Penta-O-galloyl-beta-D-glucose suppresses tumor growth via inhibition of angiogenesis and stimulation of apoptosis: roles of cyclooxygenase-2 and mitogen-activated protein kinase pathways

Jeong-Eun Huh, Eun-Ok Lee, Min-Seok Kim 1, Kyung-Sun Kang 2, Cheol-Ho Kim 3, Bae-Cheon Cha 4, Young-Joon Surh 5 and Sung-Hoon Kim *

Graduate School of East-West Medical Science, KyungHee University, Yongin 449-701, 1 Department of Molecular Biology, University of Wisconsin, Madison, Wisconsin 53706, USA, 2 Department of Veterinary Public Health, College of Veterinary Medicine, Seoul 151-742, 3 Department of Biochemistry and Molecular Biology, College of Oriental Medicine, Dongguk University, Kyungju 780-714, 4 Department of Applied Animal Sciences, College of Life Sciences and Natural Resources, Sangji University, Wonju 220-702 and 5 College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea

* To whom correspondence should be addressed Email: sungkim7{at}khu.ac.kr


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Recent studies have revealed that 1,2,3,4,6-penta-O-galloyl-beta-D-glucose (PGG) has anti-tumorigenic activity in vitro. In the present work, we evaluated the in vitro and in vivo antiangiogenic and antitumor activities of PGG and examined its molecular mechanisms. PGG significantly inhibited the proliferation and tube formation in basic fibroblast growth factor (bFGF)-treated human umbilical vein endothelial cells (HUVECs) at non-cytotoxic concentrations. PGG effectively disrupted the bFGF-induced neo-vascularization in chick chorioallantoic membrane (CAM) and in Matrigel plugs in the mice. When mice were intraperitoneally injected, PGG also significantly inhibited tumor angiogenesis induced by Lewis lung carcinoma (LLC) and the growth of LLC by 57 and 91% of control tumor weight at 4 and 20 mg/kg, respectively. Immunohistochemical analysis revealed decreased microvessel density, decreased expression of cyclooxygenase-2 (COX-2) and vascular endothelial growth factor (VEGF), reduced tumor cell proliferation and increased tumor cell apoptosis. Similarly, PGG significantly attenuated the expression of COX-2 and VEGF and reduced the secretion of VEGF and prostaglandin E2 in bFGF-treated HUVECs. Furthermore, the COX-2 inhibitor NS398 significantly inhibited tube formation and neo-vascularization in CAM, supporting the role of COX-2 in PGG inhibition of angiogenesis. PGG diminished the phosphorylation of extracellular signal regulated kinase 1/2, Jun NH2-terminal kinase and activated phospho-p38 mitogen-activated protein kinase (MAPK) in a dose-dependent manner in bFGF-treated HUVECs. In addition, p38 inhibitor SB203580 abolished the downregulation of COX-2, VEGF and the antiproliferative activity by PGG. Taken together, our data demonstrate that PGG exerts antitumor activity primarily via inhibition of angiogenesis through COX-2 and MAPK- dependent pathways.

Abbreviations: bFGF, basic fibroblast growth factor; CAM, Chick chorioallantoic membrane; COX-2, cyclooxygenase-2; ERK, extracellular signal regulated kinase 1/2; HUVECs, human umbilical vein endothelial cells; JNK, Jun NH2-terminal kinase; LLC, Lewis lung carcinoma; MAPK, mitogen activated protein kinase; PCNA, proliferating cell nuclear antigen; PGE2, prostaglandin E2; PGG, 1,2,3,4,6-penta-O-galloyl-beta-D-glucose; VEGF, vascular endothelial growth factor


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Angiogenesis, the formation of new capillaries by budding from existing vessels, occurs in tumors and permits their growth, invasiveness, and metastasis (1,2). Pathological angiogenesis occurs in diseases such as cancer, rheumatoid arthritis, endometriosis and diabetic retinopathy (3,4).

