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Ligustrazine inhibits B16F10 melanoma metastasis and suppresses angiogenesis induced by Vascular Endothelial Growth Factor
Biochemical and Biophysical Research Communications 386 (2009) 374–379
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Ligustrazine inhibits B16F10 melanoma metastasis and suppresses angiogenesis induced by Vascular Endothelial Growth Factor Lei Chen a, Yin Lu a,b,*, Jia-ming Wu a, Bo Xu a, Li-juan Zhang a, Ming Gao a, Shi-zhong Zheng a, Ai-yun Wang a, Chang-bin Zhang a, Wei-wei Zhang a, Na Lei a a b
College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210029, China Jiangsu Key Laboratory for Traditional Chinese Medicine Formulae Research, Nanjing University of Chinese Medicine, Nanjing 210046, China
a r t i c l e
i n f o
Article history: Received 7 June 2009 Available online 11 June 2009
Keywords: Ligustrazine Tumor metastasis Angiogenesis VEGF
a b s t r a c t Angiogenesis is crucial for tumor metastasis, with many compounds that inhibit tumor metastasis acting through suppression of angiogenesis. We investigated anti-angiogenic properties of Ligustrazine in a series of in vitro and in vivo models. Ligustrazine inhibited VEGF-induced HUVECs migration and tube formation in a dose-dependent manner in vitro, and had limited cytotoxicity to HUVECs and normal fibroblasts even at a dose up to 100 lg/ml. Ligustrazine also suppressed VEGF-induced rat aortic ring sprouting dose-dependently. In vivo, Ligustrazine reduced the Hb content in a Matrigel plug implanted in mice and inhibited new vessel formation in CAM. In addition, in a B16F10 spontaneous metastasis model, Ligustrazine decreased the expression of CD34 and VEGF in primary tumor tissue and reduced the number of metastasis nodi on the lung surface. Our data suggests that Ligustrazine may inhibit tumor metastasis, at least in part, through its anti-angiogenic activity. Ó 2009 Elsevier Inc. All rights reserved.
Introduction Tumor metastasis is a complex process by which tumor cells escape from primary locus, intravasate into, and disseminate through, the blood and lymphatic vessels. It also involves extravasation from vessels into tissues at various secondary sites [1]. Mammalian cells require oxygen and nutrients for their survival and are therefore located within 100–200 lm of blood vessels, i.e. the diffusion limit for oxygen. Thus angiogenesis is essential for the increased growth required for tumor growth and metastasis [2–4]. Angiogenesis consists of: release of angiogenic factors; alteration in endothelial cells (EC) morphology; release of proteolytic enzymes; EC migration and capillary morphogenesis; EC proliferation; neovascularization and microvessel differentiation, and morphology [5]. This process is regulated by a balance between pro- and anti-angiogenic molecules, which is disregulated in various diseases, especially cancer [6,7]. Thus anti-angiogenesis strategies, aimed at the inappropriate angiogenesis required for tumor growth and metastasis, are of potential importance in tumor therapeutics. The identification of new drugs from plants has a long and successful history, and certain proangiogenic and anti-angiogenic plant * Corresponding author. Address: College of Pharmacy, Jiangsu Key Laboratory for Traditional Chinese Medicine Formulae Research, Nanjing University of Chinese Medicine, Nanjing 210029, China. Fax: +86 25 86798188. E-mail address: [email protected] (Y. Lu). 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.06.042
components have been used in traditional Chinese medicine (TCM) for thousands of years [8]. Ligustrazine (2,3,5,6-tetramethylpyrazine, TMP, relative molecular mass: 208.5), a constituent of Rhizoma Chuanxiong, has been isolated, purified, and chemically synthesized. It has been widely used, especially in the treatment of patients with cerebral and cardiac ischemic diseases. Meanwhile, many actions of Ligustrazine have been found, including capillary dilation, organ blood volume augmentation, microcirculation improvement, and protection against free radicals [9,10]. Tumor angiogenesis plays an important role in tumor metastasis, and Ligustrazine has been reported to exhibit anti-thrombosis and anti-platelet aggregation activity. In this report, we investigated the in vitro and in vivo effects of Ligustrazine on tumor metastasis and angiogenesis in a series of models. Our data indicated that Ligustrazine inhibits B16F10 melanoma metastasis and suppresses angiogenesis both in vitro and in vivo.
L. Chen et al. / Biochemical and Biophysical Research Communications 386 (2009) 374–379
Materials and methods Animals. Female C57BL/6 mice (6–8 weeks old) were purchased from the Slac Animal Inc. (Shanghai, China). Throughout the experiments mice were maintained in plastic cages at 21 ± 2 °C on a 12 h light/dark cycle and with free access to food and water. Animal welfare and experimental procedures were performed strictly in accordance with the care and use of laboratory animals, and the related ethics regulations of our University. All possible efforts were made to minimize the animals’ suffering and to reduce the number of animals used. Male Sprague–Dawley rats (6 weeks old), purchased from the Animal Center of Nanjing University of Chinese Medicine, were used in the aortic ring assay. Cell lines and culture condition. Human umbilical vein endothelial cells (HUVECs) were isolated from fresh umbilical cords using a modification of a previously described method [11,12]. HUVECs were cultured in tissue culture flasks coated with 1% w/v gelatin (Sigma). The growth media consisted of Medium 199 (Sigma, St. Louis, MO, USA) supplemented with LSGS (Cascade biologics, USA). HUVECs were used at passages 2–4. NIH-3T3 fibroblasts and B16F10 melanoma cells, kindly provided by Nanjing University, were cultured in a monolayer in DMEM (Gibco, Grand Island, NY, USA), containing 10% v/v calf serum (Gibco). All cells were grown in a humidified atmosphere, containing 5% CO2 at 37 °C. Cell proliferation assay. The growth inhibition effect of Ligustrazine on cells was carried out using the MTT assay. Briefly, exponentially growing HUVECs or NIH-3T3 fibroblasts were seeded in 96well plates (5 103 cells/well) and incubated for 24 h in complete medium. Then HUVECs or NIH-3T3 fibroblasts were incubated for 24–72 h in the presence of indicated doses of Ligustrazine (Hangzhou Zhongxiang Chemical Co. Ltd., purity > 99%). Twenty microliters of 5 mg/ml MTT (Amresco, USA) stock solution was added to each well, and plates were gently shaken and incubated at 37 °C. After 4 h incubation, cells were lysed with dimethyl sulfoxide and quantified at OD490 with an enzyme-linked immunosorbent assay reader. HUVEC migration assay. HUVEC motility was tested in a Transwell Boyden Chamber (Costar, Bethesda, MD, USA) using a polycarbonate filter (8 lm pores) coated with 0.1% w/v gelatin in the upper chamber. Briefly, HUVECs (1 106) were added to the upper chamber, in the absence or presence of increasing concentrations of Ligustrazine (from 0.8 to 100 lg/ml), while the lower chamber contained 600 ll of M199 with 1% v/v FBS with, or