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Suppression of angiogenesis by the plant alkaloid, sanguinarine
BBRC Biochemical and Biophysical Research Communications 317 (2004) 618–624 www.elsevier.com/locate/ybbrc
Suppression of angiogenesis by the plant alkaloid, sanguinarine Jong-Pil Euna and Gou Young Kohb,* b
a Department of Neurosurgery, Chonbuk National University Hospital, Jeonju 560-180, Republic of Korea Biomedical Research Center and Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Republic of Korea
Received 7 February 2004
Abstract Sanguinarine is a benzophenanthridine alkaloid derived from the root of Sanguinaria canadensis. Its principal pharmacologic use is in dental products where it has antibacterial, antifungal, and anti-inflammatory activities that reduce gingival inflammation and supragingival plaque formation. Angiogenesis is indispensable for inflammation, and most angiogenesis is dependent on vascular endothelial growth factor (VEGF). However, the effect of sanguinarine on angiogenesis is not known. In the present study, we examined the effect of sanguinarine on VEGF-induced angiogenesis in vitro and in vivo. Interestingly, sanguinarine markedly suppressed VEGF-induced endothelial cell migration, sprouting, and survival in vitro in a dose-dependent manner at nanomolar concentrations. Furthermore, sanguinarine potently suppressed blood vessel formation in vivo in mouse Matrigel plugs and the chorioallantoic membrane of chick embryos. Our biochemical assays indicated that sanguinarine strongly suppressed basal and VEGF-induced Akt phosphorylation, while it did not produce any changes in VEGF-induced activation of ERK1/2 and PLCc1. Therefore, we conclude that sanguinarine is a potent antiangiogenic natural product, and its mode of action could involve the blocking of VEGF-induced Akt activation. Thus, in addition to antibacterial, antifungal, and anti-inflammatory activities, sanguinarine has a novel antiangiogenic role. Ó 2004 Elsevier Inc. All rights reserved.
Sanguinarine (13-methyl[1,3]benzodioxolo[5,6-c]-1,3dioxolo[4,5-i]phenanthridinium) is derived from the root of Sanguinaria canadensis and is also found in poppy and Fumaria species [1] (Fig. 1). Sanguinarine has been shown to have antimicrobial [2] and anti-inflammatory activities in animals [3], to inhibit neutrophil function, including degranulation and phagocytosis in vitro [4], and to have antioxidant properties [5]. To date, the principal pharmacologic use of sanguinarine is in dental products. Its antibacterial, antifungal, and anti-inflammatory activities reduce gingival inflammation and supragingival plaque formation [6]. In fact, sanguinarine at micromolar concentrations is also known to potently inhibit the activation of nuclear transcription factor NF-jB, which has been implicated to play a key role in regulation of inflammation [7]. A recent study [8] has also shown that sanguinarine, at micromolar concentrations, inhibits the growth of human squamous carcinoma (A431) cells through the * Corresponding author. Fax: +82-42-869-2610. E-mail address: [email protected] (G.Y. Koh).
0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.03.077
induction of apoptosis. In sharp contrast, normal human embryonic kidney cells treated with high concentrations of sanguinarine do not show any evidence of apoptosis, but undergo necrotic cell death. Thus, sanguinarine may be able to affect the steady-state cell population and thus has the potential for development as an agent against skin cancer and possibly against other cancer types as well [8]. Blood vessels form through two distinct processes, vasculogenesis and angiogenesis [9–11]. In vasculogenesis, endothelial cells differentiate de novo from mesodermal precursors, whereas in angiogenesis new vessels are generated from pre-existing ones [9–11]. In embryos, both processes are essential for normal development. In adults, pathologic angiogenesis is an unwanted process in certain disease states, including cancer, diabetic retinopathy, granulation tissue formation, rheumatoid arthritis, and psoriasis [12,13]. VEGF plays a key role in normal blood vessel formation as well as in pathologic angiogenesis [14–16]. Many endogenous factors and pharmacological agents that regulate cancer progression and inflammation can also affect angiogenesis [17,18].
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Fig. 1. Structure of sanguinarine.
However, there is no information regarding the effect of sanguinarine on angiogenesis. In this study, we assayed the effect of sanguinarine on VEGF-induced endothelial cell migration, sprouting, and survival in vitro and on angiogenesis in vivo. We also tried to clarify the mechanism of how sanguinarine suppresses VEGF-induced angiogenesis. We found that sanguinarine suppresses basal and VEGF-induced angiogenesis, possibly through blocking Akt activation in endothelial cells.
Materials and methods Materials and cell culture. Sanguinarine chloride (Sigma–Aldrich, St. Louis, MO) was dissolved in ethanol:Cremophore (1:1) solution and then diluted with the cell culture medium to the indicated concentrations. Recombinant human vascular endothelial growth factor165 (VEGF) was purchased from R&D Systems (Minneapolis, MN). Antibodies for phospho-specific ERK1/2 (Thr202/Tyr204), ERK1/2, phospho-specific Akt (Ser473), and Akt were purchased form Cell Signaling (Beverly, MA). Antibodies for PLCc1 and phospho-specific PLCc1 (Tyr783) were obtained from Dr. Pann-Gill Suh (Postech, Pohang, Korea). Matrigel, cell culture reagents, and most other biochemical reagents were purchased from Sigma–Aldrich, unless otherwise specified. Human umbilical vein endothelial cells (HUVECs) and porcine pulmonary arterial endothelial cells (PPAECs) were prepared from human umbilical cords and porcine pulmonary arteries by collagenase digestion, as described previously [19]. HUVECs were maintained in M-199 medium supplemented with 20% fetal bovine serum (FBS), and PPAECs were maintained in DMEM supplemented with 10% FBS at 37 °C in 5% CO2 . The primary cultured cells used in this study were between passages 2 and 3. Assays for migration, sprouting, apoptosis, and DNA synthesis. The migration assay in HUVECs was performed using a modified Boyden chamber (Neuroprobe, Cabin John, MD) as previously described [20]. Briefly, the indicated reagents in M199 containing 1% FBS were placed in the bottom wells of the chamber. Polycarbonate filters with 8 lm pores (Poretics, Livermore, CA) were coated with 50 lg/ml fibronectin and 0.2% gelatin and placed between the test substances and the upper chambers. Cells were trypsinized, washed twice in M199, and resuspended in M199 containing 1% FBS. We placed 2 105 cells into each well in the upper chamber and then incubated for 6 h at 37 °C in a humidified chamber with 5% CO2 . After incubation, the non-migrated cells were removed from the upper side of the filters with a cotton ball.