Neo-angiogenesis is now considered essential for solid tumor growth and progression. Any significant increase in tumor mass beyond 2–3 mm must be preceded by an increase in the vascular supply to deliver nutrients and oxygen to the tumor cells and the neo-vascularization of endothelial cells (5). Thus, angiogenesis inhibitors, which are not directly cytotoxic to tumor cells, can suppress tumor growth by inhibiting endothelial proliferation and migration and/or by inducing endothelial apoptosis in the vascular bed of tumors (6).

Cyclooxygenase-2 (COX-2) has been implicated in tumor growth, metastasis and angiogenesis (7), and is closely correlated with the microvessel density within tumors grown in animals (8,9). However, the adverse effects of synthetic COX-2 inhibitors Vioxx and celebrex have received much publicity, lately, and highlight the need for novel COX-2 inhibitors with less side effects for cancer prevention use.

Recently agents that inhibit angiogenesis and COX-2 have been identified from plants, with little side effects. Medicinal plants with anticancer activity have long been used for the treatment or prevention of various human disorders in folk medicine (10). Among biologically active phytochemicals, tannins and polyphenolic compounds have been in the spotlight with preventive effects for heart disease (11), chronic inflammation (12) and cancer (13).

By activity-guided fractionation, we isolated 1,2,3,4,6-penta-O-galloyl-beta-D-glucose (PGG) from Galla Rhois, gallnut of Rhus chinensis MILL used for the treatment of thrombosis and cancer in Oriental medicine (14). Recently, PGG was reported to inhibit in vitro capillary differentiation (tube formation) in endothelial cells (15), interieukin-8 gene expression in U937 cells (16), the proliferation of SK-HEP-1 cells (17), and the expression of inducible nitric oxide synthase and production of prostaglandin E2 (PGE2) in Raw 264.7 cells (18). It was also reported to protect rat neuronal cells from oxidative damage (19). However, the in vivo antiangiogenic and anticancer activities of PGG and its underlying mechanisms still remain unclear, despite substantial in vitro data. This prompted us to evaluate the in vivo as well as in vitro antiangiogenic and anticancer activities of PGG and to elucidate its molecular mechanisms with special focus on COX-2 and MAPK pathways in human umbilical vein endothelial cells (HUVECs) and Lewis lung carcinoma (LLC) cells. With respect to COX-2 targeting actions, we used a known selective inhibitor, NS398, as a reference to compare the anti-angiogenic and antitumor efficacies and to compare and contrast mechanistic commonalities and differences.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Isolation and identification of PGG from gallnut of Rhus chinensis MILL
Gallnut of Rhus chinensis MILL was obtained from the Oriental Medical Hospital of Kyunghee University in Seoul, and kindly authenticated by Professor Nam-In Baek of Department of Oriental Herbal Materials, Kyunghee University. The methanol (MeOH) extract (252 g) was dissolved in distilled water (800 ml), and successively fractionated with equal volumes of n-hexane, ethyl acetate (EtOAc) and butanol with water. The butanol fraction (35 g), which was most effective in terms of antiangiogenic activity, was subject to silica gel column chromatography and eluted by chloroform, MeOH and H2O (65:35:10) and EtOAc, MeOH and H2O (100:15.6:13.5), followed by purification using HPLC (J'sphere ODS-HP80, 250 x 20 mm ID, S-4 um, 80A, EtOAc:MeOH:H2O = 6:3:1). The active compound was identified as PGG (MW = 986) by NMR and FAB–MS analysis (Figure 1A). The purity of PGG was estimated to be >98%.



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Fig. 1. Structures of PGG (A) and NS398 (B).