without, 10 ng VEGF/ml. After being incubated for 4 h, cells that had migrated to the lower surface of the filter membrane were fixed in ethanol and stained with hematoxylin and eosin. Migrated cells were counted under a microscope. Five random fields were observed for each membrane. Tube formation assay. Tube formation assay was performed using a modification of a previously described method [13]. Briefly, a mixture of fibrinogen and thrombin were pipetted into a 48-well culture plate and polymerized for 1 h at 37 °C. HUVECs were seeded at a density of 1 104 cells/well in 400 ll of complete culture medium. After incubation for 12 h culture medium was removed, without disturbing the gel, and another layer of fibrinogen and thrombin solution was added. After polymerization Ligustrazine and culture medium were added onto the upper layer of the fibrin gel and the plate incubated for a further 48 h. The morphological changes of the cells and any tubes formed were observed and recorded under a microscope. Five random fields were observed for each well. Rat aortic ring assay. The rat aortic ring assay was performed using a modification of a previously described method [14]. Thoracic aortas were excised from 6-week-old Sprague–Dawley male rats, cut into cross-section, 1 mm wide rings and flushed with DMEM/F12 medium (Hyclone, USA). Rings were immediately
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placed into wells of a 48-well plate containing 400 ll fibrinogen/ thrombin solution and then incubated at 37 °C until the fibrinogen/thrombin solution polymerized. The wells were then overlaid with 600 ll DMEM/F12 medium containing 10 ng/ml VEGF and various concentrations of Ligustrazine (ranging from 4 to 100 lg/ ml). On the seventh day vascular sprouting from each ring was examined using a Leica Inverted Microscope (100) and digital images taken. Chick chorioallantoic membrane assay. The chick allantoic membrane (CAM) assay was carried out using a modification of a previously described method [15]. A window was cut through the shell over the dropped CAM and a gelatin sponge (4 4 4 mm), saturated with 10 ng/ml VEGF or Ligustrazine (at concentrations of either 25, 50, or 100 lg/egg), placed on the CAM. After 72 h the area around the loaded gelatin sponge was photographed using a Canon digital camera. The angiogenic index was defined as the mean number of visible blood vessel branch points within the defined area of the gelatin sponge. Assays for each test sample were carried out using 10 eggs. In vivo Matrigel plug assay. Matrigel plug assay was performed as using a modification of a previously described method [16]. Briefly, 500 ll of precooled liquid Matrigel (Beckon Dickinson Labware, Bedford, MA, USA), containing 100 ng bFGF (Invitrogen, Carlsbad, CA, USA) and 20 U/ml heparin, were subcutaneously injected into the flank of 8-week-old C57BL/6 mice. The mice were divided into three groups, each of 8 mice. Two treatment groups received intraperitoneal injections of either 20 or 40 mk/kg, and a control group normal saline. After 7 days, the Matrigel plug was carefully removed and the angiogenic response evaluated by measuring the hemoglobin (Hb) content of the Matrigel plugs. The experiment was repeated twice. Spontaneous metastasis model. Spontaneous metastasis model was established using a modification of a previously described method [17,18]. B16F10 cells (5 105 cells in 0.05 ml PBS per mouse) were injected into the right hind footpads of female, 6– 8 weeks old, C57BL/6 mice. Ligustrazine of different concentration (6.7, 20, and 60 mg/kg body weight), dissolved in normal saline, was then given by intraperitoneal injection daily. The control group was injected with normal saline only (all five mice per group). Twenty-three days later the right footpads were resected and fixed with formalin. After a period of 23 days the mice were sacrificed. The number of lung metastasis nodules were measured to analyze the effects on tumor spontaneous metastasis. Immunohistochemistry analysis. Immunohistochemistry was carried out as described previously [19]. In brief, the paraffinembedded primary melanoma tissues were sectioned with a microtome, deparaffined with xylene, rehydrated and stained immunohistochemically using the labeled-(strept) avidin–biotin method. The slides incubated with the appropriate antibody at 4 °C overnight. These consisted of a rabbit anti-mouse CD34 (BA0532,1:50) antibody (Boster Biological Technology Co., Ltd., Wuhan, China), and a goat anti-VEGF (sc-152,1:50) polyclonal antibody (Santa Cruz Biotech, Santa Cruz, CA, USA). The next day the tissue sections were incubated in blocking buffer, containing the corresponding secondary antibody, at RT for 15 min. After washing with PBS, immunohistoreactivity was visualized using diaminobenidine (DAB) under a light microscope at the indicated magnification. Hematoxyline/eosin (H and E) staining analysis. Hematoxylin/eosin staining analysis was performed as previously described [20]. Specimens were examined and photographed under a microscope after staining. Immunoreactive areas were measured from ten randomly selected fields of lung tissue sections per animal using the Optimas image analyzer (Optimas Corporation USA). Three animals were examined per group.
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L. Chen et al. / Biochemical and Biophysical Research Communications 386 (2009) 374–379
Fig. 1. Ligustrazine has no effect on HUVECs and normal fibroblasts proliferation. Proliferating HUVECs and NIH-3T3 fibroblasts were treated with various concentrations (0, 0.16, 0.8, 4, 20, 100 lg/ml) of Ligustrazine. After TMP was added for 24, 48, and 72 h, cell growth and viability were measured by MTT assay. The reading of control was normalized to 100%, and readings from Ligustrazine-treated cells were expressed as% of control. (A) Ligustrazine has no effect on HUVECs proliferation. (B) Ligustrazine has no effect on NIH-3T3 fibroblasts proliferation.
Fig. 2. Ligustrazine inhibits VEGF-induced migration, tube formation and vessel sprouting in vitro. (A) Ligustrazine inhibited VEGF-induced migration of HUVECs. Chemotaxis was quantified by counting the cells that migrated to the lower side of the filter after fixation and staining (n = 3). Taken at 100 magnification, the photographs show the lower chamber of different groups (a–f). Migrated cells were counted by an inverted light microscope at 400 magnification (g). (B) Ligustrazine inhibits VEGF-induced tube formation of HUVECs. Micrographs were taken at 40 magnification (a–f). The mean number of tubes was counted (g) (n = 3). (C) Ligustrazine inhibits VEGF-induced vessel sprouting. Representative aortic rings were photographed (a–e). The numbers of sprouting vessels of each ring were counted by an inverted microscope at a 100 magnification. The data are presented as mean (n = 6); The arrows indicate the newly formed microvessels. Five random fields were chosen for each count. Experiments were repeated three times and values are thus the means of triplicates. (*P < 0.05; **P < 0.01 Ligustrazine + VEGF group versus VEGF group, DDP < 0.01 versus control); bars,±s.e.m.