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The filters were fixed with methanol, mounted onto microscope slides, and stained with Diff-Quik solution. The migrated cells were counted at 100 magnification using a microscope. A sprouting assay was performed as previously described [19]. Briefly, PCAECs were grown to confluence on microcarrier (MC) beads (diameter 175 lm; Sigma) and placed in a 2.5 mg/ml fibrinogen gel containing 200 U/ml Trasylol (Bayer). Fibrin gels containing the MC beads were incubated in M-199 medium supplemented with 2% FBS and VEGF with the indicated amount of sanguinarine. The fibrin gels were incubated in the same medium with a daily addition of the same amount of reagents. After 3 days, two independent, blinded investigators counted the number of sprouts using an inverted microscope. The number of endothelial sprouts with length exceeding the diameter of the MC beads (175 lm) per 50 MC beads was counted. For the apoptosis assay, HUVECs were plated onto gelatinized 24well plates (7 104 cells/well) in M-199 containing 20% FBS and incubated for 12 h. Then the wells were extensively washed with PBS, and the medium was changed to serum-free M-199 containing control buffer, VEGF, and the indicated amount of sanguinarine, and incubated for 24 h. Floating apoptotic cells were collected with two washes in PBS. Adherent cells were collected by trypsinization. All cells were stained with the annexin-V-fluos staining kit (Roche Molecular Biochemicals, Mannheim, Germany) for 15 min at 20 °C. Following staining of annexin-V and propidium iodide, the cells were analyzed on a flow cytometer and data were analyzed with CellQuest software (Becton–Dickinson). The DNA synthetic activity of HUVECs was measured as previously described [20]. Briefly, HUVECs were plated onto gelatinized 24-well plates at a density of 2 104 cells/cm2 in M-199 medium supplemented with 2% (vol/vol) FBS. The cells were cultured for 12 h. Then the medium was changed and indicated amounts of VEGF and sanguinarine were added. After 24 h, fresh medium and indicated amounts of VEGF and sanguinarine were added and the cells were incubated for an additional 24 h. The cells were washed twice with PBS, and the DNA amount was measured with PicoGreen fluorescent reagent (Molecular Probes) using a fluorescence spectrometer equipped with a microplate reader (Molecular Device). In vivo blood vessel formation. A Matrigel plug assay was performed as previously described [21]. In brief, C57/BL6 mice were injected subcutaneously with 0.5 ml Matrigel with heparin (50 U/ml) that contained control buffer, VEGF, or VEGF plus the indicated amount of sanguinarine. All three gels were injected into the same animal at different locations on the flank in order to avoid variations between individual mice. Each portion of the injected Matrigel rapidly formed a single solid gel plug. After 2 weeks, the mice were killed, and the Matrigel plugs were recovered, fixed with 4% paraformaldehyde in PBS, embedded in paraffin, sectioned, and stained with Mason-Trichrome solution for microscopic observation. To quantify the formation of functional neovessels in Matrigel, the amount of hemoglobin in each plug was assayed according to the manufacturer’s protocol (Drabkin reagent kit 525, Sigma–Aldrich). For the chorioallantoic membrane (CAM) assay, fertilized chick embryos were preincubated for 9 days at 38 °C with 70% humidity. A hole was drilled over the air sac at the end of the eggs and an avascular zone was identified on the CAMs. A 1 1-cm window in the shell was made to expose the CAM. Thermanox discs were sterilized and loaded with control buffer or the indicated amount of sanguinarine. After airdrying under a laminar flow hood, the discs were applied to the CAM surface. The windows were sealed with clear tape and the eggs were incubated for 60 h. Blood vessels were viewed, counted, and photographed with an Axioskope2 plus microscope (Carl Zeiss, Germany) equipped with color CCD camera (ProgResC14, Jenoptik, Germany) and monitor. Two independent, blinded investigators counted blood vessels for each group. Inter-investigator variation was <5%. Biochemical analyses. HUVECs were incubated in M-199 medium with 1% fetal bovine serum (FBS) for 16 h before addition of control buffer, VEGF, and sanguinarine. To measure ERK1/2 (Thr202/
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Tyr204), Akt (Ser473), and PLCc1 (Tyr783) phosphorylation, HUVECs were treated with VEGF and sanguinarine for the indicated times and doses. The treated HUVECs were washed two times with PBS, dissolved in sample buffer (50 mM Tris–HCl, 100 mM NaCl, 0.1% SDS, 1% NP-40, 50 mM NaF, 1 mM Na3 VO4 , 1 lg/ml aprotinin, and 1 lg/ml pepstatin, and 1 lg/ml leupeptin), boiled, separated by SDS–PAGE, and transferred to nitrocellulose membrane. The membranes were treated and the phosphorylation levels were analyzed according to the manufacturer’s protocol (Cell Signaling). All signals were visualized and analyzed by densitometric scanning (LAS-1000, Fuji Film, Tokyo). Data analyses. Data are expressed as means standard deviation (SD). Statistical significance was tested using one-way ANOVA followed by the Student–Newman–Keuls test. Statistical significance was set at p < 0:05.
Results and discussion Sanguinarine suppresses VEGF-induced migration, sprouting, and survival in endothelial cells in a dosedependent manner Endothelial cell migration and sprouting from preexisting blood vessels are hallmarks of angiogenesis during pathologic processes [9–11]. In addition, endothelial cell survival and proliferation are prerequisite for migration, sprouting, and angiogenesis [9–11]. VEGF is known to be a strong migration, sprouting, survival, and proliferation factor for endothelial cells during pathologic angiogenesis [14–16]. We modeled these in vivo processes using primary cultured endothelial cells. Then, we first examined the effect of sanguinarine on VEGFinduced endothelial cell migration, sprouting, survival, and proliferation in vitro. VEGF (10 ng/ml) increased migration approximately 5.1-fold, while sanguinarine
(100 nM) slightly decreased migration (Fig. 2). Although a very low concentration of sanguinarine (1 nM) did not significantly suppress VEGF-induced migration, higher concentrations of sanguinarine (10–300 nM) significantly suppressed VEGF-induced migration in a dosedependent manner (Fig. 2). VEGF (10 ng/ml) also increased sprouting approximately 3.1-fold, while sanguinarine (100 nM) reduced sprouting approximately 25% (Fig. 3). Even at the lowest concentration of sanguinarine (1 nM), VEGF-induced sprouting was significantly suppressed. Higher concentrations of sanguinarine (10–300 nM) markedly suppressed VEGF-induced sprouting in a dose-dependent manner (Fig. 3). Serum-deprivation for 24 h produced approximately 33.3% endothelial cell apoptosis (Fig. 4). Addition of sanguinarine (50 nM) slightly increased apoptosis (Fig. 4). VEGF (10 ng/ml) prevented approximately 75% of the apoptotic events. Sanguinarine (10–100 nM) markedly suppressed the VEGF-induced antiapoptotic effect in a dose-dependent manner (Fig. 4). Notably, addition of sanguinarine (300 nM) produced severe apoptosis, up to 96%, even in the presence of VEGF (10 ng/ml). Thus, sanguinarine is a potent inhibitor of VEGF-induced endothelial cell survival. VEGF (10 ng/ ml) also increased DNA synthesis approximately 2.2-fold. However, sanguinarine (10–100 nM) did not produce any significant effect on VEGF-induced DNA synthesis (data not shown), while addition of sanguinarine (300 nM) reduced VEGF-induced DNA synthesis approximately 18%. Thus, sanguinarine is a relatively mild inhibitor of VEGF-induced DNA synthesis in endothelial cell. To assess any possible toxicity of sanguinarine to HUVECs that were maintained in M-199 medium supplemented with 10% FBS, we performed
Fig. 2. Sanguinarine suppresses VEGF-induced endothelial cell migration in a dose-dependent manner. (A) Representative photographs of migrated HUVECs after addition of control buffer (CB), sanguinarine (SG, 100 nM), VEGF (10 ng/ml), or sanguinarine (100 nM) plus VEGF (10 ng/ml) (SG + VEGF) for 6 h in a modified Boyden chamber. After incubation, migrated cells were fixed with methanol, mounted onto microscope slides, and stained with Diff-Quik solution. Irregular circles indicate pores of the chambers, while deep-purple colors indicate nuclei of migrated cells. Note that VEGF increased the number of migrated cells, while sanguinarine suppressed this effect. (B) Quantification of migrated HUVECs. The migrated cells were counted at 100 magnification using a microscope. Bars represent the means SD from five experiments. *p < 0:05 versus CB; # p < 0:05 versus CB plus VEGF165 (10 ng/ml). (For interpretation of the references to colours in this figure legend, the reader is referred to the web version of this paper.)