 
Cell culture
HUVECs were isolated from fresh human umbilical cord veins by collagenase treatment as described previously (20). The cells were cultured in M199 (Invitrogen, Carlsbad, CA) supplemented with 20% fetal bovine serum (FBS), 3 ng/ml basic fibroblast growth factor (bFGF) (R&D Systems, Minneapolis, MN), 5 U/ml heparin and 100 U/ml antibiotic–antimycotic in 0.1% gelatin coated flasks. Mouse LLC cells were kindly provided by Dr Ikuo Saiki (Toyama Medical and Pharmaceutical Univ., Toyama, Japan) and cultured in EMEM (Invitrogen) supplemented with 10% FBS, 100 U/ml antibiotic–antimycotic and 2.2 g/l sodium bicarbonate. All cells were grown at 37°C in a humidified atmosphere containing 5% CO2.

Proliferation assay
Cell proliferation was determined using a 5-bromo-2'-deoxyuridine (BrdU) colorimetric assay kit (Roche, Sanhofer, Mannheim) according to the manufacturer's protocols. HUVECs (5 x 103 cells/well) were seeded onto 0.1% gelatin coated 96-well plates and incubated in a humidified incubator for 24 h. After being starved for 6 h in M199 containing 5% heat-inactivated FBS, the cells were exposed to various concentrations of PGG (1, 5 and 10 µM) in the presence or absence of bFGF (10 ng/ml) incubated for 48 h at 37°C. Then, 10 µl of BrdU (100 µM) was added to each well, and the cells were further incubated for 6 h at 37°C. The cells were fixed and incubated with anti-BrdU and then detected by the substrate reaction. The reaction was stopped by the addition of 25 µl of 1 M H2SO4 and the absorbance was measured using a microplate reader (Molecular Devices Co., USA) at 450 nm with 690 nm correction.

Tube formation assay
Tube formation assay was performed on Matrigel (Becton Dickinson Labware, Bedford, MA) as described previously (21). Matrigel (250 µl) was added to 24-well plates and allowed to solidify for 1 h at 37°C. HUVECs (1 x 105 cells/well) were treated with various concentrations of PGG (1, 5 and 10 µM) or NS398 (20, 40 and 80 µM) in the absence or presence of bFGF (10 ng/ml). After 18 h, randomly chosen fields were photographed under an Axiovert S 100 light microscope (Carl Zeiss, USA) at 100x magnification. Tube network was quantified using NIH Scion image program.

Chorioallantoic membrane assay
The ex vivo angiogenic activity was assayed using chorioallantoic membrane (CAM) assay as described previously (22). PGG (1 µg/egg) or NS398 (1.5 µg/egg) and bFGF (100 ng) were loaded onto one-fourth piece of thermonox disk (Nunc, Naperville, IL). The dried thermonox disk was applied to the CAM of a 10-day-old embryo. After a 72-h incubation, a fat emulsion was injected under the CAM for better visualization of the blood vessels. The number of newly formed blood vessels was counted. The experiment was repeated twice with 15 eggs for each group.

Matrigel plug assay
The Matrigel plug assay was performed as described previously (23). Briefly, 6-week-old C57BL/6 mice (Daehan Biolink, Chungbuk) were given subcutaneous injection of 0.5 ml of growth factor reduced Matrigel containing PGG (80 µg) or NS398 (80 µg), bFGF (300 ng/mouse) and heparin (5 U). After 7 days, mice were killed, and the Matrigel plugs were removed. To quantify the formation of functional blood vessels, the amount of hemoglobin (Hb) was measured using the Drabkin reagent kit 525 (Sigma, St Louis, MO).

Tumor-induced angiogenesis
LLC cells (5 x 105/50 µl) were intradermally inoculated on the back of 6-week-old C57BL/6 mice (Daehan Biolink). Three days later, PGG (4 and 20 mg/kg) or NS398 (4 mg/kg) were intraperitoneally administered for four consecutive days. Seven days after tumor inoculation, the mice were killed and the tumor-inoculated skin was separated from the underlying tissues. Tumor-induced angiogenensis was quantified by counting the newly formed blood vessels around LLC cells under a dissecting microscope.