L. Chen et al. / Biochemical and Biophysical Research Communications 386 (2009) 374–379
Statistical analysis. The data obtained from at least three independent tests are presented as means ± s.e.m. and statistical comparisons between groups performed using 1-way ANOVA followed by Student’s t-test at P values of < 0.01() or <0.05(). Results Ligustrazine has no effect on HUVECs and normal fibroblasts proliferation To determine the cytotoxicity of Ligustrazine, its inhibitory effect on proliferation of HUVECs and NIH-3T3 fibroblasts was evaluated by MTT assay. The data showed that Ligustrazine didn’t inhibit HUVECs and normal fibroblasts proliferation (Fig. 1). These results indicate that Ligustrazine may have limited cytotoxicity to HUVECs and normal fibroblasts even at a dose up to 100 lg/ml. Ligustrazine inhibits VEGF-induced migration, tube formation and vessel sprouting in vitro The migration of endothelial cells through basement membranes is a crucial step in the development of new blood vessels [21,22]. We thus first examined the effect of Ligustrazine on VEGF-induced migration of endothelial cells in vitro. In this experiment, we found that Ligustrazine (0.8–100 lg/ml) inhibited HUVECs migration dose-dependently (Fig. 2A). In order to study the effect on endothelial cell differentiation and spontaneous formation of capillary-like structures, functions crucial in blood vessel formation, we next tested whether Ligustrazine decreased the formation of tubes by HUVECs on fibrin gel in vitro. In the control group HUVECs formed a mesh of tubes, whereas those treated with Ligustrazine (from 0.8 to 100 lg/ml) were unable to form
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intact tubes (Fig. 2B a–f). Moreover, the inhibition effect was dose-dependent (Fig. 2B g). The sprouting of vessels from rat aortic rings was investigated to determine whether Ligustrazine inhibits VEGF-induced angiogenesis in vitro. Sprouts start to form within three days, with a complex three-dimensional network emerging by day 7. VEGF (10 ng/ml) significantly stimulated vessel sprouting above that seen with medium alone. The presence of Ligustrazine (20, 100 lg/ml) resulted in a significant reduction of VEGF-induced vessel sprouting in a dose-dependent manner (Fig. 2C). Ligustrazine inhibits angiogenesis in CAM and vessel formation in Matrigel Plug in vivo We found that Ligustrazine inhibited VEGF-induced migration, tube formation and vessel sprouting in vitro. To investigate the effect of Ligustrazine on angiogenesis in vivo, a modified chick chorioallantoic membrane assay was carried out. The control and VEGF-treated CAMs showed well-developed zones of neo-vascularization surrounding the sponge (Fig. 3A a–b). In contrast, CAM neovascularization was significantly suppressed by addition of Ligustrazine (25, 50, 100 lg/egg; Fig. 3A c–e, f). The Matrigel plug assay was employed to determine whether Ligustrazine was capable of blocking bFGF-induced angiogenesis in vivo. Compared with the control group, plugs from the Ligustrazine treated groups were pale in their color, indicating less blood vessel formation (Fig. 3B a–c). Furthermore, the mean hemoglobin content in the control group was 2.41 ± 1.11 mg/ml compared to 1.38 ± 0.96(20 mg/kg/day) and 0.91 ± 0.54 mg/ml (40 mg/kg/day) (P < 0.01) in in Ligustrazine-treated groups (Fig. 3B d). These results indicate that Ligustrazine is capable of inhibiting bFGF-induced angiogenesis in vivo.
Fig. 3. Ligustrazine inhibits angiogenesis in CAM and vessel formation in Matrigel Plug in vivo. (A) Ligustrazine (25, 50, or 100 lg/egg) or saline (control) were loaded on gelatin sponges which were loaded on the chick chorioallantoic membranes of chick embryos. After 72 h incubation, 10% v/v formaldehyde was added onto the surface of CAMs to fix the blood. The disc and surrounding CAMs were incised carefully and photographed; representative photographs show the CAMs of different groups (a–e). Quantification of newly formed blood vessels were shown in (f). Ten eggs were used for each data point, and mean is shown. bars,±s.e.m (*P < 0.05, **P < 0.01 versus VEGF, D P < 0.05 versus control). (B) Ligustrazine reduces vessel formation in a Matrigel plug. C57BL/6 mice injected with Matrigel plugs containing bFGF were treated intraperitoneally with Ligustrazine (20 or 40 mg/kg) or the control group received saline only. After 7 days mice were killed and the neovascularization and hemoglobin content of Matrigel plugs were evaluated. Macroscopic analysis of Matrigel from one representative experiment shown (a–c). Hemoglobin content of Matrigel plugs is shown in (d). The data are presented as mean, **P < 0.01, versus control; bars,±s.e.m. The experiment was repeated twice (n = 8).
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Ligustrazine inhibits angiogenesis in B16F10 melanoma primary tumor, lung metastasis, and microvessels in lung As we have shown, Ligustrazine has an obvious inhibitory effect on angiogenesis in vitro and in vivo. We thus investigated whether Ligustrazine plays a role in tumor angiogenesis in vivo by establishing a spontaneous B16F10 melanoma metastasis model. After injection of B16F10 cells followed by the administration of Ligustrazine (6.7, 20, or 60 mg/kg) or normal saline only (control group),
on day 23, the primary tumor was resected from the control and Ligustrazine treatment groups and fixed with formalin. Immunohistochemistry analyses of CD34 and VEGF were carried out as previously described. We found that the expression of CD34 (Fig. 4A) and VEGF (Fig. 4B) in the Ligustrazine treated groups were lower than that seen in the control group. Angiogenesis is crucial for tumor metastasis and we found that Ligustrazine inhibited angiogenesis in a primary tumor. Thus we then tested the ability of Ligustrazine to inhibit tumour metastasis using spontaneous a
Fig. 4. Ligustrazine inhibits angiogenesis in B16F10 melanoma primary tumor, lung metastasis and microvessels. In spontaneous metastasis model, on day 23 the footpads bearing the local tumors were amputated. VEGF and CD34-positive immunohistochemical staining of tumor cross-sections in the control and Ligustrazine (TMP)-treated mice were determined in the primary tumor tissue by a light microscope at 100 magnification. (A) Immunohistochemical analysis of CD34 protein expression in tumor tissue; (B) immunohistochemical analysis of VEGF protein expression in tumor tissues. After 23 days post-excision of the primary tumor, mice were killed and lungs were removed. The metastatic colonies were counted under a dissecting microscope. The lungs were photographed to show the effects of Ligustrazine on tumor spontaneous metastasis. (C) Lung metastasis nodules in a model of spontaneous metastasis. (a) Saline control, (b) Mice treated with TMP 6.7 mg/kg, (c) Mice treated with TMP 20 mg/kg, (d) Mice treated with TMP 60 mg/kg. (e) The number of lung metastases foci in each group. (D) H and E staining for blood vessels in lung tissues, arrows indicate microvessels (magnification, 100). Columns, mean (n = 5); bars,±s.e.m., *P < 0.05 versus control and **P < 0.01 versus control.