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Fig. 3. Sanguinarine suppresses VEGF-induced endothelial cell sprouting in a dose-dependent manner. (A) Representative phase-contrast photographs of sprouting activity. PPAECs grown on MC beads were placed in fibrin gels containing control buffer (CB), sanguinarine (SG, 100 nM), VEGF (10 ng/ml), or VEGF (10 ng/ml) plus sanguinarine (100 nM) (SG + VEGF), and incubated with daily supplementation with the same amount of reagents. Note that longer sprouts are formed on VEGF-treated beads than on CB- or SG-treated beads. Addition of SG suppressed VEGFinduced sprouting activity. Magnifications are 200. (B) Quantification of the sprouting activities. The number of endothelial sprouts with length exceeding the diameter of the MC beads (175 lm) per 50 MC beads was counted after 3 days. Bars represent means SD from five experiments. *p < 0:05 versus CB; # p < 0:05 versus CB plus VEGF (10 ng/ml).
Fig. 4. Sanguinarine suppresses VEGF-induced endothelial cell survival in a dose-dependent manner. (A) Representative phase-contrast photographs of apoptotic activity. Control buffer (CB), sanguinarine (SG, 50 nM), VEGF (10 ng/ml), or VEGF (10 ng/ml) plus sanguinarine (50 nM) (SG + VEGF) was added to cells after changing to serum-derived medium. Apoptosis was examined 24 h after serum deprivation. Note that there are fewer adherent cells and more floating cells in the CB-treated culture. There are more floating cells among those exposed to SG, while there are more adherent cells among those exposed to VEGF. Addition of SG suppresses the VEGF-induced antiapoptotic effect. Magnification: 200. (B) Quantification of apoptosis. Bars represent means SD of five experiments. *p < 0:05 versus CB; # p < 0:05 versus CB plus VEGF (10 ng/ml).
careful microscopic observation of the cells and an apoptosis assay. In the range from 1 to 300 nM, sanguinarine exposure for 24 h did not produce any notable effects on HUVECs. However, at concentrations of 1.0 and 3.0 lM, sanguinarine for 24 h induced apoptosis approximately 5.6% and 9.5% in the cells. Thus, sanguinarine at less than 300 nM is not toxic, while concentrations of sanguinarine above 1 lM may have toxic effects with mild induction of apoptosis. Thus, in the in vitro systems, sanguinarine at nanomolar concentrations suppressed VEGF-induced endothelial cell migration, sprouting, and survival in a dose-dependent manner without a notable toxic effect.
Sanguinarine suppresses blood vessel formation in vivo Second, to determine whether sanguinarine is capable of suppressing blood vessel formation in vivo, two angiogenesis models, the mouse Matrigel plug assay and CAM assay, were used. At day 14, macroscopic and histochemical analyses indicated that Matrigel containing VEGF (100 ng) produced a greater formation of blood vessels containing red blood cells within the gel than Matrigel containing control buffer (Figs. 5A and B). Accordingly, Matrigel containing VEGF (100 ng) had more hemoglobin, approximately 5.2-fold higher than Matrigel containing control buffer (Fig. 5C).
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Fig. 5. Sanguinarine suppresses blood vessel formation in vivo. (A) Representative photographs of Matrigel plugs that contained control buffer (CB), VEGF (100 ng), or VEGF (100 ng) plus sanguinarine (100 nM) (SG + VEGF). The mice were injected subcutaneously with 0.5 ml Matrigel containing the indicated agents. After 14 days, mice were sacrificed, and Matrigel plugs were excised and fixed. (B) Representative photograph of the gels shown in (A) cross-sectioned and stained with Trichrome-Mason stain. Note that there is abundant blood vessel formation containing red blood cells in the VEGF-containing plug. Addition of SG suppressed the VEGF-induced blood vessel formation. Magnification: 100. (C) Quantification of blood vessel formation by measurement of hemoglobin in the Matrigel. Bars represent means SD. For each group, n ¼ 7. *p < 0:05 versus CB; # p < 0:05 versus VEGF (100 ng/ml). (D) Representative photographs of CAM treated with CB or sanguinarine (SG, 100 ng). A coverslip containing CB or SG was placed on the CAMs of 9-day-old chicken embryos. After 60 h, the blood vessel formation in the area under the coverslip was photographed. Magnification: 33. (E) Quantification of inhibition of blood vessel formation. The inhibition of blood vessel formation was determined as a percentage of the number of blood vessels for the coverslip containing the indicated amount of sanguinarine over the number of blood vessels under the CB-containing coverslip areas. Bars represent means SD. For each group, n ¼ 9. *p < 0:05 versus CB.
Addition of sanguinarine (100 nM) suppressed VEGFinduced blood vessel formation in the gel (Figs. 5A and B). Sanguinarine (100 nM) suppressed the VEGF-induced hemoglobin amount approximately 67% (Fig. 5C). In the CAM assay, sanguinarine (1, 10, and 100 ng) suppressed blood vessel formation in a dosedependent manner (Figs. 5D and E). These results indicate that sanguinarine at nanomolar concentrations is a potent inhibitor of blood vessel formation in vivo. Sanguinarine suppresses VEGF-induced Akt phosphorylation but not ERK 1/2 or PLCc1 phosphorylation VEGF binds to receptor tyrosine kinase receptors of which VEGFR1 (flt-1) and VEGFR2 (flk-1/KDR) are well characterized [14–16]. Once VEGF binds to VEGFR2, in endothelial cells, several intracellular kinases are activated [22–26]. Of these, activation of ERK1/2, PI 30 -kinase/Akt, and PLCc1 are three major signaling cascades for endothelial cell migration, survival, and proliferation [22–26]. Therefore, we third examined how sanguinarine suppressed VEGF-induced
endothelial cell migration, sprouting, and survival at the level of intercellular signaling. Preliminary analysis indicated that VEGF maximally induced phosphorylation of ERK1/2 at 5 min, and Akt and PLCc1 at 10 min. Sanguinarine (100 nM) itself did not produce any change in the basal phosphorylation of ERK1/2 and PLCc1 (Y783), while it suppressed basal phosphorylation of Akt (Fig. 6). Pretreatment with sanguinarine (100 nM) did not produce significant changes in the VEGF-induced ERK1/2 or PLCc1 phosphorylation, while it completely abolished VEGF-induced Akt phosphorylation (Fig. 6). Our preliminary experiments indicated that pretreatment with sanguinarine (100 nM) suppressed VEGF-induced phosphorylation of the p85 subunit of PI 30 -kinase (data not shown). These results suggest that the suppression of basal and VEGF-induced angiogenesis by sanguinarine could occur partly through blocking basal and VEGF-induced PI 30 -kinase/ Akt activations in endothelial cells. In summary, saunguinarine is a natural product and it is currently used in toothpastes and oral rinses. However, its effective concentrations of antimicrobial
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partly through suppression of basal and VEGF-induced Akt activation in endothelial cells. Therefore, it would be worthwhile to test sanguinarine at low dosages as an antiangiogenic agent for angiogenic dependent diseases.
Acknowledgments We thank S.-N. So, J.S. Eun, H.J. Kwak, C-H. Cho, and H.Y. Choi for critical comments and helpful discussion. We thank Jennifer Macke for help in preparing the manuscript. This work was supported by the Brain Korea 21 Project.