Immunoassay for PGE2
PGE2, a product of arachidonic acid by prostaglandin synthase COX-2, was determined using an enzyme linked immunosorbant assay kit (Cayman, Ann Arbor, MI) according to the manufacturer's protocol. HUVECs were starved for 6 h in M199 containing 5% FBS and then treated with bFGF (10 ng/ml) containing PGG (0.01, 0.1, 1 and 10 µM). After a 24-h culture, the cells were rinsed and incubated in Hanks' HEPES buffer with 50 µM arachidonic acid (Cayman) for 15 min at 37°C. The Hanks' HEPES buffer samples were collected in triplicate for the PGE2 assay as described above.

Measurement of hVEGF
The level of hVEGF in the supernatant of HUVECs was measured with a commercially available ELISA kit (R&D systems, Minneapolis, MN). Briefly, HUVECs were starved for 6 h in M199 containing 5% FBS and then treated with bFGF (50 ng/ml) containing PGG (1, 5 and 10 µM). After a 48-h incubation, the supernatant was individually collected and measured by ELISA kit.

Western blotting
HUVECs exposed to various concentrations of PGG (1, 5, 10 and 20 µM) with or without bFGF (10 ng/ml) for 24 h were harvested and washed with cold PBS. The cells were incubated in lysis buffer (50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 0.1% SDS and 1 mM EDTA), supplemented with protease inhibitors (10 µg/ml leupeptin, 10 µg/ml aprotinin, 10 µg/ml pepstatin A and 1 mM of 4-(2-aminoethyl) benzenesulfonyl fluoride) and phosphatase inhibitors (1 mM NaF and 1mM Na3VO4) for 20 min on ice. Lysates were centrifuged at 14 000 g for 20 min at 4°C. The lysates containing 20 µg of protein were fractionated by SDS–PAGE and electrotransferred to a Hybond ECL transfer membrane (Amersham Pharmacia, Arlington Heights, IL). The blocked membranes were then immunoblotted with primary antibodies (1:1000 dilution) of COX-1 (Santa Cruz Biotechnology, Santa Cruz, CA), COX-2 (Becton Dickinson, Bedford, MA), VEGF (Santa Cruz Biotechnology), ß-actin (Sigma), phospho-ERK-1/2, phospho-p38, phospho-JNK, ERK, p38, JNK (Cell Signaling Tech., Beverly, MA). The proteins were visualized using enhanced chemiluminescence.

Mouse tumor model
LLC cells in subconfluent condition were harvested, resuspended in sterile PBS. LLC cells (2 x 104 in 100 µl PBS) were subcutaneously injected into the right flank of C57BL/6 mice (Daehan Biolink). Three days after tumor inoculation, mice were given intraperitoneal injection of PGG (4 or 20 mg/kg) or NS398 (4 mg/kg) every other day. Tumor volumes were measured every 3 days with a caliper, and calculated according to the formula [(1 x w2)/2], where l and w stand for length and width, respectively (2426). All mice were killed 18 days after tumor inoculation and the tumors were excised and weighed.

Immunohistochemistry
Tumor specimens were immediately removed from killed mice and prepared for histological examination. Tumors were fixed overnight in 10% neutral buffered formalin, embedded in paraffin, and sectioned to 4 µm thickness. The tumor sections were immobilized and deparaffinized by immersing in xylene, dehydrated in a graded series of ethanol and washed with distilled water. For antigen retrieval, the tumor sections were boiled in 10 mM sodium citrate buffer (pH 6.0) for 10 min and cooled at room temperature. After washing with Tris Buffered Saline (TBS), endogenous peroxidase activity was blocked by incubation in 3% H2O2–methanol for 10 min at room temperature. The sections were stained for proliferating cell nuclear antigen (PCNA) (Dako A/S, Glostrup, Denmark), and von Willebrand factor (vWF) (Dako A/S), COX-1, COX-2 and VEGF antibodies overnight at 4°C using ABC and DAB kits (Vector Lab., Burlingame, CA) and counterstained with Mayer's hematoxylin solution (Sigma). Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining was performed using a TdT-FragELTM DNA fragmentation kit (Oncogene, Boston, MA) and counterstained with Mayer's hematoxylin solution. All stained sections were photographed under an Axiovert S 100 light microscope (Carl Zeiss) at 400x magnification.