L. Chen et al. / Biochemical and Biophysical Research Communications 386 (2009) 374–379
B16F10 melanoma metastasis model. After 23 days post-excision of the primary tumor, the lungs of mice were removed, photographed, and the metastatic colonies counted under a dissecting microscope. Excitingly, compared with the control group, Ligustrazine remarkably decreased the number of tumor nodules on the lung surface in a dose-dependent fashion (6.7 mg/kg, P < 0.05; 20 mg/kg, P < 0.01; 60 mg/kg, P < 0.01; Fig. 4C). We also observed the density of blood vessels in the lung tissues by H and E staining. The number of microvessels in lung of the Ligustrazine group was less than that in the control group (Fig. 4D). Discussion In this study, we attempted to elucidate the effect of Ligustrazine on angiogenesis and tumour metastasis. Our results show that systemic treatment of C57BL/6 mice with intraperitoneally injected Ligustrazine suppresses metastasis in the B16F10 melanoma model. This effect of Ligustrazine is likely to be due to its angiogenic activity as follows. We first investigated the anti-angiogenic activity of Ligustrazine in vitro. VEGF, generated from a variety of tumors, is the most important angiogenic factor associated closely with induction and maintenance of the neovasculature in human tumors [23,24]. We found that Ligustrazine inhibited VEGF-induced endothelial migration, capillary formation and vessel sprouting, and had limited cytotoxicity to HUVECs and normal fibroblasts even at a dose up to 100 lg/ml. The in vitro activity of Ligustrazine across all phases of the angiogenic process was confirmed using in vivo angiogenic assays. Ligustrazine inhibited neovascularization in the chick chorioallantoic membrane and significantly reduced vessel formation, while inhibiting the propensity of vessels to grow toward the Matrigel plug. In addition, Ligustrazine decreased the expression of CD34 and VEGF in tumor tissues. From systemic treatments of tumor-bearing mice with Ligustrazin (6.7, 20, and 60 mg/kg), we discovered that Ligustrazine has a potent antimetastatic activity in spontaneous metastasis. We found that Ligustrazine decreased metastasis nodi on the lung surface in a dose-dependent manner. Our results indicate that the inhibitory effect of Ligustrazine is to some extent mediated through its anti-angiogenic activity. Such anti-angiogenic effects are likely to have been primarily responsible for the inhibition of the tumor metastasis, for the microvessel density was only about 20% of that in the control group. As vascularization is a prerequisite of tumor growth [25], the lower number of blood vessels must have been a major impediment to tumor metastasis in the treated mice. Thus inhibition of angiogenesis, with a resulting suppressive effect on tumor metastases, thus possibly underlies the antimetastatic effect of Ligustrazine. For example, we found that the number of metastases correlated with the number of blood vessels. The reduced vasculature in tumors could make it more difficult for tumor cells to enter the circulation. In conclusion, we have demonstrated that Ligustrazine can inhibit B16F10 melanoma metastasis through inhibition of neovascularization. Our findings suggest that Ligustrazine could potentially be beneficial in inhibiting tumor metastasis and angiogenesis. Acknowledgments We are grateful for financial support from National Nature Science Foundation of China (Project Nos. 30371727 and 30772766)
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and Nature Science Foundation of Jiangsu Province (Project Nos. BK2003113 and BK2007239). We also appreciate Prof. Neville Punchard for critical reading and kind help in editing of the manuscript. References [1] R. Bicknell, A.L. Harris, Mechanisms and therapeutic implications of angiogenesis, Curr. Opin. Oncol. 8 (1996) 60–65. [2] I.J. Fidler, L.M. Ellis, The implications of angiogenesis for the biology and therapy of cancer metastasis, Cell 79 (1994) 185–188. [3] J. Folkman, What is the evidence that tumours are angiogenesis dependent?, J Nutl. Cancer Inst. 82 (1990) 4–6. [4] I.R. Hart, A. Saini, Biology of tumour metastasis, Lancet 339 (1992) 1453–1457. [5] E. Ruoslahti, Specialization of tumour vasculature, Nat. Rev. Cancer 2 (2002) 83–90. [6] Y. Cao, Tumor angiogenesis and therapy, Biomed. Pharmacother. 59 Suppl 2 (2005) S340–S343. [7] P. Carmeliet, R.K. Jain, Angiogenesis in cancer and other disease, Nature 406 (2000) 249–257. [8] T.P. Fan, J.C. Yeh, K.W. Leung, P.Y. Yue, R.N. Wong, Angiogenesis: from plants to blood vessels, Trends Pharmacol. Sci. 27 (2006) 297–309. [9] C.Y. Kwan, E.E. Daniel, M.C. Chen, Inhibition of vasoconstriction by tetramethylpyrazine, Does it act by blocking the voltage-dependent Ca channel, J. Cardiovasc. Pharmacol. 15 (1990) 157–162. [10] W. Peng, D. Hucks, R.M. Priest, Y.M. Kan, J.P. Ward, Ligustrazine-induced endothelium-dependent relaxation in pulmonary arteries via an NO-mediated and exogenous L-arginine-dependent mechanism, Br. J. Pharmacol. 119 (1996) 1063–1071. [11] E.A. Jaffe, R.L. Nachman, C.G. Becker, C.R. Minick, Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria, J. Clin. Invest. 52 (1973) 2745–2756. [12] V. Marin, G. Kaplanski, S. Gres, C. Farnarier, P. Bongrand, Endothelial cell culture: protocol to obtain and cultivate human umbilical endothelial cells, J. Immunol. Methods 254 (2001) 183–190. [13] J.R. Gamble, L.J. Matthias, G. Meyer, P. Kaur, G. Russ, R. Faull, M.C. Berndt, M.A. Vadas, Regulation of in vitro capillary tube formation by anti-integrin antibodies, J. Cell. Biol. 121 (1993) 931–943. [14] R.F. Nicosia, A. Ottinetti, Modulation of microvascular growth and morphogenesis by reconstituted basement membrane gel in threedimensional cultures of rat aorta: a comparative study of angiogenesis in matrigel, collagen, fibrin, and plasma clot, In Vitro Cell Dev. Biol. 26 (1990) 119–128. [15] C.W. Kim, H.M. Lee, T.H. Lee, C. Kang, H.K. Kleinman, Y.S. Gho, Extracellular membrane vesicles from tumor cells promote angiogenesis via sphingomyelin, Cancer Res. 62 (2002) 6312–6317. [16] N. Akhtar, E.B. Dickerson, R. Auerbach, The sponge/Matrigel angiogenesis assay, Angiogenesis 5 (2002) 75–80. [17] R.G. Chirivi, A. Garofalo, M.J. Crimmin, L.J. Bawden, A. Stoppacciaro, P.D. Brown, R. Giavazzi, Inhibition of the metastatic spread and growth of B16-BL6 murine melanoma by a synthetic matrix metalloproteinase inhibitor, Int. J. Cancer 58 (1994) 460–464. [18] R. Hirayama, K. Hirokawa, T. Makinodan, Change in the metastatic mode of B16 malignant melanoma in C57BL/6 mice with ageing and sex, IARC Sci. Publ. (1985) 85–96. [19] K.I. O’Rourke, T.V. Baszler, T.E. Besser, J.M. Miller, R.C. Cutlip, G.A. Wells, S.J. Ryder, S.M. Parish, A.N. Hamir, N.E. Cockett, A. Jenny, D.P. Knowles, Preclinical diagnosis of scrapie by immunohistochemistry of third eyelid lymphoid tissue, J. Clin. Microbiol. 