References
Fig. 6. Effect of sanguinarine on VEGF-induced activation of ERK, Akt, and PLCc1 in HUVECs. HUVECs were incubated for 16 h in 1% serum-containing M-199 medium and then incubated with control buffer (CB), VEGF (V, 10 ng/ml), sanguinarine (SG, 100 nM), or sanguinarine (100 nM) plus VEGF (10 ng/ml) (SG + V) for 5 min (for ERK1/2) or 10 min (for PLCc1 and Akt). After treatment, cell lysates were harvested. Each lane contains 50 lg of total protein from the cell lysates. Blots were probed with anti-phospho-ERK1/2 antibody (A), anti-phospho-Akt (Ser473) antibody (B) or anti-phospho-PLCc1 (Y783) (C) (upper panels). The membrane was stripped and reprobed with anti-ERK1/2, anti-Akt antibody, or anti-PLCc1 antibody (lower panels) to verify equal loading of protein in each lane. Fold: densitometric analyses are presented as the relative ratio of phospho-ERK2 to ERK2, phospho-Akt to Akt, or phospho-PLCc1 to PLCc1. The relative ratio to CB is arbitrarily presented as 1. Numbers represent means SD from three experiments. *p < 0:05 versus CB; # p < 0:05 versus VEGF (10 ng/ml).
and anti-inflammatory are in the micromolar range. In comparison, our study indicated that sanguinarine has antiangiogenic activity at nanomolar concentrations. Sanguinarine-induced antiangiogenic activity occurs
[1] M. Shamma, H. Guinaudeau, Anorphinoid alkaloids, Nat. Prod. Rep. 3 (1986) 345–351. [2] L.A. Mitscher, Y.H. Park, D. Clark, G.W. Clark, P.D. Hammesfahr, W.N. Wu, J.L. Beal, Antimicrobial agents from higher plants. An investigation of Hunnemannia fumariaefolia pseudoalcoholates of sanguinarine and chelerythrine, Lloydia 41 (1978) 145–150. [3] J. Lenfeld, M. Kroutil, E. Marsalek, J. Slavik, V. Preininger, V. Simanek, Antiinflammatory activity of quaternary benzophenanthridine alkaloids from Chelidonium majus, Planta Med. 43 (1981) 161–165. [4] S. Agarwal, M.A. Reynolds, S. Pou, D.E. Peterson, J.A. Charon, J.B. Suzuki, The effect of sanguinarine on human peripheral blood neutrophil viability and functions, Oral Microbiol. Immunol. 6 (1991) 51–61. [5] C. Vavreckova, J. Ulrichova, M. Hajduch, F. Grambal, E. Weigl, V. Simanek, Effect of quaternary benzo[c]phenanthridine alkaloids sanguinarine, chelerythrine and fagaronine on some mammalian cells, Acta Univ. Palacki Olomuc. Fac. Med. 138 (1994) 7– 10. [6] J.J. Hannah, J.D. Johnson, M.M. Kuftinec, Long-term clinical evaluation of toothpaste and oral rinse containing sanguinaria extract in controlling plaque, gingival inflammation, and sulcular bleeding during orthodontic treatment, Am. J. Orthod. Dentofacial Orthop. 96 (1989) 199–207. [7] M.M. Chaturvedi, A. Kumar, B.G. Darnay, G.B.N. Chainy, S. Agarwal, B.B. Aggarwal, Sanguinarine (pseudochelerythrine) is a potent inhibitor of NF-jB activation, IjBa phosphorylation, and degradation, J. Biol. Chem. 272 (1997) 30129–30134. [8] N. Ahmad, S. Gupta, M.M. Husain, K.M. Heiskanen, H. Mukhtar, Differential antiproliferative and apoptotic response of sanguinarine for cancer cells versus normal cells, Clin. Cancer Res. 6 (2000) 1524–1528. [9] W. Risau, Mechanisms of angiogenesis, Nature 386 (1997) 671– 674. [10] J. Folkman, P.A. D’Amore, Blood vessel formation: what is its molecular basis? Cell 87 (1996) 1153–1155. [11] P. Carmeliet, Mechanisms of angiogenesis and arteriogenesis, Nat. Med. 6 (2000) 389–395. [12] J. Folkman, Angiogenesis in cancer, vascular, rheumatoid and other disease, Nat. Med. 1 (1995) 27–31. [13] P. Carmeliet P, R.K. Jain, Angiogenesis in cancer and other diseases, Nature 407 (2000) 249–257. [14] N. Ferrara, T. Davis-Smyth, The biology of vascular endothelial growth factor, Endocr. Rev. 18 (1997) 4–25. [15] T. Veikkola, K. Alitalo, VEGFs, receptors and angiogenesis, Semin. Cancer Biol. 9 (1999) 211–220. [16] N. Ferrara, VEGF: an update on biological and therapeutic aspects, Curr. Opin. Biotechnol. 11 (2000) 617–624.
624
J.-P. Eun, G.Y. Koh / Biochemical and Biophysical Research Communications 317 (2004) 618–624
[17] J. Folkman, Angiogenesis inhibitors: a new class of drugs, Cancer Biol. Ther. 2 (4 Suppl. 1) (2003) S127–S133. [18] M.K. Jones, H. Wang, B.M. Peskar, E. Levin, R.M. Itani, I.J. Sarfeh, A.S. Tarnawski, Inhibition of angiogenesis by nonsteroidal anti-inflammatory drugs: insight into mechanisms and implications for cancer growth and ulcer healing, Nat. Med. 5 (1999) 1418–1423. [19] I. Kim, H.G. Kim, S.O. Moon, S.W. Chae, J.N. So, K.N. Koh, B.C. Ahn, G.Y. Koh, Angiopoietin-1 induces endothelial cell sprouting through the activation of focal adhesion kinase and plasmin secretion, Circ. Res. 86 (2000) 952–959. [20] I. Kim, Y.S. Ryu, H.J. Kwak, S.Y. Ahn, J.L. Oh, G.D. Yancopoulos, N.W. Gale, G.Y. Koh, EphB ligand, ephrinB2, suppresses the VEGF- and angiopoietin-1-induced ras/mitogenactivated protein kinase pathway in venous endothelial cells, FASEB J. 16 (2002) 1126–1128. [21] H.S. Kim, H.S. Shin, H.J. Kwak, C.H. Cho, C.O. Lee, G.Y. Koh, Betacellulin induces angiogenesis through activation of mitogenactivated protein kinase and phosphatidylinositol 30 -kinase in endothelial cells, FASEB J. 17 (2003) 318–320. [22] J. Kroll, J. Waltenberger, The vascular endothelial cell growth factor receptor KDR activates multiple signal transduction
[23]
[24]
[25]
[26]
pathways in porcine aortic endothelial cells, J. Biol. Chem. 272 (1997) 32521–32527. G.D. Thakker, D.P. Hajjar, W.A. Muller, T.K. Rosengart, The role of phosphatidylinositol 3-kinase in vascular endothelial growth factor signaling, J. Biol. Chem. 274 (1999) 10002– 10007. H.P. Gerber, A. McMurtrey, J. Kowalski, M. Yan, B.Y. Keyt, V. Dixit, N. Ferrara, Vascular endothelial growth factor regulates endothelial cell survival through the phosphatidylinositol 30 -kinase/Akt signal transduction pathway, J. Biol. Chem. 273 (1998) 30336–30343. T. Takahashi, H. Ueno, M. Shibuya, VEGF activates protein kinase C-dependent, but Ras-independent Raf-MEK-MAP kinase pathway for DNA synthesis in primary endothelial cells, Oncogene 18 (1999) 2221–2230. L.W. Wu, L.D. Mayo, J.D. Dunbar, K.M. Kessler, M.R. Baerwald, E.A. Jaffe, D. Wang, R.S. Warren, D.B. Donner, Utilization of distinct signaling pathways by receptors for vascular endothelial cell growth factor and other mitogens in the induction of endothelial cell proliferation, J. Biol. Chem. 275 (2000) 5096– 5103.
