic acid with various concentrations of either EEBP or artepillin C (0, 3.13, 12.5 and 50 lg/ml) were added to the wells and incubated for 36 h. The resulting web-like capillary structure was viewed with a microscope under 100· magnification and captured with a Olympus C4040ZOOM digital camera. The tube formation was quantified by determining the pixel number of tubes in each image using NIH Image program (developed at the U.S. National Institutes of Health and available on the Internet at http://rsb.info.nih.gov/nih-image/). 2.6. Measurement of proliferation inhibition of cells HUVECs were seeded onto gelatin-coated 24-multiwell plates at a density of 1.0 · 104 cells/cm2 in HUVEC growth media. After a 24-h incubation at 37 C in a 5% CO2 incubator, artepillin C was added to the wells (3.13–50 lg/ml) and the cells were further cultured for 3 days. The number of cells was counted with a Coulter Counter (Coulter Electronics, Hialeah, USA). 2.7. Statistical analysis The significance of difference between different groups was analyzed by the multiple comparison test. Results were expressed as means ± SE. obtained from three experiments. Comparisons between control and treatments were performed using Student’s unpaired t-test (*P < 0.05; **P < 0.005). 3. Results We previously analyzed the chemical composition of propolis from various geographic origins [20,41,42]. Table 1 shows the main constituents of the ethanol extract of Brazilian propolis which we used in the current study. The composition of propolis primarily depends upon the vegetation of the area where it was collected and secondarily upon the methods for its extraction [20]. It has been shown that Baccharis dracunculifolia is an important plant source of Brazilian propolis collected in Minas Gerais [43]. A distinctive feature of Brazilian propolis is that it lacks flavonoids, such as quercetin and kaempferol, and phenolic acid esters, such
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Table 1 Qualitative and quantitative analysis of Brazilian propolis Main constituents
Contenta (mg/g of EEP)
Caffeic acid p-Coumaric acid Artepillin C
1.6 27.4 43.9
a Each value (mg/g of EEP) is expressed as a mean of triplicate analyses for each sample. EEP, ethanol extracts of propolis. Data in this table are derived from our previous report [20].
as caffeic acid phenethyl ester and cinnamyl caffeate, both of which are commonly contained in propolis from other origins. On the other hand, artepillin C (Fig. 1), a unique substance found only in propolis from Brazilian origin, is the major constituent of EEBP. In our previous study, we found over 40 mg of artepillin C per g of EEBP [20]. The next most abundant components were p-coumaric acid at under 30 mg per g and caffeic acid at under 2 mg per g of EEBP, which can also be found in propolis from other origins. 3.1. Suppression of tumor-induced angiogenesis in vivo by oral administration of Brazilian propolis
2 mm
Angiogenesis index
To evaluate the effect of Brazilian propolis on the angiogenesis in vivo, we first performed a DAS assay using ICR mice. Oral administration of Brazilian propolis significantly suppressed tumor-induced angiogenesis in a dose-dependent manner (Fig. 2). Angiogenesis indexes (the numbers of newly formed blood vessels) were 5.4, 3.4 and 2.8 by positive control (group I-2), 2.5% (group I-3) and 5% EEBP (group I-4), respectively, while that of negative control (group I-1) was about 0.5. The numbers of newly formed blood vessels in mice treated with foods containing 2.5% and 5% EEBP were suppressed to 59% and 46%, respectively, compared to the positive control group. In addition, no signs of toxicity were observed in any ICR mice (body weight change during the feeding period; data not shown).
* *
Negative control
Fig. 1. Chemical structure of artepillin C.