Proliferative index (%) = (No. of PCNA positive cells/total cells) x 100
Angiogenic index = No. of vWF positive vessels/area of region of interest (ROI) (mm2)
Apoptotic index (%) = (No. of TUNEL positive cells/total cells) x 100
COX-1 index (%) = (No. of COX-1 positive cells/total cells) x 100
COX-2 index (%) = (No. of COX-2 positive cells/total cells) x 100
VEGF index (%) = (No. of VEGF positive vessels/total cells) x 100

Statistical analysis
All values represent means ± SD. The statistically significant differences between control and sample groups were calculated by the Student's t-test.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
PGG inhibits bFGF-induced proliferation and tube formation of HUVECs
To assess the antiangiogenic activity of PGG, its inhibitory effects on proliferation and tube formation in bFGF-treated HUVECs were examined. PGG significantly inhibited the proliferation of bFGF-treated HUVECs in a dose-dependent manner with IC50 of 8 µM (Figure 2A). This inhibitory effect was not due to cytotoxicity of PGG in HUVECs, because PGG did not show any significant cytotoxic effect on HUVECs up to 20 µM (data not shown). The IC50 of cytotoxicity by PGG against HUVECs and LLC cells was over 75 µM (data not shown). Next, the effect of PGG and NS398 on capillary differentiation of HUVECs seeded on Matrigel was examined. PGG significantly reduced the formation of capillary-like structures in bFGF-stimulated HUVECs in a dose-dependent manner with IC50 of ~3 µM (Figure 2B and C). In comparison, NS398 showed a moderate inhibitory effect on bFGF-induced tube formation of HUVECs with IC50 of ~50 µM (Figure 2B and D).



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Fig. 2. PGG inhibits bFGF-induced proliferation and tube formation of HUVECs in vitro. (A) Proliferation assay using BrdU incorporation method. HUVECs were exposed to various concentrations of PGG in the presence or absence of bFGF (10 ng/ml) for 48 h. (B, C and D) Tube formation assay on Matrigel. HUVECs were treated with various concentrations of PGG or NS 398 in the absence or presence of bFGF (10 ng/ml). (B) Tube formation images were photographed under a microscope at 100x magnification. (C) and (D) Tube networks were quantified using NIH Scion image program. Experiments were repeated twice in triplicates. Values represent means ± SD. ###P < 0.001 versus unstimulated control; *P < 0.05, **P < 0.01 and ***P < 0.001 versus bFGF control.

 
PGG inhibits bFGF-induced angiogenesis in vivo
To evaluate the in vivo antiangiogenic activity of PGG and NS398, CAM and Matrigel plug assays were performed. PGG and NS398 significantly decreased the bFGF-induced vascularization in the CAMs at 1 and 1.5 µg/egg, respectively (Figure 3). In Matrigel plug assay, bFGF-loaded plugs from mice exhibited a reddish color indicating abundant red blood cells and, therefore, hemoglobin in the newly formed vasculature, whereas a light yellowish color was shown in the PGG/bFGF- or NS398/bFGF-loaded plugs (Figure 4A). PGG and NS398 significantly reduced the hemoglobin content to 30 and 45% of untreated control, respectively, at a dose of 80 µg per plug (Figure 4B).



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Fig. 3. (A) PGG disrupts capillary formation in CAMs. PGG or NS398 with or without bFGF was loaded on the CAMs of day 10 chick embryos. After 72 h incubation, a fat emulsion was injected into the CAMs for better visualization of the blood vessels. Thermanox and surrounding CAMs were photographed. (B) Numbers of newly formed blood vessels in CAMs were counted. n = 15. Values represent means ± SD. ##P < 0.01 versus unstimulated control; **P < 0.01 versus bFGE control.