Suppression of angiogenesis by the plant alkaloid, sanguinarine Jong-Pil Euna and Gou Young Kohb,* b
a Department of Neurosurgery, Chonbuk National University Hospital, Jeonju 560-180, Republic of Korea Biomedical Research Center and Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Republic of Korea
Received 7 February 2004
Abstract Sanguinarine is a benzophenanthridine alkaloid derived from the root of Sanguinaria canadensis. Its principal pharmacologic use is in dental products where it has antibacterial, antifungal, and anti-inflammatory activities that reduce gingival inflammation and supragingival plaque formation. Angiogenesis is indispensable for inflammation, and most angiogenesis is dependent on vascular endothelial growth factor (VEGF). However, the effect of sanguinarine on angiogenesis is not known. In the present study, we examined the effect of sanguinarine on VEGF-induced angiogenesis in vitro and in vivo. Interestingly, sanguinarine markedly suppressed VEGF-induced endothelial cell migration, sprouting, and survival in vitro in a dose-dependent manner at nanomolar concentrations. Furthermore, sanguinarine potently suppressed blood vessel formation in vivo in mouse Matrigel plugs and the chorioallantoic membrane of chick embryos. Our biochemical assays indicated that sanguinarine strongly suppressed basal and VEGF-induced Akt phosphorylation, while it did not produce any changes in VEGF-induced activation of ERK1/2 and PLCc1. Therefore, we conclude that sanguinarine is a potent antiangiogenic natural product, and its mode of action could involve the blocking of VEGF-induced Akt activation. Thus, in addition to antibacterial, antifungal, and anti-inflammatory activities, sanguinarine has a novel antiangiogenic role. Ó 2004 Elsevier Inc. All rights reserved.
Sanguinarine (13-methyl[1,3]benzodioxolo[5,6-c]-1,3dioxolo[4,5-i]phenanthridinium) is derived from the root of Sanguinaria canadensis and is also found in poppy and Fumaria species [1] (Fig. 1). Sanguinarine has been shown to have antimicrobial [2] and anti-inflammatory activities in animals [3], to inhibit neutrophil function, including degranulation and phagocytosis in vitro [4], and to have antioxidant properties [5]. To date, the principal pharmacologic use of sanguinarine is in dental products. Its antibacterial, antifungal, and anti-inflammatory activities reduce gingival inflammation and supragingival plaque formation [6]. In fact, sanguinarine at micromolar concentrations is also known to potently inhibit the activation of nuclear transcription factor NF-jB, which has been implicated to play a key role in regulation of inflammation [7]. A recent study [8] has also shown that sanguinarine, at micromolar concentrations, inhibits the growth of human squamous carcinoma (A431) cells through the * Corresponding author. Fax: +82-42-869-2610. E-mail address: [email protected] (G.Y. Koh).
0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.03.077
induction of apoptosis. In sharp contrast, normal human embryonic kidney cells treated with high concentrations of sanguinarine do not show any evidence of apoptosis, but undergo necrotic cell death. Thus, sanguinarine may be able to affect the steady-state cell population and thus has the potential for development as an agent against skin cancer and possibly against other cancer types as well [8]. Blood vessels form through two distinct processes, vasculogenesis and angiogenesis [9–11]. In vasculogenesis, endothelial cells differentiate de novo from mesodermal precursors, whereas in angiogenesis new vessels are generated from pre-existing ones [9–11]. In embryos, both processes are essential for normal development. In adults, pathologic angiogenesis is an unwanted process in certain disease states, including cancer, diabetic retinopathy, granulation tissue formation, rheumatoid arthritis, and psoriasis [12,13]. VEGF plays a key role in normal blood vessel formation as well as in pathologic angiogenesis [14–16]. Many endogenous factors and pharmacological agents that regulate cancer progression and inflammation can also affect angiogenesis [17,18].
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Fig. 1. Structure of sanguinarine.
However, there is no information regarding the effect of sanguinarine on angiogenesis. In this study, we assayed the effect of sanguinarine on VEGF-induced endothelial cell migration, sprouting, and survival in vitro and on angiogenesis in vivo. We also tried to clarify the mechanism of how sanguinarine suppresses VEGF-induced angiogenesis. We found that sanguinarine suppresses basal and VEGF-induced angiogenesis, possibly through blocking Akt activation in endothelial cells.
Materials and methods Materials and cell culture. Sanguinarine chloride (Sigma–Aldrich, St. Louis, MO) was dissolved in ethanol:Cremophore (1:1) solution and then diluted with the cell culture medium to the indicated concentrations. Recombinant human vascular endothelial growth factor165 (VEGF) was purchased from R&D Systems (Minneapolis, MN). Antibodies for phospho-specific ERK1/2 (Thr202/Tyr204), ERK1/2, phospho-specific Akt (Ser473), and Akt were purchased form Cell Signaling (Beverly, MA). Antibodies for PLCc1 and phospho-specific PLCc1 (Tyr783) were obtained from Dr. Pann-Gill Suh (Postech, Pohang, Korea). Matrigel, cell culture reagents, and most other biochemical reagents were purchased from Sigma–Aldrich, unless otherwise specified. Human umbilical vein endothelial cells (HUVECs) and porcine pulmonary arterial endothelial cells (PPAECs) were prepared from human umbilical cords and porcine pulmonary arteries by collagenase digestion, as described previously [19]. HUVECs were maintained in M-199 medium supplemented with 20% fetal bovine serum (FBS), and PPAECs were maintained in DMEM supplemented with 10% FBS at 37 °C in 5% CO2 . The primary cultured cells used in this study were between passages 2 and 3. Assays for migration, sprouting, apoptosis, and DNA synthesis. The migration assay in HUVECs was performed using a modified Boyden chamber (Neuroprobe, Cabin John, MD) as previously described [20]. Briefly, the indicated reagents in M199 containing 1% FBS were placed in the bottom wells of the chamber. Polycarbonate filters with 8 lm pores (Poretics, Livermore, CA) were coated with 50 lg/ml fibronectin and 0.2% gelatin and placed between the test substances and the upper chambers. Cells were trypsinized, washed twice in M199, and resuspended in M199 containing 1% FBS. We placed 2 105 cells into each well in the upper chamber and then incubated for 6 h at 37 °C in a humidified chamber with 5% CO2 . After incubation, the non-migrated cells were removed from the upper side of the filters with a cotton ball.