38 (2000) 3254–3259. [20] E.A. Ariazi, Y. Satomi, M.J. Ellis, J.D. Haag, W. Shi, C.A. Sattler, M.N. Gould, Activation of the transforming growth factor beta signaling pathway and induction of cytostasis and apoptosis in mammary carcinomas treated with the anticancer agent perillyl alcohol, Cancer Res. 59 (1999) 1917– 1928. [21] S. Bao, Q. Wu, S. Sathornsumetee, Y. Hao, Z. Li, A.B. Hjelmeland, Q. Shi, R.E. McLendon, D.D. Bigner, J.N. Rich, Stem cell-like glioma cells promote tumor angiogenesis through vascular endothelial growth factor, Cancer Res. 66 (2006) 7843–7848. [22] K.A. Hotchkiss, A.W. Ashton, R. Mahmood, R.G. Russell, J.A. Sparano, E.L. Schwartz, Inhibition of endothelial cell function in vitro and angiogenesis in vivo by docetaxel (Taxotere): association with impaired repositioning of the microtubule organizing center, Mol. Cancer Ther. 1 (2002) 1191–1200. [23] G. McMahon, VEGF receptor signaling in tumor angiogenesis, Oncologist 5 (Suppl. 1) (2000) 3–10. [24] T. Veikkola, K. Alitalo, VEGFs, receptors and angiogenesis, Semin. Cancer Biol. 9 (1999) 211–220. [25] D. Hanahan, J. Folkman, Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis, Cell 86 (1996) 353–364.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Ligustrazine inhibits B16F10 melanoma metastasis and suppresses angiogenesis induced by Vascular Endothelial Growth Factor Lei Chen a, Yin Lu a,b,*, Jia-ming Wu a, Bo Xu a, Li-juan Zhang a, Ming Gao a, Shi-zhong Zheng a, Ai-yun Wang a, Chang-bin Zhang a, Wei-wei Zhang a, Na Lei a a b
College of Pharmacy, Nanjing University of Chinese Medicine, Nanjing 210029, China Jiangsu Key Laboratory for Traditional Chinese Medicine Formulae Research, Nanjing University of Chinese Medicine, Nanjing 210046, China
a r t i c l e
i n f o
Article history: Received 7 June 2009 Available online 11 June 2009
Keywords: Ligustrazine Tumor metastasis Angiogenesis VEGF
a b s t r a c t Angiogenesis is crucial for tumor metastasis, with many compounds that inhibit tumor metastasis acting through suppression of angiogenesis. We investigated anti-angiogenic properties of Ligustrazine in a series of in vitro and in vivo models. Ligustrazine inhibited VEGF-induced HUVECs migration and tube formation in a dose-dependent manner in vitro, and had limited cytotoxicity to HUVECs and normal fibroblasts even at a dose up to 100 lg/ml. Ligustrazine also suppressed VEGF-induced rat aortic ring sprouting dose-dependently. In vivo, Ligustrazine reduced the Hb content in a Matrigel plug implanted in mice and inhibited new vessel formation in CAM. In addition, in a B16F10 spontaneous metastasis model, Ligustrazine decreased the expression of CD34 and VEGF in primary tumor tissue and reduced the number of metastasis nodi on the lung surface. Our data suggests that Ligustrazine may inhibit tumor metastasis, at least in part, through its anti-angiogenic activity. Ó 2009 Elsevier Inc. All rights reserved.
Introduction Tumor metastasis is a complex process by which tumor cells escape from primary locus, intravasate into, and disseminate through, the blood and lymphatic vessels. It also involves extravasation from vessels into tissues at various secondary sites [1]. Mammalian cells require oxygen and nutrients for their survival and are therefore located within 100–200 lm of blood vessels, i.e. the diffusion limit for oxygen. Thus angiogenesis is essential for the increased growth required for tumor growth and metastasis [2–4]. Angiogenesis consists of: release of angiogenic factors; alteration in endothelial cells (EC) morphology; release of proteolytic enzymes; EC migration and capillary morphogenesis; EC proliferation; neovascularization and microvessel differentiation, and morphology [5]. This process is regulated by a balance between pro- and anti-angiogenic molecules, which is disregulated in various diseases, especially cancer [6,7]. Thus anti-angiogenesis strategies, aimed at the inappropriate angiogenesis required for tumor growth and metastasis, are of potential importance in tumor therapeutics. The identification of new drugs from plants has a long and successful history, and certain proangiogenic and anti-angiogenic plant * Corresponding author. Address: College of Pharmacy, Jiangsu Key Laboratory for Traditional Chinese Medicine Formulae Research, Nanjing University of Chinese Medicine, Nanjing 210029, China. Fax: +86 25 86798188. E-mail address: [email protected] (Y. Lu). 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.06.042
components have been used in traditional Chinese medicine (TCM) for thousands of years [8]. Ligustrazine (2,3,5,6-tetramethylpyrazine, TMP, relative molecular mass: 208.5), a constituent of Rhizoma Chuanxiong, has been isolated, purified, and chemically synthesized. It has been widely used, especially in the treatment of patients with cerebral and cardiac ischemic diseases. Meanwhile, many actions of Ligustrazine have been found, including capillary dilation, organ blood volume augmentation, microcirculation improvement, and protection against free radicals [9,10]. Tumor angiogenesis plays an important role in tumor metastasis, and Ligustrazine has been reported to exhibit anti-thrombosis and anti-platelet aggregation activity. In this report, we investigated the in vitro and in vivo effects of Ligustrazine on tumor metastasis and angiogenesis in a series of models. Our data indicated that Ligustrazine inhibits B16F10 melanoma metastasis and suppresses angiogenesis both in vitro and in vivo.