Positive control
2.5% EEBP
5% EEBP
Fig. 2. Suppressive effects of Brazilian propolis on tumorinduced angiogenesis in DAS assay (in vivo angiogenesis assay). Chamber filled with 0.15 ml of S180 cell suspension (3.0 · 106 cells) were inoculated subcutaneously to ICR mice. Arrowheads point to newly formed vessels with a zigzagging character. S180treated positive control mice (b) induced strong angiogenic responses compared to negative control mice (a). Oral administration of 2.5% (c) and 5.0% (d) Brazilian propolis (EEBP) reduced the number of newly formed vessels. Angiogenesis index was defined as the number of newly formed blood vessels above 3 mm in length and 0.075 mm in diameter. Brazilian propolis significantly reduced Angiogenesis index in a dose-dependent manner (e). Scale bar, 2 mm. Values are expressed as means ± SE (n = 5). *P < 0.05, as compared to the positive control.
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3.2. Inhibition of HUVEC tube formation, an in vitro model of angiogenesis, by EEBP We examined the effect of EEBP on angiogenesis in vitro using a tube formation model of HUVECs cultured in a 2-D system. After induction of tube formation, the endothelial cells formed a network of capillary-like tubes, which were composed of multiple cells that gathered together and adhered to each other. Fig. 3 shows the inhibitory effects of EEBP on tube formation of endothelial cells. At concentrations of 3.13 and 12.5 lg/ml, EEBP slightly reduced the width of the tubes. At a concentration of 50 lg/ml, EEBP completely inhibited elongation of HUVECs. 3.3. Inhibition of HUVEC tube formation, an in vitro model of angiogenesis, by artepillin C We then examined the effects of three constituents of EEBP on angiogenesis in vitro using a tube formation model of HUVECs. First, the inhibitory effects of artepillin C, the most abundant constituents of EEBP, on tube formation of endothelial cells cultured in a 2-D system were tested (Fig. 4). At a concentration of 3.13 lg/ml, artepillin C slightly reduced the width of the tubes. At a concentration of 12.5 lg/ml, artepillin C reduced both the width and the length of the tubes. At a concentration of 50 lg/ml, artepillin C completely inhibited tube formation in a concentration-dependent manner. Area ratios of the tubes per pictured field were 49.1%, 35.6%, 17.8% and 5.3% at concentrations of 0, 3.13, 12.5 and 50 lg/ml, respectively, which were calculated to be 72.5%, 36.2% and 10.8% (% of control) at concentrations of 3.13, 12.5 and 50 lg/ml, respectively (Fig. 5). On the other hand, both p-coumaric acid and caffeic acid did not show any significant inhibitory effects on tube formation (data not shown). 3.4. Inhibition of HUVEC proliferation, another in vitro model of angiogenesis, by artepillin C We then examined the effects of artepillin C on endothelial cell proliferation, another in vitro model of angiogenesis (Fig. 6). Artepillin C significantly reduced cell proliferation to 94.6%, 71.0% and 36.9% (% of control) at concentrations of 3.13, 12.5 and 50 lg/ml, respectively. Thus, artepillin C was shown to inhibit cell proliferation in a concentration-dependent manner, with a projected IC50 of 37.2 lg/ml. 3.5. Suppression of tumor-induced angiogenesis in vivo by oral administration of artepillin C
Fig. 3. Inhibitory effects of Brazilian propolis on tube formation of endothelial cells. Cell were sandwiched between two layers of collagen gels at a density of 6.0 · 104 cells/cm2 and induced to form blood vessel-like tubes. The experiment was repeated three times and representative data are shown. HUVECs were seeded and cultured between two collagen gel layers in the presence of various concentrations of Brazilian propolis. (a) control, (b) 3.13 lg/ml, (c) 12.5 lg/ml, (d) 50 lg/ml. Scale bar, 100 lm.
Oral administration of artepillin C was tested for effects on the angiogenesis in vivo. Artepillin C significantly suppressed tumor-induced angiogenesis in a dosedependent manner (Fig. 7). Angiogenesis indexes (the
numbers of newly formed blood vessels) were 5.2, 2.4, 2.0 and 1.75 by positive control (group II-2), 0.125% (group II-3), 0.25% (group II-4) and 0.5% (group II-5)
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Tube area (%)
50 40
*
30
*
20 10
*
0 control
3.13
12.5
50
Concentration (μg/ml)
Fig. 5. Quantification of inhibitory effects of artepillin C on tube formation of endothelial cells. Cells were treated with artepillin C and the areas of formed tubes were measured as described in materials and methods. At least four different pictures were used for each measurement. Values are expressed as means ± SE (n = 3). *P < 0.05, as compared to the control.