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The filters were fixed with methanol, mounted onto microscope slides, and stained with Diff-Quik solution. The migrated cells were counted at 100 magnification using a microscope. A sprouting assay was performed as previously described [19]. Briefly, PCAECs were grown to confluence on microcarrier (MC) beads (diameter 175 lm; Sigma) and placed in a 2.5 mg/ml fibrinogen gel containing 200 U/ml Trasylol (Bayer). Fibrin gels containing the MC beads were incubated in M-199 medium supplemented with 2% FBS and VEGF with the indicated amount of sanguinarine. The fibrin gels were incubated in the same medium with a daily addition of the same amount of reagents. After 3 days, two independent, blinded investigators counted the number of sprouts using an inverted microscope. The number of endothelial sprouts with length exceeding the diameter of the MC beads (175 lm) per 50 MC beads was counted. For the apoptosis assay, HUVECs were plated onto gelatinized 24well plates (7 104 cells/well) in M-199 containing 20% FBS and incubated for 12 h. Then the wells were extensively washed with PBS, and the medium was changed to serum-free M-199 containing control buffer, VEGF, and the indicated amount of sanguinarine, and incubated for 24 h. Floating apoptotic cells were collected with two washes in PBS. Adherent cells were collected by trypsinization. All cells were stained with the annexin-V-fluos staining kit (Roche Molecular Biochemicals, Mannheim, Germany) for 15 min at 20 °C. Following staining of annexin-V and propidium iodide, the cells were analyzed on a flow cytometer and data were analyzed with CellQuest software (Becton–Dickinson). The DNA synthetic activity of HUVECs was measured as previously described [20]. Briefly, HUVECs were plated onto gelatinized 24-well plates at a density of 2 104 cells/cm2 in M-199 medium supplemented with 2% (vol/vol) FBS. The cells were cultured for 12 h. Then the medium was changed and indicated amounts of VEGF and sanguinarine were added. After 24 h, fresh medium and indicated amounts of VEGF and sanguinarine were added and the cells were incubated for an additional 24 h. The cells were washed twice with PBS, and the DNA amount was measured with PicoGreen fluorescent reagent (Molecular Probes) using a fluorescence spectrometer equipped with a microplate reader (Molecular Device). In vivo blood vessel formation. A Matrigel plug assay was performed as previously described [21]. In brief, C57/BL6 mice were injected subcutaneously with 0.5 ml Matrigel with heparin (50 U/ml) that contained control buffer, VEGF, or VEGF plus the indicated amount of sanguinarine. All three gels were injected into the same animal at different locations on the flank in order to avoid variations between individual mice. Each portion of the injected Matrigel rapidly formed a single solid gel plug. After 2 weeks, the mice were killed, and the Matrigel plugs were recovered, fixed with 4% paraformaldehyde in PBS, embedded in paraffin, sectioned, and stained with Mason-Trichrome solution for microscopic observation. To quantify the formation of functional neovessels in Matrigel, the amount of hemoglobin in each plug was assayed according to the manufacturer’s protocol (Drabkin reagent kit 525, Sigma–Aldrich). For the chorioallantoic membrane (CAM) assay, fertilized chick embryos were preincubated for 9 days at 38 °C with 70% humidity. A hole was drilled over the air sac at the end of the eggs and an avascular zone was identified on the CAMs. A 1 1-cm window in the shell was made to expose the CAM. Thermanox discs were sterilized and loaded with control buffer or the indicated amount of sanguinarine. After airdrying under a laminar flow hood, the discs were applied to the CAM surface. The windows were sealed with clear tape and the eggs were incubated for 60 h. Blood vessels were viewed, counted, and photographed with an Axioskope2 plus microscope (Carl Zeiss, Germany) equipped with color CCD camera (ProgResC14, Jenoptik, Germany) and monitor. Two independent, blinded investigators counted blood vessels for each group. Inter-investigator variation was <5%. Biochemical analyses. HUVECs were incubated in M-199 medium with 1% fetal bovine serum (FBS) for 16 h before addition of control buffer, VEGF, and sanguinarine. To measure ERK1/2 (Thr202/
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Tyr204), Akt (Ser473), and PLCc1 (Tyr783) phosphorylation, HUVECs were treated with VEGF and sanguinarine for the indicated times and doses. The treated HUVECs were washed two times with PBS, dissolved in sample buffer (50 mM Tris–HCl, 100 mM NaCl, 0.1% SDS, 1% NP-40, 50 mM NaF, 1 mM Na3 VO4 , 1 lg/ml aprotinin, and 1 lg/ml pepstatin, and 1 lg/ml leupeptin), boiled, separated by SDS–PAGE, and transferred to nitrocellulose membrane. The membranes were treated and the phosphorylation levels were analyzed according to the manufacturer’s protocol (Cell Signaling). All signals were visualized and analyzed by densitometric scanning (LAS-1000, Fuji Film, Tokyo). Data analyses. Data are expressed as means standard deviation (SD). Statistical significance was tested using one-way ANOVA followed by the Student–Newman–Keuls test. Statistical significance was set at p < 0:05.
Results and discussion Sanguinarine suppresses VEGF-induced migration, sprouting, and survival in endothelial cells in a dosedependent manner Endothelial cell migration and sprouting from preexisting blood vessels are hallmarks of angiogenesis during pathologic processes [9–11]. In addition, endothelial cell survival and proliferation are prerequisite for migration, sprouting, and angiogenesis [9–11]. VEGF is known to be a strong migration, sprouting, survival, and proliferation factor for endothelial cells during pathologic angiogenesis [14–16]. We modeled these in vivo processes using primary cultured endothelial cells. Then, we first examined the effect of sanguinarine on VEGFinduced endothelial cell migration, sprouting, survival, and proliferation in vitro. VEGF (10 ng/ml) increased migration approximately 5.1-fold, while sanguinarine
(100 nM) slightly decreased migration (Fig. 2). Although a very low concentration of sanguinarine (1 nM) did not significantly suppress VEGF-induced migration, higher concentrations of sanguinarine (10–300 nM) significantly suppressed VEGF-induced migration in a dosedependent manner (Fig. 2). VEGF (10 ng/ml) also increased sprouting approximately 3.1-fold, while sanguinarine (100 nM) reduced sprouting approximately 25% (Fig. 3). Even at the lowest concentration of sanguinarine (1 nM), VEGF-induced sprouting was significantly suppressed. Higher concentrations of sanguinarine (10–300 nM) markedly suppressed VEGF-induced sprouting in a dose-dependent manner (Fig. 3). Serum-deprivation for 24 h produced approximately 33.3% endothelial cell apoptosis (Fig. 4). Addition of sanguinarine (50 nM) slightly increased apoptosis (Fig. 4). VEGF (10 ng/ml) prevented approximately 75% of the apoptotic events. Sanguinarine (10–100 nM) markedly suppressed the VEGF-induced antiapoptotic effect in a dose-dependent manner (Fig. 4). Notably, addition of sanguinarine (300 nM) produced severe apoptosis, up to 96%, even in the presence of VEGF (10 ng/ml). Thus, sanguinarine is a potent inhibitor of VEGF-induced endothelial cell survival. VEGF (10 ng/ ml) also increased DNA synthesis approximately 2.2-fold. However, sanguinarine (10–100 nM) did not produce any significant effect on VEGF-induced DNA synthesis (data not shown), while addition of sanguinarine (300 nM) reduced VEGF-induced DNA synthesis approximately 18%. Thus, sanguinarine is a relatively mild inhibitor of VEGF-induced DNA synthesis in endothelial cell. To assess any possible toxicity of sanguinarine to HUVECs that were maintained in M-199 medium supplemented with 10% FBS, we performed
Fig. 2. Sanguinarine suppresses VEGF-induced endothelial cell migration in a dose-dependent manner. (A) Representative photographs of migrated HUVECs after addition of control buffer (CB), sanguinarine (SG, 100 nM), VEGF (10 ng/ml), or sanguinarine (100 nM) plus VEGF (10 ng/ml) (SG + VEGF) for 6 h in a modified Boyden chamber. After incubation, migrated cells were fixed with methanol, mounted onto microscope slides, and stained with Diff-Quik solution. Irregular circles indicate pores of the chambers, while deep-purple colors indicate nuclei of migrated cells. Note that VEGF increased the number of migrated cells, while sanguinarine suppressed this effect. (B) Quantification of migrated HUVECs. The migrated cells were counted at 100 magnification using a microscope. Bars represent the means SD from five experiments. *p < 0:05 versus CB; # p < 0:05 versus CB plus VEGF165 (10 ng/ml). (For interpretation of the references to colours in this figure legend, the reader is referred to the web version of this paper.)