 


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Fig. 4. PGG inhibits angiogenesis in bFGF-treated Matrigel plugs and tumor-induced angiogenesis in vivo. (A) C57BL/6 mice were subcutaneously injected with 0.5 ml of growth factor reduced Matrigel containing PGG (80 µg) or NS398 (80 µg), 300 ng bFGF, and 5 unit heparin. After 7 days, the Matrigel plugs were removed and photographed. (B) Functional blood vessels estimated by hemoglobin (Hb) content. n = 6. ##P < 0.01 versus normal control; ***P < 0.001 versus bFGF control. (C) Mice were intradermally inoculated with LLC cells and intraperitoneally administered with PGG (4, 20 mg/kg) for 4 days from day 3 post-tumor inoculation. Tumor inoculated sites were isolated from mice 7 days after tumor inoculation. The macroscopic observation of neo-vessel formation is shown. (D) Tumor-supplying vessels were counted. n = 7. Values represent means ± SD. ***P < 0.001 versus PBS-treated control.

 
PGG suppresses tumor-induced angiogenesis in vivo
To assess the effect on the formation of new blood vessels induced by tumor cells (27), LLC cells were intradermally inoculated on the back skin for tumor angiogenesis assay. As shown in Figure 4C and D, PGG and NS398 significantly reduced the number of vessels around LLC cells compared with untreated control.

PGG inhibits tumor growth in LLC-bearing mice
PGG (4 and 20 mg/kg/day) or NS398 (4 mg/kg) were intraperitoneally administered on alternate days for 17 days from day 3 after subcutaneous inoculation with LLC on the flank region of the mice. PGG did not cause any significant body weight loss during the experiment (final body weight for the control group was 20.11 ± 1.34 g, and with 4 and 20 mg/kg PGG it was 19.49 ± 1.27 and 18.69 ± 1.21 g, respectively). PGG significantly inhibited tumor volume over time (Figure 5A) and also suppressed the tumor weight to 43 and 9% of PBS-treated control at 4 and 20 mg/kg, respectively (Figure 5B) while NS398-inhibited tumor volume to 26% of PBS-treated control at 4 mg/kg.



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Fig. 5. PGG inhibits tumor growth in LLC-bearing mice. LLC cells (2 x 104 in 100 µl PBS) were subcutaneously injected into the right flank of C57BL/6 mice. Three days after tumor inoculation, mice were given intraperitoneal injection of PGG (4 and 20 mg/kg) or NS398 (4 mg/kg). (A) Tumor volumes measured with a caliper. (B) Tumor weight after necropsy. n = 10. Values represent means ± SD. *P < 0.05 and ***P < 0.001 versus PBS-treated control.

 
PGG treatment results in a decrease of microvessel density and tumor cell proliferation and an increase of tumor cell apoptosis in the LLC tumor model
To confirm that antiangiogenic activity and apoptosis were involved in the anticancer action of PGG, immunohistochemical examination of the tumor sections was carried out (Figure 6A). PGG significantly decreased the percentage of tumor epithelial cells positive for PCNA staining, a marker of proliferation (Figure 6B). PGG-treated tumors showed much decreased staining for vWF (Figure 6C), a marker of microvessel density, in comparison with PBS-treated control tumors. In contrast, PGG significantly increased the number of TUNEL positive tumor epithelial cells indicating apoptosis (Figure 6D).



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Fig. 6. PGG treatment results in a decrease of microvessel density and tumor cell proliferation and an increase of tumor cell apoptosis in LLC tumor model. (A) Representative immunohistochemical staining for PCNA (column 1), vWF (column 2), and TUNEL (column 3). The proliferative (B), angiogenic (C) and apoptotic (D) indices were calculated by the ratio of positive cells to total stained cells in tumor sections. Values represent means ± SD. **P < 0.01 and ***P < 0.001 versus PBS-treated control.

 
PGG inhibits COX-2 and VEGF expression in the LLC tumor model
The expression of COX-1, COX-2, and VEGF in the LLC tumor model was examined by immunohistochemical analysis (Figure 7A). PGG treatment resulted in a significant decrease in the expression of COX-2 and VEGF (Figure 7C and D), while PGG did not affect the COX-1 expression in the LLC cells (Figure 7B). The selective COX-2 inhibitor NS398 also inhibited VEGF expression as well as COX-2 expression.