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Materials and methods Animals. Female C57BL/6 mice (6–8 weeks old) were purchased from the Slac Animal Inc. (Shanghai, China). Throughout the experiments mice were maintained in plastic cages at 21 ± 2 °C on a 12 h light/dark cycle and with free access to food and water. Animal welfare and experimental procedures were performed strictly in accordance with the care and use of laboratory animals, and the related ethics regulations of our University. All possible efforts were made to minimize the animals’ suffering and to reduce the number of animals used. Male Sprague–Dawley rats (6 weeks old), purchased from the Animal Center of Nanjing University of Chinese Medicine, were used in the aortic ring assay. Cell lines and culture condition. Human umbilical vein endothelial cells (HUVECs) were isolated from fresh umbilical cords using a modification of a previously described method [11,12]. HUVECs were cultured in tissue culture flasks coated with 1% w/v gelatin (Sigma). The growth media consisted of Medium 199 (Sigma, St. Louis, MO, USA) supplemented with LSGS (Cascade biologics, USA). HUVECs were used at passages 2–4. NIH-3T3 fibroblasts and B16F10 melanoma cells, kindly provided by Nanjing University, were cultured in a monolayer in DMEM (Gibco, Grand Island, NY, USA), containing 10% v/v calf serum (Gibco). All cells were grown in a humidified atmosphere, containing 5% CO2 at 37 °C. Cell proliferation assay. The growth inhibition effect of Ligustrazine on cells was carried out using the MTT assay. Briefly, exponentially growing HUVECs or NIH-3T3 fibroblasts were seeded in 96well plates (5 103 cells/well) and incubated for 24 h in complete medium. Then HUVECs or NIH-3T3 fibroblasts were incubated for 24–72 h in the presence of indicated doses of Ligustrazine (Hangzhou Zhongxiang Chemical Co. Ltd., purity > 99%). Twenty microliters of 5 mg/ml MTT (Amresco, USA) stock solution was added to each well, and plates were gently shaken and incubated at 37 °C. After 4 h incubation, cells were lysed with dimethyl sulfoxide and quantified at OD490 with an enzyme-linked immunosorbent assay reader. HUVEC migration assay. HUVEC motility was tested in a Transwell Boyden Chamber (Costar, Bethesda, MD, USA) using a polycarbonate filter (8 lm pores) coated with 0.1% w/v gelatin in the upper chamber. Briefly, HUVECs (1 106) were added to the upper chamber, in the absence or presence of increasing concentrations of Ligustrazine (from 0.8 to 100 lg/ml), while the lower chamber contained 600 ll of M199 with 1% v/v FBS with, or without, 10 ng VEGF/ml. After being incubated for 4 h, cells that had migrated to the lower surface of the filter membrane were fixed in ethanol and stained with hematoxylin and eosin. Migrated cells were counted under a microscope. Five random fields were observed for each membrane. Tube formation assay. Tube formation assay was performed using a modification of a previously described method [13]. Briefly, a mixture of fibrinogen and thrombin were pipetted into a 48-well culture plate and polymerized for 1 h at 37 °C. HUVECs were seeded at a density of 1 104 cells/well in 400 ll of complete culture medium. After incubation for 12 h culture medium was removed, without disturbing the gel, and another layer of fibrinogen and thrombin solution was added. After polymerization Ligustrazine and culture medium were added onto the upper layer of the fibrin gel and the plate incubated for a further 48 h. The morphological changes of the cells and any tubes formed were observed and recorded under a microscope. Five random fields were observed for each well. Rat aortic ring assay. The rat aortic ring assay was performed using a modification of a previously described method [14]. Thoracic aortas were excised from 6-week-old Sprague–Dawley male rats, cut into cross-section, 1 mm wide rings and flushed with DMEM/F12 medium (Hyclone, USA). Rings were immediately
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placed into wells of a 48-well plate containing 400 ll fibrinogen/ thrombin solution and then incubated at 37 °C until the fibrinogen/thrombin solution polymerized. The wells were then overlaid with 600 ll DMEM/F12 medium containing 10 ng/ml VEGF and various concentrations of Ligustrazine (ranging from 4 to 100 lg/ ml). On the seventh day vascular sprouting from each ring was examined using a Leica Inverted Microscope (100) and digital images taken. Chick chorioallantoic membrane assay. The chick allantoic membrane (CAM) assay was carried out using a modification of a previously described method [15]. A window was cut through the shell over the dropped CAM and a gelatin sponge (4 4 4 mm), saturated with 10 ng/ml VEGF or Ligustrazine (at concentrations of either 25, 50, or 100 lg/egg), placed on the CAM. After 72 h the area around the loaded gelatin sponge was photographed using a Canon digital camera. The angiogenic index was defined as the mean number of visible blood vessel branch points within the defined area of the gelatin sponge. Assays for each test sample were carried out using 10 eggs. In vivo Matrigel plug assay. Matrigel plug assay was performed as using a modification of a previously described method [16]. Briefly, 500 ll of precooled liquid Matrigel (Beckon Dickinson Labware, Bedford, MA, USA), containing 100 ng bFGF (Invitrogen, Carlsbad, CA, USA) and 20 U/ml heparin, were subcutaneously injected into the flank of 8-week-old C57BL/6 mice. The mice were divided into three groups, each of 8 mice. Two treatment groups received intraperitoneal injections of either 20 or 40 mk/kg, and a control group normal saline. After 7 days, the Matrigel plug was carefully removed and the angiogenic response evaluated by measuring the hemoglobin (Hb) content of the Matrigel plugs. The experiment was repeated twice. Spontaneous metastasis model. Spontaneous metastasis model was established using a modification of a previously described method [17,18]. B16F10 cells (5 105 cells in 0.05 ml PBS per mouse) were injected into the right hind footpads of female, 6– 8 weeks old, C57BL/6 mice. Ligustrazine of different concentration (6.7, 20, and 60 mg/kg body weight), dissolved in normal saline, was then given by intraperitoneal injection daily. The control group was injected with normal saline only (all five mice per group). Twenty-three days later the right footpads were resected and fixed with formalin. After a period of 23 days the mice were sacrificed. The number of lung metastasis nodules were measured to analyze the effects on tumor spontaneous metastasis. Immunohistochemistry analysis. Immunohistochemistry was carried out as described previously [19]. In brief, the paraffinembedded primary melanoma tissues were sectioned with a microtome, deparaffined with xylene, rehydrated and stained immunohistochemically using the labeled-(strept) avidin–biotin method. The slides incubated with the appropriate antibody at 4 °C overnight. These consisted of a rabbit anti-mouse CD34 (BA0532,1:50) antibody (Boster Biological Technology Co., Ltd., Wuhan, China), and a goat anti-VEGF (sc-152,1:50) polyclonal antibody (Santa Cruz Biotech, Santa Cruz, CA, USA). The next day the tissue sections were incubated in blocking buffer, containing the corresponding secondary antibody, at RT for 15 min. After washing with PBS, immunohistoreactivity was visualized using diaminobenidine (DAB) under a light microscope at the indicated magnification. Hematoxyline/eosin (H and E) staining analysis. Hematoxylin/eosin staining analysis was performed as previously described [20]. Specimens were examined and photographed under a microscope after staining. Immunoreactive areas were measured from ten randomly selected fields of lung tissue sections per animal using the Optimas image analyzer (Optimas Corporation USA). Three animals were examined per group.