Viable number (% of control)
12 120
**
100
**
80 60
**
40 20 0 control
3.13
12.5
50
Concentration (μg/ml)
Fig. 6. Inhibitory effects of artepillin C on growth of endothelial cells. Cells were seeded on gelatin-coated plates at a density of 1.0 · 104 cells/cm2 in 1.0 ml of MCDB-104 with 10% FBS. After 24 h incubation at 37 C in a 5% CO2 incubator, artepillin C was added to the wells at the indicated concentrations and the cells were cultured further for 3 days before conducting cell count. Values are expressed as means ± SE (n = 3). **P < 0.005, as compared to the control.
Fig. 4. Inhibitory effects of artepillin C on tube formation of endothelial cells. Cell were sandwiched between two layers of collagen gels at a density of 6.0 · 104 cells/cm2 and induced to form blood vessel-like tubes. The experiment was repeated three times and representative data are shown. HUVECs were seeded and cultured between two collagen gel layers in the presence of various concentrations of artepillin C. (a) control, (b) 3.13 lg/ml, (c) 12.5 lg/ml, (d) 50 lg/ml. Scale bar, 100 lm.
artepillin C, respectively, while that of negative control (group II-1) did not have any incidence of newly formed blood vessels.
Thus, the oral dose of 0.125%, 0.25% and 0.5% artepillin C significantly suppressed the numbers of newly formed blood vessels in mice to 46.2%, 38.5% and 33.7%, respectively, compared to the positive control. In addition, no signs of toxicity were observed in any ICR mice (body weight change during the feeding period; data not shown).
4. Discussion In this report, we showed for the first time that artepillin C, a major component of Brazilian propolis, possesses antiangiogenic activity. Although some investigators have reported that propolis can
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a
b
c
d
e
2mm
f
7
Angiogenesis index
6 5 4
*
3 2
*
*
1 0
Negative control
Positive control
Artepillin C Artepillin C Artepillin C 0.125% 0.25% 0.5%
Fig. 7. Suppressive effects of artepillin C on tumor-induced angiogenesis in DAS assay (in vivo angiogenesis assay). Chamber filled with 0.15 ml of S180 cell suspension (3.0 · 106 cells) were inoculated subcutaneously to ICR mice. Arrowheads point to newly formed vessels with a zigzagging character. S180-treated positive control mice (b) induced strong angiogenic responses compared to negative control mice (a). Oral administration of 0.125% (c) 0.25% (d) and 0.5% (e) artepillin C reduced the number of newly formed vessels. Angiogenesis index was defined as the number of newly formed blood vessels above 3 mm in length and 0.1 mm in diameter. Artepillin C significantly reduced Angiogenesis index in a dose-dependent manner (f). Scale bar, 2 mm. Values are expressed as means ± SE (n = 5). *P < 0.05, as compared to the positive control.