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Fig. 7. PGG inhibits COX-2 and VEGF expression in LLC tumor model. (A) Representative immunohistochemical staining for COX-1 (column 1), COX-2 (column 2), and VEGF (column 3). The COX-1 (B), COX-2 (C) and VEGF (D) indices as the ratio of positive cells to total cells in tumor sections. Values represent means ± SD. **P < 0.01 versus bFGF control.

 
PGG downregulates COX-2 expression and reduces the levels of PGE2 and VEGF in bFGF-induced HUVECs
PGE2, a product of COX-2 and VEGF, has been linked to cancer (28,29). Therefore, the effects of PGG on COX-2 and VEGF expression were examined by western blotting. In addition, the levels of secreted PGE2 and VEGF were measured by ELISA in bFGF-treated HUVECs. PGG downregulated COX-2 and VEGF expression without affecting COX-1 in bFGF-treated HUVECs (Figure 8A). Likewise, PGG significantly decreased the PGE2 release and secreted VEGF level in a concentration-dependent manner compared with untreated control (Figure 8B and C).



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Fig. 8. PGG inhibits COX-2 and VEGF expression and reduces the secretion of PGE2 and VEGF in bFGF-induced HUVECs. (A) Expression of COX-1, COX-2, and VEGF in bFGF-treated HUVECs, detected by Western blot. HUVECs were treated with PGG with or without bFGF for 24 h. (B) bFGF-induced PGE2 release in HUVECs. The cells were exposed to PGG in M199 containing 5% FBS plus heparin and bFGF (10 ng/ml) for 24 h, and then PGE2 release was measured by ELISA. (C) VEGF secretion in bFGF-treated HUVECs. HUVECs were exposed to PGG for 48 h, the level of VEGF in conditioned medium was measured by ELISA. Values represent means ± SD. ###P < 0.001 versus unstimulated control; **P < 0.01 and ***P < 0.001 versus bFGF control.

 
PGG inhibits bFGF-induced angiogenesis via MAPK-dependent pathway
bFGF, a member of the heparin-binding multifunctional polypeptides, is one of the most potent angiogenic factors. It is also involved in proliferation and differentiation of a variety of normal and malignant cells and tissues (30,31). To understand the molecular mechanism by which PGG inhibits bFGF-induced angiogenesis, the potential involvement of MAPK pathway during antiangiogenic process by PGG was investigated by western blotting. PGG diminished the phosphorylation of extracellular signal regulated kinase (ERK) 1/2 in a dose-dependent manner. Interestingly, PGG completely blocked bFGF-induced phosphorylation of phospho-Jun NH2-terminal kinase (JNK) at a concentration of 1 µM. On the contrary, PGG activated the expression of p38 MAPK in a concentration-dependent manner in bFGF-treated HUVECs (Figure 9A). The p38 specific inhibitor SB203580, which did not affect JNK and ERK (Figure 9B), effectively blocked the downregulation of COX-2 and VEGF and the antiproliferative activity induced by PGG in bFGF-treated HUVECs at non-toxic concentrations (Figure 9C and D). These results support a critical role of p38 MAPK pathway in inhibiting COX-2 and VEGF expression and angiogenesis.