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Fig. 1. Ligustrazine has no effect on HUVECs and normal fibroblasts proliferation. Proliferating HUVECs and NIH-3T3 fibroblasts were treated with various concentrations (0, 0.16, 0.8, 4, 20, 100 lg/ml) of Ligustrazine. After TMP was added for 24, 48, and 72 h, cell growth and viability were measured by MTT assay. The reading of control was normalized to 100%, and readings from Ligustrazine-treated cells were expressed as% of control. (A) Ligustrazine has no effect on HUVECs proliferation. (B) Ligustrazine has no effect on NIH-3T3 fibroblasts proliferation.
Fig. 2. Ligustrazine inhibits VEGF-induced migration, tube formation and vessel sprouting in vitro. (A) Ligustrazine inhibited VEGF-induced migration of HUVECs. Chemotaxis was quantified by counting the cells that migrated to the lower side of the filter after fixation and staining (n = 3). Taken at 100 magnification, the photographs show the lower chamber of different groups (a–f). Migrated cells were counted by an inverted light microscope at 400 magnification (g). (B) Ligustrazine inhibits VEGF-induced tube formation of HUVECs. Micrographs were taken at 40 magnification (a–f). The mean number of tubes was counted (g) (n = 3). (C) Ligustrazine inhibits VEGF-induced vessel sprouting. Representative aortic rings were photographed (a–e). The numbers of sprouting vessels of each ring were counted by an inverted microscope at a 100 magnification. The data are presented as mean (n = 6); The arrows indicate the newly formed microvessels. Five random fields were chosen for each count. Experiments were repeated three times and values are thus the means of triplicates. (*P < 0.05; **P < 0.01 Ligustrazine + VEGF group versus VEGF group, DDP < 0.01 versus control); bars,±s.e.m.
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Statistical analysis. The data obtained from at least three independent tests are presented as means ± s.e.m. and statistical comparisons between groups performed using 1-way ANOVA followed by Student’s t-test at P values of < 0.01() or <0.05(). Results Ligustrazine has no effect on HUVECs and normal fibroblasts proliferation To determine the cytotoxicity of Ligustrazine, its inhibitory effect on proliferation of HUVECs and NIH-3T3 fibroblasts was evaluated by MTT assay. The data showed that Ligustrazine didn’t inhibit HUVECs and normal fibroblasts proliferation (Fig. 1). These results indicate that Ligustrazine may have limited cytotoxicity to HUVECs and normal fibroblasts even at a dose up to 100 lg/ml. Ligustrazine inhibits VEGF-induced migration, tube formation and vessel sprouting in vitro The migration of endothelial cells through basement membranes is a crucial step in the development of new blood vessels [21,22]. We thus first examined the effect of Ligustrazine on VEGF-induced migration of endothelial cells in vitro. In this experiment, we found that Ligustrazine (0.8–100 lg/ml) inhibited HUVECs migration dose-dependently (Fig. 2A). In order to study the effect on endothelial cell differentiation and spontaneous formation of capillary-like structures, functions crucial in blood vessel formation, we next tested whether Ligustrazine decreased the formation of tubes by HUVECs on fibrin gel in vitro. In the control group HUVECs formed a mesh of tubes, whereas those treated with Ligustrazine (from 0.8 to 100 lg/ml) were unable to form
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intact tubes (Fig. 2B a–f). Moreover, the inhibition effect was dose-dependent (Fig. 2B g). The sprouting of vessels from rat aortic rings was investigated to determine whether Ligustrazine inhibits VEGF-induced angiogenesis in vitro. Sprouts start to form within three days, with a complex three-dimensional network emerging by day 7. VEGF (10 ng/ml) significantly stimulated vessel sprouting above that seen with medium alone. The presence of Ligustrazine (20, 100 lg/ml) resulted in a significant reduction of VEGF-induced vessel sprouting in a dose-dependent manner (Fig. 2C). Ligustrazine inhibits angiogenesis in CAM and vessel formation in Matrigel Plug in vivo We found that Ligustrazine inhibited VEGF-induced migration, tube formation and vessel sprouting in vitro. To investigate the effect of Ligustrazine on angiogenesis in vivo, a modified chick chorioallantoic membrane assay was carried out. The control and VEGF-treated CAMs showed well-developed zones of neo-vascularization surrounding the sponge (Fig. 3A a–b). In contrast, CAM neovascularization was significantly suppressed by addition of Ligustrazine (25, 50, 100 lg/egg; Fig. 3A c–e, f). The Matrigel plug assay was employed to determine whether Ligustrazine was capable of blocking bFGF-induced angiogenesis in vivo. Compared with the control group, plugs from the Ligustrazine treated groups were pale in their color, indicating less blood vessel formation (Fig. 3B a–c). Furthermore, the mean hemoglobin content in the control group was 2.41 ± 1.11 mg/ml compared to 1.38 ± 0.96(20 mg/kg/day) and 0.91 ± 0.54 mg/ml (40 mg/kg/day) (P < 0.01) in in Ligustrazine-treated groups (Fig. 3B d). These results indicate that Ligustrazine is capable of inhibiting bFGF-induced angiogenesis in vivo.