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Fig. 3. Sanguinarine suppresses VEGF-induced endothelial cell sprouting in a dose-dependent manner. (A) Representative phase-contrast photographs of sprouting activity. PPAECs grown on MC beads were placed in fibrin gels containing control buffer (CB), sanguinarine (SG, 100 nM), VEGF (10 ng/ml), or VEGF (10 ng/ml) plus sanguinarine (100 nM) (SG + VEGF), and incubated with daily supplementation with the same amount of reagents. Note that longer sprouts are formed on VEGF-treated beads than on CB- or SG-treated beads. Addition of SG suppressed VEGFinduced sprouting activity. Magnifications are 200. (B) Quantification of the sprouting activities. The number of endothelial sprouts with length exceeding the diameter of the MC beads (175 lm) per 50 MC beads was counted after 3 days. Bars represent means SD from five experiments. *p < 0:05 versus CB; # p < 0:05 versus CB plus VEGF (10 ng/ml).
Fig. 4. Sanguinarine suppresses VEGF-induced endothelial cell survival in a dose-dependent manner. (A) Representative phase-contrast photographs of apoptotic activity. Control buffer (CB), sanguinarine (SG, 50 nM), VEGF (10 ng/ml), or VEGF (10 ng/ml) plus sanguinarine (50 nM) (SG + VEGF) was added to cells after changing to serum-derived medium. Apoptosis was examined 24 h after serum deprivation. Note that there are fewer adherent cells and more floating cells in the CB-treated culture. There are more floating cells among those exposed to SG, while there are more adherent cells among those exposed to VEGF. Addition of SG suppresses the VEGF-induced antiapoptotic effect. Magnification: 200. (B) Quantification of apoptosis. Bars represent means SD of five experiments. *p < 0:05 versus CB; # p < 0:05 versus CB plus VEGF (10 ng/ml).
careful microscopic observation of the cells and an apoptosis assay. In the range from 1 to 300 nM, sanguinarine exposure for 24 h did not produce any notable effects on HUVECs. However, at concentrations of 1.0 and 3.0 lM, sanguinarine for 24 h induced apoptosis approximately 5.6% and 9.5% in the cells. Thus, sanguinarine at less than 300 nM is not toxic, while concentrations of sanguinarine above 1 lM may have toxic effects with mild induction of apoptosis. Thus, in the in vitro systems, sanguinarine at nanomolar concentrations suppressed VEGF-induced endothelial cell migration, sprouting, and survival in a dose-dependent manner without a notable toxic effect.
Sanguinarine suppresses blood vessel formation in vivo Second, to determine whether sanguinarine is capable of suppressing blood vessel formation in vivo, two angiogenesis models, the mouse Matrigel plug assay and CAM assay, were used. At day 14, macroscopic and histochemical analyses indicated that Matrigel containing VEGF (100 ng) produced a greater formation of blood vessels containing red blood cells within the gel than Matrigel containing control buffer (Figs. 5A and B). Accordingly, Matrigel containing VEGF (100 ng) had more hemoglobin, approximately 5.2-fold higher than Matrigel containing control buffer (Fig. 5C).
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Fig. 5. Sanguinarine suppresses blood vessel formation in vivo. (A) Representative photographs of Matrigel plugs that contained control buffer (CB), VEGF (100 ng), or VEGF (100 ng) plus sanguinarine (100 nM) (SG + VEGF). The mice were injected subcutaneously with 0.5 ml Matrigel containing the indicated agents. After 14 days, mice were sacrificed, and Matrigel plugs were excised and fixed. (B) Representative photograph of the gels shown in (A) cross-sectioned and stained with Trichrome-Mason stain. Note that there is abundant blood vessel formation containing red blood cells in the VEGF-containing plug. Addition of SG suppressed the VEGF-induced blood vessel formation. Magnification: 100. (C) Quantification of blood vessel formation by measurement of hemoglobin in the Matrigel. Bars represent means SD. For each group, n ¼ 7. *p < 0:05 versus CB; # p < 0:05 versus VEGF (100 ng/ml). (D) Representative photographs of CAM treated with CB or sanguinarine (SG, 100 ng). A coverslip containing CB or SG was placed on the CAMs of 9-day-old chicken embryos. After 60 h, the blood vessel formation in the area under the coverslip was photographed. Magnification: 33. (E) Quantification of inhibition of blood vessel formation. The inhibition of blood vessel formation was determined as a percentage of the number of blood vessels for the coverslip containing the indicated amount of sanguinarine over the number of blood vessels under the CB-containing coverslip areas. Bars represent means SD. For each group, n ¼ 9. *p < 0:05 versus CB.
Addition of sanguinarine (100 nM) suppressed VEGFinduced blood vessel formation in the gel (Figs. 5A and B). Sanguinarine (100 nM) suppressed the VEGF-induced hemoglobin amount approximately 67% (Fig. 5C). In the CAM assay, sanguinarine (1, 10, and 100 ng) suppressed blood vessel formation in a dosedependent manner (Figs. 5D and E). These results indicate that sanguinarine at nanomolar concentrations is a potent inhibitor of blood vessel formation in vivo. Sanguinarine suppresses VEGF-induced Akt phosphorylation but not ERK 1/2 or PLCc1 phosphorylation VEGF binds to receptor tyrosine kinase receptors of which VEGFR1 (flt-1) and VEGFR2 (flk-1/KDR) are well characterized [14–16]. Once VEGF binds to VEGFR2, in endothelial cells, several intracellular kinases are activated [22–26]. Of these, activation of ERK1/2, PI 30 -kinase/Akt, and PLCc1 are three major signaling cascades for endothelial cell migration, survival, and proliferation [22–26]. Therefore, we third examined how sanguinarine suppressed VEGF-induced
endothelial cell migration, sprouting, and survival at the level of intercellular signaling. Preliminary analysis indicated that VEGF maximally induced phosphorylation of ERK1/2 at 5 min, and Akt and PLCc1 at 10 min. Sanguinarine (100 nM) itself did not produce any change in the basal phosphorylation of ERK1/2 and PLCc1 (Y783), while it suppressed basal phosphorylation of Akt (Fig. 6). Pretreatment with sanguinarine (100 nM) did not produce significant changes in the VEGF-induced ERK1/2 or PLCc1 phosphorylation, while it completely abolished VEGF-induced Akt phosphorylation (Fig. 6). Our preliminary experiments indicated that pretreatment with sanguinarine (100 nM) suppressed VEGF-induced phosphorylation of the p85 subunit of PI 30 -kinase (data not shown). These results suggest that the suppression of basal and VEGF-induced angiogenesis by sanguinarine could occur partly through blocking basal and VEGF-induced PI 30 -kinase/ Akt activations in endothelial cells. In summary, saunguinarine is a natural product and it is currently used in toothpastes and oral rinses. However, its effective concentrations of antimicrobial
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partly through suppression of basal and VEGF-induced Akt activation in endothelial cells. Therefore, it would be worthwhile to test sanguinarine at low dosages as an antiangiogenic agent for angiogenic dependent diseases.
Acknowledgments We thank S.-N. So, J.S. Eun, H.J. Kwak, C-H. Cho, and H.Y. Choi for critical comments and helpful discussion. We thank Jennifer Macke for help in preparing the manuscript. This work was supported by the Brain Korea 21 Project.