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Fig. 9. PGG inhibits bFGF-induced angiogenesis through MAPK pathways. (A) Effect of PGG on MAPK proteins. (B) Effect of p38 specific inhibitor, SB203580, on the expression of JNK and ERK. (C) Effect of SB203580 on COX-2 and VEGF expression by PGG in bFGF-treated HUVECs. (D) Effect of PGG on the proliferation of bFGF-treated HUVECs with or without SB203580 by BrdU assay. PGG treatment was for 54 h (48 + 6 h). Values represent means ± SD. +++P < 0.001 versus unstimulated control; ***P < 0.001 versus bFGF control; ##P < 0.01 versus PGG and bFGF treated group.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Angiogenesis plays a vital role in tumor growth, intravasation and metastasis (32,35). This process involves the proliferation, migration and tube formation of endothelial cells and can be initiated by angiogenic cytokines such as bFGF and VEGF (33). Our data showed that PGG, one of the well-known gallotannin precursors (34), significantly inhibited the proliferation and tube formation of bFGF-treated HUVECs with IC50 of 8 µM. This result was similar to the in vitro antiangiogenic activity of PGG in VEGF-treated HUVECs (15). PGG significantly inhibited the bFGF-stimulated capillary vessel formation in the CAM and also suppressed the vascularization in bFGF-treated Matrigel plugs in mice. These data support a potent antiangiogenic effect in vitro and in vivo. In addition, PGG suppressed LLC-induced angiogenesis without any adverse effect on body weight.

The strong antiangiogenic activity of PGG predicts antitumor activity in vivo, which we demonstrated with LLC cells inoculated into the flank of C57BL/6 mice. PGG suppressed the growth of LLC tumors by 57 and 91% at 4 and 20 mg/kg, respectively. Immunohistochemical analysis has revealed the decreased microvessel density, reduced tumor cell proliferation (PCNA index) and increased tumor cell apoptosis from the excised tumor sections. While our data are consistent with the hypothesis that PGG can exert antitumor activity via a primary antiangiogenesis effect, leading to tumor cell growth inhibition and apoptosis, the experimental design did not exclude a direct apoptotic effect of PGG on tumor cells. There is ample evidence in the literature that the apoptosis of endothelial cells in the vascular bed of tumors precedes apoptosis of tumor cells indicating the close relationship between antiangiogenesis and tumor cell apoptosis (6). Thus, additional works are required to sort out the cause–effect relationship between antiangiogenesis and tumor apoptogenic effects of PGG.

Mechanistically, our work examined the role of COX-2 in PGG-induced antiangiogenic process. PGG significantly attenuated the expression of COX-2 and VEGF as well as decreased secretion of VEGF and PGE2 in bFGF-treated HUVECs. COX-2 is known to be involved in tumor angiogenesis by promoting the expression of pro-angiogenic factors (3638). Hence, the decreased expression of COX-2 and VEGF found in tumor sections provided evidence that PGG is a COX-2 inhibitor with antiangiogenic activity in vivo. The COX-2 inhibitor NS398 significantly inhibited tube formation and neo-vascularization in CAM, much like another COX-2 inhibitor celecoxib (39), supporting the important role of COX-2 pathway as a target in angiogenesis inhibition by PGG.

In our molecular work to identify the role of MAPK pathway, PGG diminished ERK 1/2, and JNK phosphorylation and increased phospho-p38 MAPK in a dose-dependent manner in bFGF-treated HUVECs. Our data showed that p38 MAPK inhibitor blocked the anti-proliferative activity and the decreased COX-2 and VEGF expression by PGG in bFGF-stimulated HUVECs, supporting a critical role of p38 MAPK in the inhibition of COX-2 and angiogenesis by PGG with the other MAPK pathways also contributing to PGG-induced antiangiogenesis. Considering that p38 MAPK is generally activated by genotoxic agents or apoptosis (40), activation of p38 MAPK by PGG may mediate the apoptosis of endothelial cells during antiangiogenic process. Thus, it will be necessary to study further the apoptotic mechanism of PGG in HUVECs and tumor cells.

In conclusion, these results demonstrate that PGG exerts anticancer activity chiefly via the inhibition of angiogenesis through COX-2 and MAPK-dependent pathways and suggest that PGG can be a novel non-toxic cancer chemopreventive agent.


    Acknowledgments
 
This study was supported by grants from KOSEF, Ministry of Health and Welfare and Biogreen 21 program, Republic of Korea.

Conflict of Interest Statement: None declared.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received February 8, 2005; revised March 31, 2005; accepted April 10, 2005.





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