Fig. 3. Ligustrazine inhibits angiogenesis in CAM and vessel formation in Matrigel Plug in vivo. (A) Ligustrazine (25, 50, or 100 lg/egg) or saline (control) were loaded on gelatin sponges which were loaded on the chick chorioallantoic membranes of chick embryos. After 72 h incubation, 10% v/v formaldehyde was added onto the surface of CAMs to fix the blood. The disc and surrounding CAMs were incised carefully and photographed; representative photographs show the CAMs of different groups (a–e). Quantification of newly formed blood vessels were shown in (f). Ten eggs were used for each data point, and mean is shown. bars,±s.e.m (*P < 0.05, **P < 0.01 versus VEGF, D P < 0.05 versus control). (B) Ligustrazine reduces vessel formation in a Matrigel plug. C57BL/6 mice injected with Matrigel plugs containing bFGF were treated intraperitoneally with Ligustrazine (20 or 40 mg/kg) or the control group received saline only. After 7 days mice were killed and the neovascularization and hemoglobin content of Matrigel plugs were evaluated. Macroscopic analysis of Matrigel from one representative experiment shown (a–c). Hemoglobin content of Matrigel plugs is shown in (d). The data are presented as mean, **P < 0.01, versus control; bars,±s.e.m. The experiment was repeated twice (n = 8).
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Ligustrazine inhibits angiogenesis in B16F10 melanoma primary tumor, lung metastasis, and microvessels in lung As we have shown, Ligustrazine has an obvious inhibitory effect on angiogenesis in vitro and in vivo. We thus investigated whether Ligustrazine plays a role in tumor angiogenesis in vivo by establishing a spontaneous B16F10 melanoma metastasis model. After injection of B16F10 cells followed by the administration of Ligustrazine (6.7, 20, or 60 mg/kg) or normal saline only (control group),
on day 23, the primary tumor was resected from the control and Ligustrazine treatment groups and fixed with formalin. Immunohistochemistry analyses of CD34 and VEGF were carried out as previously described. We found that the expression of CD34 (Fig. 4A) and VEGF (Fig. 4B) in the Ligustrazine treated groups were lower than that seen in the control group. Angiogenesis is crucial for tumor metastasis and we found that Ligustrazine inhibited angiogenesis in a primary tumor. Thus we then tested the ability of Ligustrazine to inhibit tumour metastasis using spontaneous a
Fig. 4. Ligustrazine inhibits angiogenesis in B16F10 melanoma primary tumor, lung metastasis and microvessels. In spontaneous metastasis model, on day 23 the footpads bearing the local tumors were amputated. VEGF and CD34-positive immunohistochemical staining of tumor cross-sections in the control and Ligustrazine (TMP)-treated mice were determined in the primary tumor tissue by a light microscope at 100 magnification. (A) Immunohistochemical analysis of CD34 protein expression in tumor tissue; (B) immunohistochemical analysis of VEGF protein expression in tumor tissues. After 23 days post-excision of the primary tumor, mice were killed and lungs were removed. The metastatic colonies were counted under a dissecting microscope. The lungs were photographed to show the effects of Ligustrazine on tumor spontaneous metastasis. (C) Lung metastasis nodules in a model of spontaneous metastasis. (a) Saline control, (b) Mice treated with TMP 6.7 mg/kg, (c) Mice treated with TMP 20 mg/kg, (d) Mice treated with TMP 60 mg/kg. (e) The number of lung metastases foci in each group. (D) H and E staining for blood vessels in lung tissues, arrows indicate microvessels (magnification, 100). Columns, mean (n = 5); bars,±s.e.m., *P < 0.05 versus control and **P < 0.01 versus control.
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B16F10 melanoma metastasis model. After 23 days post-excision of the primary tumor, the lungs of mice were removed, photographed, and the metastatic colonies counted under a dissecting microscope. Excitingly, compared with the control group, Ligustrazine remarkably decreased the number of tumor nodules on the lung surface in a dose-dependent fashion (6.7 mg/kg, P < 0.05; 20 mg/kg, P < 0.01; 60 mg/kg, P < 0.01; Fig. 4C). We also observed the density of blood vessels in the lung tissues by H and E staining. The number of microvessels in lung of the Ligustrazine group was less than that in the control group (Fig. 4D). Discussion In this study, we attempted to elucidate the effect of Ligustrazine on angiogenesis and tumour metastasis. Our results show that systemic treatment of C57BL/6 mice with intraperitoneally injected Ligustrazine suppresses metastasis in the B16F10 melanoma model. This effect of Ligustrazine is likely to be due to its angiogenic activity as follows. We first investigated the anti-angiogenic activity of Ligustrazine in vitro. VEGF, generated from a variety of tumors, is the most important angiogenic factor associated closely with induction and maintenance of the neovasculature in human tumors [23,24]. We found that Ligustrazine inhibited VEGF-induced endothelial migration, capillary formation and vessel sprouting, and had limited cytotoxicity to HUVECs and normal fibroblasts even at a dose up to 100 lg/ml. The in vitro activity of Ligustrazine across all phases of the angiogenic process was confirmed using in vivo angiogenic assays. Ligustrazine inhibited neovascularization in the chick chorioallantoic membrane and significantly reduced vessel formation, while inhibiting the propensity of vessels to grow toward the Matrigel plug. In addition, Ligustrazine decreased the expression of CD34 and VEGF in tumor tissues. From systemic treatments of tumor-bearing mice with Ligustrazin (6.7, 20, and 60 mg/kg), we discovered that Ligustrazine has a potent antimetastatic activity in spontaneous metastasis. We found that Ligustrazine decreased metastasis nodi on the lung surface in a dose-dependent manner. Our results indicate that the inhibitory effect of Ligustrazine is to some extent mediated through its anti-angiogenic activity. Such anti-angiogenic effects are likely to have been primarily responsible for the inhibition of the tumor metastasis, for the microvessel density was only about 20% of that in the control group. As vascularization is a prerequisite of tumor growth [25], the lower number of blood vessels must have been a major impediment to tumor metastasis in the treated mice. Thus inhibition of angiogenesis, with a resulting suppressive effect on tumor metastases, thus possibly underlies the antimetastatic effect of Ligustrazine. For example, we found that the number of metastases correlated with the number of blood vessels. The reduced vasculature in tumors could make it more difficult for tumor cells to enter the circulation. In conclusion, we have demonstrated that Ligustrazine can inhibit B16F10 melanoma metastasis through inhibition of neovascularization. Our findings suggest that Ligustrazine could potentially be beneficial in inhibiting tumor metastasis and angiogenesis. Acknowledgments We are grateful for financial support from National Nature Science Foundation of China (Project Nos. 30371727 and 30772766)
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