References
Fig. 6. Effect of sanguinarine on VEGF-induced activation of ERK, Akt, and PLCc1 in HUVECs. HUVECs were incubated for 16 h in 1% serum-containing M-199 medium and then incubated with control buffer (CB), VEGF (V, 10 ng/ml), sanguinarine (SG, 100 nM), or sanguinarine (100 nM) plus VEGF (10 ng/ml) (SG + V) for 5 min (for ERK1/2) or 10 min (for PLCc1 and Akt). After treatment, cell lysates were harvested. Each lane contains 50 lg of total protein from the cell lysates. Blots were probed with anti-phospho-ERK1/2 antibody (A), anti-phospho-Akt (Ser473) antibody (B) or anti-phospho-PLCc1 (Y783) (C) (upper panels). The membrane was stripped and reprobed with anti-ERK1/2, anti-Akt antibody, or anti-PLCc1 antibody (lower panels) to verify equal loading of protein in each lane. Fold: densitometric analyses are presented as the relative ratio of phospho-ERK2 to ERK2, phospho-Akt to Akt, or phospho-PLCc1 to PLCc1. The relative ratio to CB is arbitrarily presented as 1. Numbers represent means SD from three experiments. *p < 0:05 versus CB; # p < 0:05 versus VEGF (10 ng/ml).
and anti-inflammatory are in the micromolar range. In comparison, our study indicated that sanguinarine has antiangiogenic activity at nanomolar concentrations. Sanguinarine-induced antiangiogenic activity occurs
[1] M. Shamma, H. Guinaudeau, Anorphinoid alkaloids, Nat. Prod. Rep. 3 (1986) 345–351. [2] L.A. Mitscher, Y.H. Park, D. Clark, G.W. Clark, P.D. Hammesfahr, W.N. Wu, J.L. Beal, Antimicrobial agents from higher plants. An investigation of Hunnemannia fumariaefolia pseudoalcoholates of sanguinarine and chelerythrine, Lloydia 41 (1978) 145–150. [3] J. Lenfeld, M. Kroutil, E. Marsalek, J. Slavik, V. Preininger, V. Simanek, Antiinflammatory activity of quaternary benzophenanthridine alkaloids from Chelidonium majus, Planta Med. 43 (1981) 161–165. [4] S. Agarwal, M.A. Reynolds, S. Pou, D.E. Peterson, J.A. Charon, J.B. Suzuki, The effect of sanguinarine on human peripheral blood neutrophil viability and functions, Oral Microbiol. Immunol. 6 (1991) 51–61. [5] C. Vavreckova, J. Ulrichova, M. Hajduch, F. Grambal, E. Weigl, V. Simanek, Effect of quaternary benzo[c]phenanthridine alkaloids sanguinarine, chelerythrine and fagaronine on some mammalian cells, Acta Univ. Palacki Olomuc. Fac. Med. 138 (1994) 7– 10. [6] J.J. Hannah, J.D. Johnson, M.M. Kuftinec, Long-term clinical evaluation of toothpaste and oral rinse containing sanguinaria extract in controlling plaque, gingival inflammation, and sulcular bleeding during orthodontic treatment, Am. J. Orthod. Dentofacial Orthop. 96 (1989) 199–207. [7] M.M. Chaturvedi, A. Kumar, B.G. Darnay, G.B.N. Chainy, S. Agarwal, B.B. Aggarwal, Sanguinarine (pseudochelerythrine) is a potent inhibitor of NF-jB activation, IjBa phosphorylation, and degradation, J. Biol. Chem. 272 (1997) 30129–30134. [8] N. Ahmad, S. Gupta, M.M. Husain, K.M. Heiskanen, H. Mukhtar, Differential antiproliferative and apoptotic response of sanguinarine for cancer cells versus normal cells, Clin. Cancer Res. 6 (2000) 1524–1528. [9] W. Risau, Mechanisms of angiogenesis, Nature 386 (1997) 671– 674. [10] J. Folkman, P.A. D’Amore, Blood vessel formation: what is its molecular basis? Cell 87 (1996) 1153–1155. [11] P. Carmeliet, Mechanisms of angiogenesis and arteriogenesis, Nat. Med. 6 (2000) 389–395. [12] J. Folkman, Angiogenesis in cancer, vascular, rheumatoid and other disease, Nat. Med. 1 (1995) 27–31. [13] P. Carmeliet P, R.K. Jain, Angiogenesis in cancer and other diseases, Nature 407 (2000) 249–257. [14] N. Ferrara, T. Davis-Smyth, The biology of vascular endothelial growth factor, Endocr. Rev. 18 (1997) 4–25. [15] T. Veikkola, K. Alitalo, VEGFs, receptors and angiogenesis, Semin. Cancer Biol. 9 (1999) 211–220. [16] N. Ferrara, VEGF: an update on biological and therapeutic aspects, Curr. Opin. Biotechnol. 11 (2000) 617–624.
624
J.-P. Eun, G.Y. Koh / Biochemical and Biophysical Research Communications 317 (2004) 618–624
[17] J. Folkman, Angiogenesis inhibitors: a new class of drugs, Cancer Biol. Ther. 2 (4 Suppl. 1) (2003) S127–S133. [18] M.K. Jones, H. Wang, B.M. Peskar, E. Levin, R.M. Itani, I.J. Sarfeh, A.S. Tarnawski, Inhibition of angiogenesis by nonsteroidal anti-inflammatory drugs: insight into mechanisms and implications for cancer growth and ulcer healing, Nat. Med. 5 (1999) 1418–1423. [19] I. Kim, H.G. Kim, S.O. Moon, S.W. Chae, J.N. So, K.N. Koh, B.C. Ahn, G.Y. Koh, Angiopoietin-1 induces endothelial cell sprouting through the activation of focal adhesion kinase and plasmin secretion, Circ. Res. 86 (2000) 952–959. [20] I. Kim, Y.S. Ryu, H.J. Kwak, S.Y. Ahn, J.L. Oh, G.D. Yancopoulos, N.W. Gale, G.Y. Koh, EphB ligand, ephrinB2, suppresses the VEGF- and angiopoietin-1-induced ras/mitogenactivated protein kinase pathway in venous endothelial cells, FASEB J. 16 (2002) 1126–1128. [21] H.S. Kim, H.S. Shin, H.J. Kwak, C.H. Cho, C.O. Lee, G.Y. Koh, Betacellulin induces angiogenesis through activation of mitogenactivated protein kinase and phosphatidylinositol 30 -kinase in endothelial cells, FASEB J. 17 (2003) 318–320. [22] J. Kroll, J. Waltenberger, The vascular endothelial cell growth factor receptor KDR activates multiple signal transduction
[23]
[24]
[25]
[26]
pathways in porcine aortic endothelial cells, J. Biol. Chem. 272 (1997) 32521–32527. G.D. Thakker, D.P. Hajjar, W.A. Muller, T.K. Rosengart, The role of phosphatidylinositol 3-kinase in vascular endothelial growth factor signaling, J. Biol. Chem. 274 (1999) 10002– 10007. H.P. Gerber, A. McMurtrey, J. Kowalski, M. Yan, B.Y. Keyt, V. Dixit, N. Ferrara, Vascular endothelial growth factor regulates endothelial cell survival through the phosphatidylinositol 30 -kinase/Akt signal transduction pathway, J. Biol. Chem. 273 (1998) 30336–30343. T. Takahashi, H. Ueno, M. Shibuya, VEGF activates protein kinase C-dependent, but Ras-independent Raf-MEK-MAP kinase pathway for DNA synthesis in primary endothelial cells, Oncogene 18 (1999) 2221–2230. L.W. Wu, L.D. Mayo, J.D. Dunbar, K.M. Kessler, M.R. Baerwald, E.A. Jaffe, D. Wang, R.S. Warren, D.B. Donner, Utilization of distinct signaling pathways by receptors for vascular endothelial cell growth factor and other mitogens in the induction of endothelial cell proliferation, J. Biol. Chem. 275 (2000) 5096– 5103.