suppress tumor growth in vivo [44], the actual mechanisms of these effects are not fully understood. EEBP showed significant antiangiogenic effects in a DAS assay (in vivo angiogenesis assay), significantly reducing the number of newly formed vessels induced by S180 sarcoma cells. When the areas of all tubes constructed in the 2-D culture model were measured, EEBP was shown to significantly inhibit
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tube formation of HUVECs in a concentrationdependent manner (3.13–50 lg/ml) and only artepillin C among three major constituents of EEBP was found to inhibit tube formation in a similar manner. Artepillin C was also found to inhibit proliferation of HUVECs in a concentration-dependent manner (3.13–50 lg/ml). Furthermore, the phenolic acid showed significant antiangiogenic effects in a DAS assay. From these results, we concluded that artepillin C, a major component of EEBP, had antiangiogenic activity in vitro and in vivo, and might be responsible, at least in part, for EEBP’s antiangiogenic activity in vivo. Propolis, a sticky material that honeybees collect from living plants, has been used for its pharmaceutical properties since ancient times [45]. Constituents of propolis are known to vary with the season and the vegetal source available in the geographic region where each propolis is produced. Propolis from Europe and China contains many flavonoids and phenolic acid esters [46]. Flavonoids are present only in small quantities in Brazilian propolis [47– 49], which suggests that its biological activities are due to constituents other than flavonoids. Fujimoto et al. analyzed various propolis samples from all over the world by UV and HPLC and classified the propolis into two groups according to the difference of their components: one is a Brazilian-type (Baccharis-type), and the other is a European-type (poplar-type) [50]. Brazilian-type propolis is rich in p-coumaric acid derivatives and is found only in Brazil. Phenolic compounds are commonly found in both edible and non-edible plants, and they have been reported to have multiple biological effects, including antioxidant and anti-proliferative activity [51,52]. Propolis contains a wide variety of phenolic compounds, mainly flavonoids. Variation in the flavonoid content of propolis is mainly attributable to the difference in the preferred regional plants collected by honeybees. Flavonoids and other phenolic substances have been suggested to play a preventive role in the development of cancer and heart disease [51]. Grunberger et al. reported on the preferential cytotoxicity on tumor cells of caffeic acid phenethyl ester isolated from propolis [53]. Propolis and its components caffeic acid, caffeic acid phenethyl ester, artepillin C, quercetin, galangin, naringenin and others are also promising candidates as antimutor agents. The major components in propolis of Brazilian origin are terpenoids and prenylated derivatives of p-coumaric acids. Artepillin C, one of the principal
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phenolic acids found in Brazilian propolis extract, exhibits various biological activities. The importance of artepillin C has been increasingly recognized and it has recently been designated as a criterion of the quality of Brazilian propolis. Brazilian propolis specifically contains artepillin C, because the bees collect materials from the Brazilian plant, Baccharis dracunculifolia, which contains a large amount of antioxidative artepillin C [27,32,47]. Kimoto et al. reported on the apoptosis and suppression of tumor growth by artepillin C [32]. They found that artepillin C exhibits direct anti-proliferative effects on human lung, gastric and colon cancers, murine and human hepatoma and murine malignant melanoma cells in vitro. Shimizu et al. reported that artepillin C suppressed the formation of colonic aberrant crypt foci (ACF) through the activation of antioxidant-responsive element (ARE) and induction of phase II enzymes in liver [54]. Our results together with these reports indicate that artepillin C has a tumor suppressing effect not only by directly inhibiting tumor cell growth but also by inhibiting angiogenesis. Such effects strongly support the possibility that we can prevent cancer by daily intake of artepillin C. We are studying further the mechanism of inhibition of angiogenesis by artepillin C at cellular and molecular levels. Acknowledgments This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of the Japanese government, and by a Grant-in-Aid from Japan Society for the Promotion of Sciences (JSPS) (to T. Ohta, Grant No. 15700470). References [1] J. Folkman, Tumor angiogenesis: therapeutic implications, N. Engl. J. Med. 285 (1971) 1182–1186. [2] A.I. Holleb, J. Folkman, Tumor angiogenesis, CA Cancer J. Clin. 22 (1972) 226–229. [3] J. Folkman, Clinical application of research on angiogenesis, N. Engl. J. Med. 333 (1995) 1757–1763. [4] F. Tosetti, N. Ferrari, S. de Flora, A. Albini, ‘Angioprevention’: angiogenesis is a common and key target for cancer chemopreventive agents, FASEB J. 16 (2002) 2–14. [5] S. Castaldo, F. Capasso, Propolis, an old remedy used in modern medicine, Fitoterapia 73 (2002) S1–S6. [6] V.S. Bankova, S.L.D. Castro, M.C. Marcucci, Propolis: recent advances in chemistry and plant origin, Apidologie 31 (2000) 3–15.
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